Quantitative Subcellular Study of Apical Pole Membranes from Chicken Oxyntic Cells in Resting ...

14 Pages·2003·4.3 MB·English

Quantitative Subcellular Study of Apical Pole Membranes from
Chicken Oxyntic Cells in Resting and ICH Secretory State
Cecilia S. Koenig, M6nica Dabik6, and Miguel Bronfman
Departmento de Biologia Celular, Universidad aci16taC de Chile, Casilla ,D-411 Santiago, Chile
Abstract. Vertebrate oxyntic cells, responsible for related to specific modifications in membrane fractions
gastric HCI production, undergo a remarkable mor- derived from the secretory pole of oxyntic cells. Mor-
phological reorganization in relation to their secretory phological and functional reorganization of oxyntic
cycle. In resting state, the luminal surface of the cells cells was closely correlated with changes in: the sedi-
is smooth; a peculiar system of endocellular mem- mentation pattern of the marker enzyme of the api- Do
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branes, the tubular system, occupies the luminal cyto- cal pole membrane (K-NPPase), the total activity of nlo
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plasm. Actin filaments frame a cortical network be- K-NPPase and nonmitochondrial Mg-ATPase, the ed
tween the tubular system and the luminal plasma valinomycin dependence of K-ATPase, and polypep- from
membrane. With the onset of 1CH secretion, the tubu- tides that cosediment in purified membrane fractions. http
lar system becomes incorporated into the luminal Changes in the distribution pattern of K-NPPase af- ://ru
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plfialsammean ts membrane. fill the secretory Villous surfacep.r ocesses This containing morphological micro- tceorns istent fractionation with the of hriestpalmaicneem-setnitm ulated of the small glands vesicles were ress.org
reorganization of membranes and cytoskeletal matrix typical of resting glands by dense membrane profiles, /jcb
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could regulate 1CH secretion by translocation of mem- analogous to the luminal processes of stimulated oxyn- rticle
branes containing the proton pump from the endocel- tic cells. SDS-PAGE showed that, in purified mem- -pd
lular compartment to the secretory surface. brane fractions of stimulated glands, the concentration f/10
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In this paper, we describe the isolation of mem- of 28-, 43-, and 200-kD polypeptides increased while /6/2
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branes that selectively belong to the tubular system or that of 95- and 250-kD polypeptides decreased. 5/1
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to the cytoplasmic processes of the secretory surface The present results define the tubular system of 55
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of chicken oxyntic cells. Chicken oxyntic cells are the oxyntic cells as an organelle with properties different 63
/2
main cellular component of the proventricular glands. from those of endoplasmic reticulum, mitochondria, 94
5
A resting state was obtained after cimetidine treat- and plasma membrane. The biochemical and physico- .pdf b
ment, whereas the HCl-secretory state was induced by chemical properties of this membraneous system changed y g
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histamine. when the organization of the membranes and the cyto- est o
We present a comparative analysis of resting and skeletal matrix of the apical pole was modified by the n 2
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stimulated chicken gastric glands by quantitative sub- onset of HCI secretion. Fe
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cellular fractionation. The 1CH secretory state was rua
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I T is widely accepted that translocation of organelles, lo- Vertebrate oxyntic cells provide an excellent model for
cal movements of the plasma membrane, and changes studying microfilament-membrane interactions during tran-
in cell shape depend on the integrated action of cellular sient events. The membranous systems proper of the secre-
membranes and the cytoskeletal network (Jacobson, ;3891 tory pole, the tubular system, and the plasma membrane
Geiger, 1983). The existence of different types of microfila- change their relationship with the actin cytoskeleton during
ment-membrane interactions, probably related to specific the secretory cycle of HCI (Vial and Garrido, ;6791 Vial et
cellular functions, has been clearly established (Tilney, al., ,9791 1985). In the resting state, the luminal surface of
;3891 Bennett and Condeelis, 1984; Jung et al., 1984, and oxyntic cells is smooth, and an extensively developed tubular
Mooseker et al., 1984). Transient microfilament-membrane system occupies the apical cytoplasm. Actin filaments com-
interactions that occur in epithelial cells during cyclic secre- prise a cortical network between the plasma membrane and
tory processes are less documented (Vial and Garrido, ;6791 the tubular system (Koenig et al., .)a1891 In cells actively
Tramontano et al., 1982; Osawa et al., .)4891 secreting HCI, the tubular system diminishes to point of dis-
appearance. At the same time the secretory surface markedly
Portions of this work have been published in abstract form .1891( Arch. BioL
Med. Exp. 14:R273; and 1982. Arch. Biol. Med. Exp. 15:R134). increases and becomes filled with cytoplasmic processes
(cid:14)9 The Rockefeller University P~ess, 0021-9525187/12/2945/14 $2.00
The Journal of Cell Biology, Volume 501 (No. 6, Pt. 2), Dec. 1987 2945-2958 2945
which expand the apical membrane area '~10-fold (Helander in vivo to obtain a defined resting or HCI secretory state
et al., .)2791 These processes contain parallel arrays of actin (Toner, 1963; Koenig, 1984).
microfilaments connected to the plasma membrane by regu- This paper presents a quantitative biochemical analysis of
larly spaced bridges (Vial and Garrido, 1976; Vial, 1982; chicken gastric glands in two secretory states: active 1CH
Vial et al., 1985). secretion induced by histamine and a resting state after
Thus, stimulation of HCI secretion induces the reorganiza- cimetidine administration. Morphological and functional
tion of both the actin cortical network and the membranous changes of oxyntic cells were closely correlated with mod-
systems of the secretory pole (Vial et al., 1985). While the ifications in the sedimentation pattern of the apical pole
membranes of the tubular system become associated with membranes. Differences were found in the total activity of
microfilaments, molecular interactions between actin and K-NPPase and Mg-ATPase associated with these membranes,
other cytoskeletal proteins of the cortical network change, as well as differences in the polypeptides that cosedimented
resulting in the reorganization of the secretory pole mem- in the purified membrane fractions.
branes (Gonz,~lez et al., ;1891 Black et al., 1982; Vial et al.,
1985; Dabik6 et al., 1986). This structural reorganization
Materials and Methods
precedes the onset of HCI secretion (Jiron et al., 1984).
Upon cessation of stimulus, the tubular system is reformed.
Animals dna Drug noitartsinimdA
These morphological changes could reflect the regulation of
the output of gastric 1CH by translocation of the membranous White Leghorn chickens (Callus domesticus) weighing ',,1.5 kg were used.
system containing the K/H-ATPase from the endocellular They were deprived of food 24 h before the experiment with water ad
libitum.
compartment to the luminal surface of the cell (Smolka et
To obtain resting glands, two doses of cimetidine were injected in- D
al., 1983). K/H-ATPase, recognized as the gastric proton traperitoneally (2 mg/kg body weight) 51 and 1 h before the experiment. own
pump (Forte et al., 1980; Sachs et al., 1982; Wallmark et al., To stimulate HCI secretion, 0.7 gan of histamine base per kg of body weight loa
d
1983), seems to be involved in the terminal steps of the acid were injected subcutaneously 40 min before the birds were killed. The doses ed
secretory process (Fellenius et al., .)1891 The catalytic cycle of cimetidine and histamine were chosen on the basis of studies correlating from
of gastric K/H-ATPase includes a K+-stimulated phospho- tcehlel' s secretory apical poles response in fasted and thec hickens. morphological organization of the oxyntic http
protein phosphatase, insensitive to ouabain, which has often ://ru
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been studied as a K+-p-hitrophenylphosphatase (K-NPPase)' noitaraperP of setanegomoH re
(Wallmark et al., 1983; Jackson et al., 1983; Hirst and Forte, ss.o
1985). Cytochemical studies have shown that, in oxyntic Gtliaonnd,u lar and the stomachs pH of thwee re gastric quickly fluid removed was measured from birds with killed pH papebyr decapita- to test rg/jcb
cceaillls,y changets he subcellular with the localization onset of acid of the secretion. K-NPPase In restindgr asti- aHnCdI the secretion. glandular The lobules opened were proventriculus surgically dissectweads left in from a petri the mduicsohs a on aincde , /article-p
cells, the K-NPPase reaction product is preferentially as- the muscle layers. Gastric glands were filtered through a tissue press (model df/1
sociated with the membranes of the tubular system, while in ;141 Harvard Apparatus Co. Inc., South Natick, MA) and weighed. Usually, 05
secreting cells it is concentrated in the plasma membrane of I-2 g of gland were obtained from each stomach. The tissue was placed in /6/2
a Teflon/glass grinder model A (Thomas Scientific, Philadelphia, PA) con- 94
the elaborated secretory surface (Koenig, 1984; Fujimoto et raining 5 ml of ice-cold homogenization medium (buffered sucrose: 0.25 M 5/1
0
al., 1986). sucrose, 3 mM imidazole-HCl, pH 7.4) per g of tissue wet weight. 55
6
1CH physiological secretagogues, acting at the basolateral Homogenization was performed with two strokes of the pestle driven at 63
plasma membrane, are known to activate the acid pump 1,000 rpm at 0~ /294
5
through second messengers (Berglindh, 1984). Although it .p
ralullecbuS noitanoitcarF d
has been found that in vivo stimulation of HCI secretion in- f by g
duces changes in the properties of membranes containing Homogenates were fractionated by differential centrifugation as described ue
K/H-ATPase (Wolosin and Forte, 1981a, b, 1983; Im et al., by de Duve et al. (1955) for rat liver, except that the nuclear (N) and heavy st o
1984) the molecular mechanism regulating the activation of mliigthotc hondriai mitochondrial (M) fract(iLo)n,s microsomal were sedimented (P), and final together supernatant (NM fraction). (S) fractions The n 22 F
HCI secretion is not yet fully understood. were obtained from the NM supernatants, essentially as described by these eb
The differences in structural organization of resting and authors. rua
secreting ceils may be used to isolate membrane fractions In other experiments, the NM supernatants were fractionated by filtration ry 20
corresponding either to the tubular system or to the secretory on Sepharose 2B. A total particulate fraction, corresponding to the sum of 23
the L and P fractions (LP), and a soluble fraction (S), containing the soluble
surface cytoplasmic processes. Chicken gastric glands are a
components of the homogenate (Bronfman and Beaufay, 1973), were ob-
suitable starting material for the preparation of oxyntic cells. tained by this method.
Although these cells secrete both acid and pepsinogen, The LP fraction was further subfractionated by isopycnic equilibration
chicken stomachs release more H + per kilogram of body in metrizamide-sucrose linear density gradients, extending from 1.05 to 1.22
g/ml. The gradients (5 ml) were prepared with a density-gradient former
weight than any mammal so far examined (Long, 1967;
(Beckman Instruments, Inc., Paio Alto, CA). The LP fraction (prepared in
Burhol, 1982). Oxynticopeptic cells account for over 80% buffered sucrose) was used for the light solution and a 39% wt/vol solution
of the cellular mass in glandular lobules (Farner, 1960). The of metrizamide in buffered sucrose was used for the heavy solution. The gra-
similar reorganization of oxyntic cells from a number of ver- dients were centrifugated for 60 min at 39,000 rpm in a SW-65 rotor (Beck-
man Instruments, Inc.) at 6-10"C. After centrifugation, 12-13 fractions were
tebrates, including chickens, has been well documented
collected by slicing the tube with a tube slicer (Beckman Instruments, Inc.)
(Vial, 1982; Vial et al., ;9791 Forte et al., .)a1891 In chickens,
fitted with a cooling jacket, at 0-2"C. Density measurements in each frac-
the morphofunctional state of oxyntic cells can be regulated tion were performed by the procedure of Beaufay et al. (1964).
Results from subcellnlar fractionation experiments are presented in the
.1 Abbreviations used in this paper: K-NPPase, K+-p-nitrophenylphospha - form of normalized histograms constructed according to procedures previ-
tase; L, light mitochondrial fraction; M, heavy mitochondrial fraction; N, ously described (de Duve et al., 1955; Beaufay et al., 1964; Leighton et al.,
nuclear mitochondrial fraction; P, microsomal fraction; S, supernatant. 1968).
The Journal of Cell Biology, Volume 105, 1987 2946
Enzyme Assays spectrin (220,000), myosin heavy chain (200,000), clathrin (180,000),
a-actinin (t00,000), IgMl (78,000), actin (43,000), DNase (31,000), IgM2
Established procedures were used for the determination of marker enzymes: (20,000), and lactoglobulin (18,400). Quantitative scanning densitometry of
catalase for peroxisomes (Leighton et al., 1968), NADPH cytochrome c Coomassie Blue-stained gels was used to determine the relative amount of
reductase for microsomal vesicles (Beaufay et al., 1974), cytochrome c oxi- polypeptides present in each fraction. The gels were scanned at 605 nm in
dase for mitochondria (Cooperstein and Lazarow, 1951), and acid phospha- a microdensitometer (KII; Canalco) with a Disc-Integrator and Printer Sys-
tase for lysosomes (Appelmans et al., 1955). Protein was measured by the tem (model A-25; Varian Associates, Inc., Palo Alto, CA).
method of Lowry et al. (1951), as modified by Wattiaux et al. (1978), using
BSA as standard. Phosphoglncomutase, a soluble-compartment marker, Results
was determined by the method of Ray and Roscelli (1964), as described by
Bronfman et al. (1984). Assay of 5'-nucleotidase was performed according
Correlation between Structure of Chicken Oxyntic
to Ray and Forte (1974). (K-EIYI'A)-ATPase, a marker of myosin (Ostlund
et al., 1978), was quantified as described by Koenig et al. (1981a). Cells and Gastric 1CH Secretory State
ATPase activities were assayed as reported for gastric glands (Ganser and
The morphological analysis of glands used for fractionation
Forte, 1973a, b; Forte et al., 1975; Saccomanni et al., 1977) with minor
modifications. The reaction mixture contained 2 mM MgC12, 2 mM ATE showed a very good correlation between oxyntic cell ultra-
0.t mM ouabain, and 01 mM PIPES-NaOH buffer, pH 6.8, in a final volume structure and gastric HCI secretory state. In each stomach,
of 0.1 ml. Incubations were performed at 3-/*C. Activation by K § was de- nearly 100% of the cells examined had the same structure.
termined in the presence of increasing KCI concentrations (from 1.0 up to
Fig. 1 typifies the ultrastructure of gastric glands isolated
50 mM). The amount of valinomycin necessary to reach a final concentra-
tion of 01 ltM was added to the incubation medium. ATPase activities were from secreting (Fig. ,1 a and b) and resting (Fig. ,1 c and
defined after Lee et al. (1979): Mg-ATPase is the activity detected in the ab- d) stomachs. After histamine stimulation, chicken oxyntic
sence of K+; K-ATPase is the increase in activity of Mg-ATPase after addi- cells showed a prominent secretory pole surrounded by an
tion of K*; valinomycin-stimulated K-ATPase is the additional increase in intercellular cleft filled with the complex processes of the lu-
activity of K-ATPase in the presence of the ionophore. K-NPPase was as- Do
minal surface (Fig. 1 a). The cytoplasmic matrix inside these w
sayed as for ATPases, except that 25 mM Tris-HCl, pH 7.2, was used as n
buffer, and 5 mM p-nitrophenylphospbate as substrate (Forte et al., .)5791 processes contained microfilaments whose associations to load
e
K-NPPase is defined as the increase in activity induced by 20 mM KCI in the plasma membrane were occasionally observed (Fig. 1 b). d
the basal activity detected in the presence of Mg .++ The kinetics of all en- In resting state, after fasting and cimetidine treatment, the from
zymes were examined in detail to find the optimum conditions of the assays h
atineds, were in particular, proportional to determine to enzyme the concentration. range within Ewnhziycmha tic the measured assays were veloci- per- prionjteecrticoenlsl.u lar The clefts tubular became system extremely filled the short cytoplasm and devoid adjacent of ttp://ru
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ftroartimoends in ofaliquots aliquots oafdd ed subcellular to the assay fractions medium suitably were: diluted. 10-300 Prote igtn~ for catalasec,o ncen- tof ramed the luminal a cortex surface interposed (Fig. between 1 c). The the cytostkuebluetlaalr system matrix and ress.o
oxid15-150 ase, gt~ 50-500 for NADPH-cytochrome g~I for acid phosphatase, c reductase, 10-150 0 .g~5I-5 for gt~phosphoglucomu- for cytochrome the luminal plasma membrane (Fig. 1 d). rg/jcb/a
tatsteg, for 50-500 ATPases, Itg foarn d 5'-nucleot5i-da2se0,0 ~g for K-NPPase. 500 gt~ for (K-EDTA)-ATPase; 1-50 ATPase, K-NPPase, and Marker Enzyme Activities rticle-p
Units of enzyme activity were defined as the amount of enzyme causing in Chicken Gastric Glands: Effect of Oxyntic Cell df/1
the transformation of 1 ~nol of substrate per min in the conditions of the Secretory State 05
assay. For catalase, I U of activity is the amount of enzyme destroying 90% /6/2
of the substrate in 1 rain in a 50-ml vol (Leighton et al., 1968). 1 U of ATPase activity in total homogenates and microsomal frac- 94
5
cytochrome c oxidase corresponds to the amount of enzyme that causes the tions was assayed under three experimental conditions: no /10
decadic logarithm of reduced cytochrome c to decrease by 1 U/min per 001 K + added (Mg-ATPase activity), K + alone, and K + plus 556
ml Substratiensc ubation and mixture enzymes (de Duve were obetta ined al., 1955). from Sigma Chemical Co. (St. valinomycin. 63/29
Louis, MO). Metrizamide (2-3-acetamido-5-N-methylacetamido-2-4-6- The Mg-ATPase activity and its sensitivity to K § and 45.p
triiodobenzamido2-deoxy-D-glucose) was obtained from Nyegaard and Co. valinomycin changed according to the secretory state of gas- df b
(Oslo, Norway). tric glands. In total homogenates from resting glands, y g
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Morphological Examination K-ATPase was not detected even in the presence of valinomy- est o
cin. In microsomal fractions, K + induced a %01 increase n
2
Lobules from all gastric glands were fixed in 3% glutaraldehyde in PES over the basal activity, whereas valinomycin produced a 20% 2 F
e
(I00 mM PIPES, pH 6.9, 001 mM KCI, 2 mM MgC12, and 0.25 M su- additional activation. In secreting glands, K-ATPase activity bru
carfotseer) extraction for 6 h at of room the proventriculutse mperature. Fixatainodn within was 51 performed rain after diismsmeecdtiaitoenl y at wmasi crosomes, detected recpornessiesntteinntg ly bota h 20% in increase total homogenates over the basal and ac- in ary 202
4~ Glands were washed overnight at 2"C in PES and postfixed for 1 h 3
tivity. In this case valinomycin was slightly inhibitory.
in I% OsO4 in 1.0 M cacodylate buffer, pH 7.2. The tissue was stained
with aqueous %1 uranyl acetate, dehydrated in acetone, and embedded in In general, K-ATPase activity was extremely labile and
Epon. Small aliquots of selected fractions were poured in an ice-cold solu- vanished after freezing or aging at 4~ On the other hand,
tion containing 3% glutaraldehyde in PES. After 2 h the fractions solidified
Mg-ATPase was very active and stable, remained unchanged
by fixation and were processed as described above. In some experiments,
for several months at -20~ and was not affected by aging
fractions were fixed in glutaraldehyde and tannic acid (Brunschwig et al.,
1982). Thin sections were stained with 2% uranyl acetate and lead citrate at 4~ hypoosmotic shock, or sonication.
and examined in a Siemens Elmiskop 201 electron microscope. The cytochemical reaction to detect Mg-ATPase on frozen
Samples of fractions from the metrizamide gradient were negatively sections of gastric glands (Fig. 2, a and b) shows that the en-
stained with %1 uranyl acetate or 0.5% phosphotungstic acid.
zyme is located at the luminal cytoplasm of resting oxyntic
cells (Fig. 2 a). Stimulation induced a change in localization
SDS-PAGE
of the Mg-ATPase reaction product, which then outlined the
SDS-PAGE was performed according to Laemmli (1970). Fractions were intercellular cleft of the apical pole of oxyntic cells (Fig. 2
dissolved in a solution containing 57 mM Tris-HCl, pH 6.8, %01 glycerol, b). The drastic change induced in the cellular distribution of
5% vol/vol 2-mercaptoethanol, 2% wt/vol SDS, and 0.002 % bromophenol
the Mg-ATPase by the onset of HCI secretion is identical to
blue, and heated for 3 min at 100~ 3 % stacking and %01 separation acryl-
amide gels were used. Molecular weight markers were filamin (250,000), that found for K-NPPase (Fig. 2 c and d) (Koenig, 1984).
Koenig et al. Subcellular Study of Chicken Oxyntic Cells 2947
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Figure .1 Ultrastructure of chicken oxyntic cells in resting and HCl-secreting glands. (a) Cross section through a glandular tubule, after
histamine stimulation. Deep intercellular clefts (c) surround the secretory pole. Cytoplasmic processes project from these clefts, especially
toward the junctional complex (j). Mitochondria (m) fill the cytoplasm. Zymogen granules are also present. (b) Intercellular cleft in a
histamine-stimulated oxyntic cell. Irregular cytoplasmic processes containing microfilamants and a dense granular matrix fill the clefts.
When sectioned at an appropriate angle, associations between microfilaments and membrane are observed (arrow). (c) Cross section
through gland tubules in resting state. The intercellular clefts (c) are very short and the luminal surface is smooth. The apical cytoplasm
contains the tubular system. (d) Luminal surface of resting oxyntic cells. The tightly packed tubules form a feltwork that excludes most
of the cytoplasmic matrix. A cortex is seen betv, v.en the tubular system and the luminal plasma membrane. Bars: (a and c) 5 I~m; (b and
d) 0.2 Ixm.
This finding suggests that at least part of the Mg-ATPase ac- was easily detected in homogenates of resting or HCI-
tivity seems to be related to an enzyme located in the mem- secreting chicken gastric glands. After addition of K § the
branous systems of the oxyntic cell's secretory pole. basal NPPase activity in homogenates increased by 75 %.
In contrast to K-ATPase, the associated K-NPPase activity In microsomal fractions this increase reached 200%. The
The Journal of Cell Biology, Volume 105, 1987 2948
Table I. Absolute Values of Enzymes and Proteins
in Homogenates from Resting and Stimulated Chicken
Gastric Glands
Absolute values
n Resting glands Stimulated glands
Cytochrome oxidase 5 46.21 + 9.63 49.71 + 9.70
Catalase 4 30.1 + 0.06 1.07 + 0.28
Acid phosphatase 3 3.69 + 0.61 3.58 + 0.93
NADPH-cytochrome c
reductase 4 0.54 + 0.12 0.49 + 0.08
5'-nucleotidase 4 1.66 + 0.58 2.13 + 0.41
Phosphoglucomutase 2 10.11 + 1.05 9.18 + 0.82
(K§ ATPase 2 0.58 + 0.03 0.77 + 0.06
esaPPN-+K 4 4.47 + 0.24 1.64 ___ 0.61"
§+gM 4 142.90 + 32.53 238.80 ___ 26.03~
Proteins 4 172.17 + 20.87 179.40 + 29.53
Absolute values refer to g 1 fresh weight of gastric glands. They are given as
means + SD, in units for enzymes and in milligrams for protein.
* Difference from resting glands significant at P < 0.006.
:~ Difference from resting glands significant at P < 0.001.
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The values of the absolute enzymatic activities and total ed
protein in homogenates of resting and secreting gastric fro
m
glands are listed in Table I. A drastic change was found in http
the absolute units of K-NPPase and Mg-ATPase as a result ://ru
of histamine stimulation. K-NPPase activity decreased to pre
one-third of that observed in resting glands, whereas the ac- ss.o
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tivity of Mg-ATPase was almost doubled. No significant /jcb
differences were observed in the activities of the other en- /a
zymes. rticle
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erugiF .2 Cytochemical staining for Mg-ATPase and K-NPPase in f/1
Differential Centrifugation 0
chicken gastric glands. (a-b) Mg-ATPase. Fixation was carried out 5/6
in 3% glutaraldehyde, 1.0 M phosphate buffer, pH 7.2. Frozen sec- The distribution patterns and the recoveries obtained in the /29
4
tions were incubated in the reaction medium containing 50 mM fractionation of resting and HCI secreting gastric glands are 5/1
0
Tris-glycylglycine buffer, pH ,0.7 1 mM MgCI, 4 mM ATP, and listed in Table II. To draw the distribution profiles shown in 55
6.3 mM Pb(NO3)2 for 5 min at room temperature. Sections were 66
rinsed in distilled water, treated with %1 yellow ammonium sulfide, Fig. 3, data were corrected for recovery as described by de 3/29
Duve et al. (1955). 4
anindd uced mounted by cimetidine. in glycerine The jelly. Mg-ATPase (a) Glandular reaction tubule prion duct resting state stains The distribution of cytochrome oxidase expresses the be- 5.pdf b
only the secretory pole of oxyntic cells and is concentrated in the havior of mitochondria in the fractionation. About 90 % of y g
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mine. luminal The cytoplasm. distribution (b) Gloandfula r Mg-ATPase tubule reaction after stimulation product drastically by hista- cthoins tained enzyme <4 wa% s of recocvyetroecd hrome in oxidase. the NM In fraction. stimulated P fracgtliaonnd s, est on
2
changes. The intercellular clefts appear heavily stained and the a small but significant increase in the average of cytochrome 2 F
secretory pole of each oxyntic cell is clearly outlined. In the inset oxidase recovered in NM fractions was observed. NADPH- eb
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ctehlel ular concentration clefts is clearly of reaction visualized. (c--d) product thien Frozen deeper sections zone of the after inter- 30 cytochrome c reductase, a microsomal marker, was concen- ary 2
trated in L and P fractions, with a high proportion in S frac- 0
2
rain incubation itnh e standard medium for gastric K-NPPase (Koe- 3
tion. This distribution was different from that found in liver,
nig, 1984). In glandular tubules in resting state, after cimetidine
where the enzyme has very low soluble activity (Beaufay et
(Fig. 3 c), the K-NPPase reaction product localized in the luminal
al., 1974). Over 50 % of catalase units were concentrated in
oxyntic cells cytoplasm. In the histamine-stimulated glands (Fig. 3
d), the K-NPPase cytochemical reactioount lined the secretoproyl e the L fraction. In contrast to what has been described in
of oxyntic ceils. It stained the rim of cytoplasm adjacent to the inter- liver, a low amount of this enzyme was present in the super-
cellular clefts. Bars, 01 .mt.t natant. This might be due to a lesser fragility of gastric
peroxisomes. A small but significant increase of catalase ac-
tivity in the S fraction was observed in stimulated glands.
Acid phosphatase, the lysosomal marker, was concentrated
K-NPPase activity was not affected by hypoosmotic shock or in the L fraction. The conventional plasma membrane marker
the presence of valinomycin, and remained stable during sev- 5'-nucleotidase was mainly distributed in fractions NM and
eral days at 4~ and for months at -20~ Based on these P. When N and M fractions were separated, the enzyme re-
results and previous cytochemical studies (Koenig, 1984; mained associated mainly to N, as it does in liver (Beaufay
Fujimoto et al., 1986) K-NPPase was chosen as a marker en- et al., 1974). The distribution of (K-EDTA)-ATPase showed
zyme for the membranous system of oxyntic ceils during that, although most of the myosin sedimented in fraction S,
quantitative subcellular fractionation. over 25 % of the enzyme was associated to fractions L and P.
Koenig et al. ralullecbuS Study of nekcihC citnyxO slleC 2949
6NtTSER SDNAL6 DETALUMITS SDNALG a differential decrease in enzyme activity in the P fraction.
This, taken together with the cytochemical redistribution of
EMORHCDTYC s ESADI(0O ;f
:l L I k K-NPPase already presented (Fig. 2, c and d), suggests that
membranes with K-NPPase activity that concentrated in P
TYC-HPDAN ESAC TCUDER fraction of resting glands sediment in the L fraction of stimu-
lated glands.
I o The changes in functional state of gastric glands were also
related to modifications in the distribution pattern of Mg-
ATPase. Most of the activity of this enzyme, both in resting
and secreting glands, was concentrated in L and P fractions.
A significant amount of activity was also found in the NM
> i i
fraction, apparently associated with mitochondria. When N
.~~ 0 2 F ~ O'C AESATAHPSOHPI
and M fractions were isolated, activity was concentrated in
i o M fraction (not shown).
w 2 ESADITOELCUN-'5
In stimulated glands, the percent of Mg-ATPase activity
sedimenting in the NM fraction decreased significantly,
~-- -*K( )ATDE - esaPTA
while it increased in the L fraction. This phenomenon may
be related to the almost twofold increase of the enzyme's ab-
6
solute activity in secreting glands (Table I). Estimations
based on the percent of Mg-ATPase activity in fractions from
D
resting glands (Table II) and on the total Mg-ATPase activi- ow
n
o r" :I ./ ties (Table I) showed that, although upon stimulation the rel- load
ative specific activity in the NM fraction decreased, the ed
specific activity (expressed as units of Mg-ATPase per milli- from
MN L P S MN L P S grams of protein) remained constant. At the same time, the http
; ~ ,;o o os 001 specific activity of Mg-ATPase sedimenting in fractions L ://ru
p
SNIETORP )%( and P increased 3.4- and 1.75-fold respectively. This suggests re
erugiF .3 Distribution pattern of constituents after fractionation of that changes in the distribution pattern of Mg-ATPase, as- ss.o
resting and stimulated chicken gastric glands by differential cen- sociated with the gland functional state, are the consequence rg/jcb
trifugation. II. sedimentation right: Fractions NM (nuclear Graphs are of plotted their were + subcellular heavy in constructed the mitochondrial), order components, from of the the average data L i.e., (light given coefficient from mitochon- in left Table otfo otchyfet o chemical a L specific and P refidrnaiccstrtieroainssb.eu t ion in Based the of Mg-AMTgP-aAsTeP ase on these results (Fisg.e dimentation 2, and a and on thbe) , in /article-pdf/10
it can be proposed that membranes with nonmitochondrial 5
drial), P (microsomal), and S (superuatant) fractions. Each fraction /6
is represented separately in the ordinate scale by the relative Mg-ATPase activity that concentrate in P fraction of resting /294
specific activity of the constituent (percentage of total amount per glands sediment in the L fraction of stimulated glands. 5/1
0
percentage of total proteins recovered in the fraction). In the ab- Finally, the distribution pattern of proteins changed with 55
6
scissa scale each fraction is represented accumulatively by sti per- the gland secretory state. Proteins present in the L fraction 63
/2
centage of protein. of stimulated glands increased significantly, while they de- 94
5
creased in the S fraction (Table II, Fig. 3). .pd
f b
y g
Morphological Examination of L and P Fractions u
The distribution of K-NPPase displayed a remarkable e
change as regards the gastric gland functional state. Fraction The high ratio of K-NPPase activity in the P fraction of rest- st on
2
P of resting glands concentrated more than 90 % of K-NPP- ing glands agrees well with their enrichment in membranous 2 F
e
ase units, with a relative specific activity over .6 Stimulation smooth vesicles (Fig. 4 a), presumably generated from the bru
by histamine induced a change in the distribution pattern of tubular system of oxyntic cells (Fig. ,1 c-d). In L and P frac- ary 2
the enzyme. In fraction P the activity was reduced to ,070 %, tions of stimulated glands, the change in sedimentation prop- 02
3
with a concomitant increase of 20% in fraction L. This be- erties of K-NPPase was associated with the presence of het-
havior can be explained by a change in the sedimentation pat- erogeneous membrane profiles that enclosed cytoplasmic
tern of the vesicles containing the enzyme. matrix (Fig. 4 b). Such profiles may correspond to fragments
However, since we have shown that the K-NPPase total ac- of the cellular processes present in stimulated oxyntic cell
tivity of the homogenate decreases by (cid:127)65 % with stimula- secretory surface (Fig. ,1 a-b).
tion (Table I), a differential decrease of enzyme activity in In P fractions of resting glands the distinctive membrane
the P fraction of stimulated glands could have occurred. structure of the tubular system was seen after tannic acid
Nevertheless, no significant increase in the relative specific staining (Fig. 5). The unit membrane of the vesicles showed
activity of this enzyme was found in NM and S fractions. a thick inner dense lamina. Most vesicles presented their cy-
Calculations based on the percent of K-NPPase activity in toplasmic side to the medium, but some of them were in-
fractions from resting glands (Table II) and on the total versely sealed. Occasionally, pentalaminar membranes were
K-NPPase activities (Table I) demonstrate that the activity in seen between closely associated vesicles, as well as cytoplas-
the L fraction could have increased at most up to 01 %, while mic fragments enclosing a poorly structured matrix and
we found that it increased by more than 22 %. This shows that membranous vesicles.
the percentage of enzyme activity only increases in L frac- In P fractions of stimulated glands, the abundant cytoplas-
tion, and that this increase is higher than that predicted by mic fragments contained microfilaments apparently associ-
The Journal of Cell Biology, Volume 105, 1987 2950
elbaT LI Enzyme noitubirtsiD after laitnereffiD noitagufirtneC of nekcihC cirtsaG sdnalG in Resting and gniterceS lCH setatS
Functional
state n NM L P S Recovery
Proteins R 4 37.8 + 1.1 7.1 4- 1.8 15.0 4- 2.7 39.9 4- 0.7 101.2 4- 7.1
S 4 39.1 -4 2.0 10.5 -4 1.8" 17.3 -4 3.7 33.0 -4 2.1" 101.3 -4 5.6
Cytochrome oxidase R 5 88.2 + 2.6 6.8 + 2.0 3.2 + 2.2 7.1 + 6.1 1.99 + 6.0
S 5 91.8 -4 1.5" 5.8 -4 0.5 9.1 -4 4.1 0.8 + 0.7 108.9 + 12.5
NADPH-cytochrome c reductase R 4 17.5 + 1.4 17.2 -4 6.7 36.6 + 6.8 28.4 -4 0.4 108.9 -4 11.9
S 4 17.4 -4 2.6 1.22 + 5.2 33.0 -4 4.0 28.5 -4 4.9 5.511 -4 3.5
Catalase R 4 14.8 + 2.9 53.2 -4 8.9 16.8 + 7.8 1.51 -4 0.9 5.301 -4 5.8
S 4 14.4 -4 2.0 56.7 -4 2.1 10.2 -4 1.1 18.7 -4 1.7" 99.9 -4 10.5
Acid phosphatase R 3 36.2 -4 7.4 18.8 + 3.7 18.3 + 6.5 26.8 -4 16.0 98.2 + 10.9
S 3 34.8 -4 4.9 21.4 5:2.9 24.3 -4 7.6 19.6 -4 12.2 106.9 -4 8.9
esaditoelcun-'5 R 4 37.5 -4 2.0 6.6 -4 0.1 28.1 + 3.9 27.8 -4 4.4 92.8 -4 9.6
S 4 20.9 -4 7.0 7.2 -4 2.5 24.9 -4 5.5 27.1 -4 0.9 88.8 -4 15.7
(K § + EDTA) ATPase R 2 9.6 -4 8.1 6.4 -4 8.1 16.3 -4 3.2 67.6 -4 3.8 114.7 -4 1.9
S 2 12.4 -4 0.9 6.5 5:0.4 14.2 -4 3.2 66.8 + 12.2 108.7 -4 1.5
esaPPN-+K R 4 2.0 -4 7.1 3.7 -4 2.1 90.3 -4 4.3 4.0 -4 2.8 108.0 -4 12.5 Do
w
S 3 1.2 -4 0.1 22.5 -4 3.1" 69.5 -4 6.0* 6.0 -4 2.9 1.901 -4 12.1 n
lo
a
d
esaPTA-++gM R 4 36.4 -4 5.4 15.9 -4 7.3 39.8 -4 9.3 7.8 -4 2.9 3.201 -4 1.8 e
d
S 4 23.4 -4 0.7* 32.4 -4 11.3" 42.0 -4 11.2 2.2 -4 0.5* 103.8 -4 4.6 fro
m
h
Results are given as means + SD, in percentage of the recovered total activity. ttp
* Statistically significant difference with respect to the same fractions from resting glands. ://ru
p
re
ss.o
rg
ated with the membrane (Fig. 6). After tannic acid staining, turn, the soluble fraction was defined by the distribution of /jcb
/a
tlhaem ina. plasma Their membrane structure of was the compfarraagmbelnet s showed to that a of thick the apical inner phosphoglucomutase.A high Mg-ATPase activity persisted in LP-Sepharose rticle-p
d
processes of stimulated oxyntic cells (Vial, 1982). fractions. The sensitivity to K + stimulation was slightly in- f/1
0
creased. Besides, a significant and reproducible activation 5/6
/2
was observed in the presence of K § and valinomycin. 9
Fractionation of Postmitochondrial Supernatants 45
K-NPPase, Mg-ATPase, and K-valinomycin-ATPase fol- /1
by Chromatography in Sepharose 2B 05
lowed exactly the distribution of cytochrome oxidase. No ac- 5
6
6
The biochemical results presented raise several issues. First, tivity of these enzymes was found in the soluble fractions. 3/2
9
the finding of membrane marker enzymes in S fractions On the other hand, NADPH-cytochrome c reductase and 45
(25 % of NADPH-cytochrome c reductase and 5'-nucleoti- 5'-nucleotidase showed that the soluble fraction contained a .pdf b
dase and 8% of K-NPPase and Mg-ATPase) suggests that proportion of these enzymes similar to that found after y g
u
stioonm.e Secpoanrdt,i culate the componenlotws or nonexistent did not seadcitimveintty of by K-ATPase centrifuga- in differential It is noteworthy centrifugation. that no activity of the soluble marker phos- est on
2
homogenates raises the question that avian gastric glands phoglucomutase was found in the particulate LP-Sepharose 2 F
e
may have a pool of K + responsible for endogenous stimula- fraction. If the decrease in proteins in the soluble fraction bru
tion of the basal Mg-ATPase activity. Quantification of K + of stimulated glands observed in differential centrifugates ary 2
content in homogenates by flame photometry gave values of had resulted from trapping of soluble proteins by sediment- 02
3
'~130 meq/liter. This figure decreased to %01 of the initial ing membrane vesicles, it might be expected that at least a
value after dialysis of the homogenates. Finally, the decrease small amount of phosphoglucomutase would be associated
in protein content of the stimulated gland S fraction might with particles. Since phosphoglucomutase was not found in
be the result of soluble proteins trapped by membrane frag- the particulate LP fraction, the decrease in soluble proteins
ments. observed in stimulated glands after differential centrifuga-
Isolation of the K-NPPase-containing vesicles by exclu- tion would correspond to specific proteins. The morphologi-
sion chromatography of postmitochondrial supernatants on cal evidence already presented in Fig. 6 suggests that part of
Sepharose 2B was used to clear up these issues. This tech- them correspond to cytoskeletal proteins.
nique gives a better separation between the soluble compo- Elution profiles similar to those shown in Fig. 7 were ob-
nents and the organelles and vesicles. tained when resting gland postmitochondrial supernatants
Fig. 7 shows the elution profiles of proteins and marker en- were filtered on Sepharose 2B. A reproducible activation of
zymes after filtration of postmitochondrial supernatants Mg-ATPase by K + and valinomycin was also observed in the
from stimulated glands on Sepharose 2B. The distribution particulate LP-Sepharose fraction of resting glands. The ab-
profile of cytochrome oxidase sharply defined the particulate solute values and percentage of recovered activities of en-
fraction, which corresponded to the sum of L and P fractions zymes and proteins were quite similar in LP-Sepharose frac-
from differential centrifugation (LP-Sepharose fraction). In tions obtained from stimulated and resting glands. However,
Koenig et al. Subcellular Study of Chicken Oxyntic Cells 2951
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
://ru
p
re
ss.o
rg
/jcb
/a
rticle
-p
d
f/1
0
5
/6
/2
9
4
Figure 4. Electron micrographs of P fractions isolated from resting and stimulated chicken gastric glands. (a) The P fractiofnr om resting 5/1
glands consists mainly of smooth membranous vesicles with diameters between 1.0 and (b) 0.2 I~m. The P fractiforno m stimulated glands 05
5
contains elongated membranous profiles with diameters between 0.2 and A 0.4 Ixm. dense granular and filamentous material,a nd occasion- 66
3
ally small vesicles, are seen inside the membranous profiles (arrow ). 1 A wef free small vesicles like those in Fig. 4 a are also observed /29
4
(arrow 2). Bar, 0.5 lam. 5.p
d
f b
y g
u
e
st o
n
2
2
F
e
b
ru
a
ry 2
0
2
3
Figure .5 A high magnification electron micrograph showing the distinguishing features of the membranes present in the P fraction of resting
glands in samples stained with tannic acid. (a) Membrane vesicles similar to those observed in Fig. 4 a. Most of them show thet hickening
of the cytoplasmic sheet of the unit membrane (arrow 1). Some vesicles with an opposite orientation are occasionally seen (arrow 2).
A dense material appears associated with the external surface of some vesicles (arrow 3). (b) Membrane-surrounded structures showing
a heterogenous content, corresponding either to vesicles (left) or to a poorly structured matrix (right), are observed. Bars: (a) 1.0 tun;
(b) 0.2 .mxI
ehT lanruoJ fo Cell ,ygoloiB ,501 emuloV 7891 2592
D
o
w
n
erugiF .6 Details of the membranous present profiles fraction in P of stimulated glands after acid tannic staining. Microfilaments are clearly loa
d
seen inside the membrane-surrounded profiles. (a, c, d, and e). When sectioned at an appropriate angle, the thickening of the cytoplasmic ed
sheet of the membrane is observed. Connections between the cytoplasmic membrane and microfilaments are seen .)sworra( Occasionally, fro
m
vesicles surrounded by a filamentous matrix were found inside these profiles (e). Bars: (a) 0.2 ~tm; (b-e) 0.05 gm. h
ttp
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p
tinh e resting difference and in HC1 activity secreting of Mg-ATPase gland homogenates and K-NPPase persisted. found oupfo n K-NPPase the sensitivity in LP-Sepharose to + K concentration. fractions of The stimulated lower activity glands ress.org
For K-NPPase, the functional state of the glands had no effect was not modified by valinomycin or deoxycholate. Yet, the /jcb
/a
sine nsitivity relation ofto the the enzgylamned secretory to substrate state. concentration changedU pon stimulation, a rticle-p
d
slight but significant difference was found between the mK f/1
2 SNIETORP and the x~mV of K-NPPase: mK increased from 1.75 to 4.75 05/6
1 and xmV changed from 0.135 to 0.130. The kinetic param- /294
5
eters of basal Mg-ATPase were not affected by the gland /1
0
5
3 ACID ESATAHPSOHP secretory state. 56
6
3
/2
9
3 Subfractionation of LP-Sepharose Fractions 45.p
d
by Centrifugation in Linear Density Gradients f b
of Metrizamide y gu
3 TYCmHPDAN C ESATCUDER The density distribution of enzymes and proteins after iso- est on
2
pycnic equilibration of LP-Sepharose fractions of resting and 2 F
stimulated glands is shown in Fig. .8 The distribution of eb
ru
ESALATAC K-NPPase demonstrated that fractionation in metrizamide ary 2
gradients clearly resolved the tubular system from micro- 0
2
3
somes (NADPH-cytochrome c reductase) and mitochondria
(cytochrome oxidase), the two other main components of the
LP-Sepharose fraction, on a protein basis. This fractionation
5'- ESADITOELCUN method also clearly separated gastric peroxisomes from the
other organelles.
After subfractionation of LP-Sepharose fractions from
o ~ ~o ~ resting glands the distribution of K-NPPase was clearly bi-
VOLUME I%) modal. A high proportion of the enzyme equilibrated be-
erugiF .7 Elution profiles of proteins and enzymes from stimulated tween densities of 1.06-1.08 g/ml, with a second peak at a
gastric gland postmitochondrial supernatant. Filtration was carded
density of,o1.125 g/rnl. Stimulation induced a drastic change
out in a Sepharose 2B column (2.5 x 0.5 cm) equilibrated with
in this distribution pattern and most of the K-NPPase ap-
0.25 M sucrose, 3 mM imidazole-HCl, pH Ordinates 7.4. represent
peared to be associated with membranes equilibrating at a
the relative concentration of each component (units/ml per C,
density of 1.12g/ml or higher. This change was quantitatively
where C is the total amount of activity recovered, divided by the
volume). recovered total In the abscissa fraction represented each is represented by an increase in the median equilibrium density
cumulatively by its percentage of the total volume eluted. The re- of K-NPPase from 1.0878 + 0.0136 g/ml in resting glands,
covered activities varied between 80 and 021 %. to 1.1310 =5 0.0129 g/ml in stimulated glands. The median
gineoK te .la ralullecbuS ydutS of nekcihC citnyxO slleC 3592
GNITSER DETALUMITS of 1.125 g/ml. Judging by the behavior of cytochrome oxi-
dase, only a small proportion of Mg-ATPase appeared to be
localized in mitochondria.
The specific activity of K-ATPase and its sensitivity to
valinomycin were determined in the low density fractions of
the gradient in one of the paired experiments shown in Fig.
.8 Results are presented in Table III. The specific activity
of Mg-ATPase found in fractions of stimulated glands was
about three times higher than that detected in resting glands.
The activation of Mg-ATPase by K + (K-ATPase), was -71
fold greater in membranes of stimulated glands, whereas the
enzyme activity in the presence of valinomycin was only 1.3-
fold greater than that in membranes of resting glands. It is
noteworthy that membranes isolated from resting glands in
the low density region of the gradient showed a purifica-
tion higher than that of stimulated glands (70 and 20 % of
w
K-NPPase total activity, respectively). It is thus possible that
the increase could have been even higher in Mg-ATPase and
K-ATPase specific activities in membranes of stimulated
glands. On the other hand, in the absence of valinomycin,
D
almost no activity of K-ATPase was detected in membranes ow
n
of resting glands, whereas in membranes of stimulated lo
a
d
glands, a 1.7-fold increase of the activity was induced by the ed
ionophore. fro
m
h
ttp
Polypeptide Composition of Membrane Fractions ://ru
Purified by Isopycnic Centrifugation pre
1,10 1.11. LIII t.22 1.tO .M1 I.III 1.22 in Metrizamide Gradients ss.org
DENSITY (g Iml) The SDS-PAGE polypeptide pattern of fractions obtained by /jcb
erugiFrose fractions .8 Distribution of gastric of glands enzymes in resting after fractionation and HCl-secreting of -ahpeS-PL states isopycnic centrifugation of LP-Sepharose preparations from /article
resting and stimulated glands is shown in Fig. 9. Distribution -p
in continuous metrizamide density gradients. Standardized and -va of the enzymes in these experiments was similar to that in df/1
erage results era shown. Ordinates represent average frequency of 05
the components of each fraction, Q/ZQ'Ap, where Q represents the Fig. .8 In resting glands, '~70% of the K-NPPase units was /6/2
activity found in the fraction, ZQ the total activity recovered, and concentrated in the light density peak corresponding to frac- 945
Ap the increment in density from top to bottom of the gradient tion .1 This fraction was enriched in polypeptides of 250, /10
5
in the centrifuge tube. Frequency is plotted against density. Numer- ,081 130, 95, 48, 40, 34, 30, and 12 kD. In stimulated glands, 56
6
als in parenthesis refer to number of experiments. Vertical lines "~20% of the K-NPPase units was associated with fractions 3/2
9
through histogram bars represent DS of frequency. The demarca- 1 and 2 of the light density region of the gradient whereas 45
.setion liforp line The at density recovery 521.1 of enzymes allows easier and proteins comparison varied of distribution between 57 65 of K-% NPPase was present units. in Only fractions slight 6 differentcoe s ,8 the heavy were density observed peak in .pdf by g
and .%031 the polypeptide patterns of the heavy density peak fractions uest o
of resting and stimulated glands when polyacrylamide gels n
2
loaded with similar amounts of protein were analyzed by 2 F
e
equilibrium densities and the distribution profiles of the densitometric scanning. brua
other marker enzymes assayed remained unaltered, except terns In contrasotf, the light major density differences peaks of were K-NPPase observed activity in the (Fig. pat- ry 202
for Mg-ATPase and total protein. Although no significant 3
.)01 The main changes were observed in polypeptides of 250,
difference was observed in the median equilibrium density
200, 180, 160, 95, 43, 40, and 28 kD. Polypeptides of 250 kD
of total protein, the change in the distribution of K-NPPase
(which co-migrated with filamin), 081 kD (which co-mi-
correlated with a change in the distribution of protein, which
was found to be bimodal in stimulated glands. The protein
peak at 521.1 g/ml seems to correspond to the increase in the
elbaT IlL Specific Activity of esaPTA-K ni
K-NPPase equilibrated at this density. A small amount of
deifiruP senarbmeM from detalumitS and Resting :sdnalG
NADPH-cytochrome c reductase also appeared to be as-
Effect of nicymonilaV
sociated with the main peak of K-NPPase in stimulated
glands. Resting glands Stimulated glands
K-ATPase activity was assayed in some of the experiments
No additions 0.092 1.576
pooled in Fig. .8 The distribution of this enzyme was quite
Plus valinomycin 2.064 2.697
similar to that of K-NPPase. Mg-ATPase partly followed the
distribution of K-NPPase. In gradients of stimulated glands, Enzymatic activities are expressed in p.mol/min per mg of protein. They were
an apparent decrease of Mg-ATPase was found at the low assayed in membranes equilibrating at density 1.06-1.08 g/ml in metrizamide
gradients (see Fig. 8), which contained respectively 70 (resting) and 20 (stimu-
density region of the gradient and an increase at a density lated) % of the total K-NPPase activity in the gradient.
The Journal of Cell Biology, Volume 105, 1987 2954