Nucleic Acids, Proteins and Carbohydrates

236 Pages·1976·6.73 MB·English

by  F. Korte (Eds.)

Methodicum
Chimicum
A Critical Survey of Proven Methods
and Their Application in Chemistry,
Natural Science, and Medicine
Editor-in-Chief
Friedhelm Korte
Volume 11
Natural Compounds
Part 1
Nucleic Acids, Proteins
and Carbohydrates
Academic Press New York - San Francisco * London 1976
Georg Thieme Publishers Stuttgart
Maruzen Co. Tokyo
Nucleic Acids,
Volume 11
Part 1
Proteins
and Carbohydrates
Edited by F. Korte and M. Goto
Contributions from
K. Anno, Tokyo K. Narita, Osaka
R. M. Flugel, Heidelberg H. Nikaido, Berkeley
Th. Godefroy-Colburn, Paris S. Nishimura, Tokyo
K. Hotta, Sagamihara K. Ohno, Sapporo
Y. Inoue, Tokyo K. Onoue, Fukuoka
S. Iwanaga, Osaka E. Ohtsuka, Osaka
Y. Iwanami, Tokyo T. Shimojo, Sapporo
I. Kato, Tokyo S. Suzuki, Nagoya
O. Minari, Sapporo M. Tsuboi, Tokyo
A. Misaki, Osaka T. Uchida, Tokyo
K. Miura, Mishima
Academic Press New York · San Francisco · London 1976
Georg Thieme Publishers Stuttgart
Maruzen Co. Tokyo
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Library of Congress Catalog Card Number: 74-21580
ISBN 0-12-460711-X (Academic Press)
ISBN 3-13-506201-5 (Thieme)
© 1976 M. Goto, Gakushuin University, Tokyo—Printed in Japan by International Academic Printing Co., Ltd.
Preface of the Series
The METHODICUM CHIMICUM is a short with particular respect to the concepts and ap­
critical description of chemical methods applied plications of theoretical chemistry essential to
in scientific research and practice. It is parti­ the practically working scientist.
cularly aimed at chemists as well as scientists Volume 3 (Types of Reactions) is designed to
working in associated areas including medicine illustrate the scope and utility of proved working
who make use of chemical methods to solve techniques and syntheses.
their 'interrelated' problems. The second part (Vols. 4-8), which is particular­
Considering the present development of science ly devoted to 'Systematic Syntheses', deals with
and the necessity for concise and unambiguous proved methods for syntheses of specific com­
information, the series provides a guide to rapid pounds. These procedures are classified accord­
and reliable detection of the method suitable for ing to functional groups linked together in the
the solution of the problem concerned. Thus, last step of reaction.
particular emphasis is placed on the description Volume 4 (Syntheses of Skeletons) describes the
of proved procedure whereby a complete and construction of hydrocarbons and heterocyclic
exhaustive compilation of all reported methods compounds.
and also a detailed description of experimental Volume 5 the formation of C-O-bonds, Volume
techniques have been deliberately omitted. 6 the formation of C-N-bonds, Volume 7 the
Newer methods as well as those which have syntheses of compounds containing main group
not yet been reported in review articles are elements, and Volume 8 compounds containing
treated more extensively, whereas conventional transition metal elements.
methods are dealt with concisely. Biological The third 'Special Part' (Volume 9-11) is con­
procedures which, in specific cases, are more cerned with the chemical aspects connected with
useful for characterizing substances than chemi­ the formulation of a question or problem.
cal or physical methods, will be discussed in the Volume 9 deals with nonmetallic synthetic fibers
analytical volume. The interrelated methods and and synthetic materials as well as their additives,
concepts which are constantly gaining impor­ Volume 10 with synthetic compounds and Vol­
tance will be fully discussed in the third 'Specific ume 11 with natural products and naturally
Part'. occurring compounds.
The METHODICUM CHIMICUM is com­ All Volumes should not contain more than 900
prised of three parts. The first, the 'General printed pages. They are intended to give the
Part' consists of Volumes 1, 2 and 3. Volume 1 chemist and any person working in fields related
(Analytical Methods) is concerned with chemical, to chemistry a sufficient answer to his problem.
physical, and biological analytical methods in­ Selected review articles or important original
cluding those necessary for the elucidation of works are cited for the sake of detailed infor­
structures of compounds. mation.
Volume 2 (Planning of Syntheses) contains a
review on fundamentals, principles, and models Friedhelm Korte
V
Preface of Volume 11
Volume 11 of METHODICUM CHIMICUM and isolation of classes of compounds are inten­
is devoted to the methods of structural deter­ tionally omitted in Volume 11. Synthetic com­
minations and syntheses of natural products pounds with industrial importance or with
which are of interest not only to the chemist physiological significance are to be discussed
but also to the scientist who works in associated in Volumes 9 and 10. In this volume, special
areas, including medicine. This compilation emphasis was layed on the structures and syn­
contains a short discussion of the principles of theses of compounds, and physiological and
well-proved procedures. This volume comprised biochemical functions of compounds are dis­
of three parts. Part 1 is concerned with the cussed secondarily.
chemistry and biochemistry of nucleic acids, The publication of this volume has been de­
proteins, and carbohydrates. Part 2 describes layed more than three years mainly due to
the different aspects of antibiotics, vitamins, unsettled market conditions. In this volume, the
enzymes and other compounds with biochemical literature has been surveyed up to 1972. Thanks
significance. Part 3 is designed to illustrate the to the close collaboration of the authors, it has
scope and utility of proved working techniques been possible to include new significant results
and syntheses in the fields of steroids, terpenes, recently published. We are grateful to the
alkaloids, and natural pigments. authors for their cooperation and their many
Separation and determination of main classes of valuable suggestions.
compounds were already discussed in Volumes
1A and IB of this series, and, to avoid duplica­ Munich, July 1976 Friedhelm Korte
tion, some important discussions on purification Tokyo, July 1976 Miki Goto
vii
1 Nucleic Acids
Contributions by
R. Flugel, Heidelberg
7%. Godefroy-Colburn, Paris
Y. Inoue, Tokyo
Y. Iwanami, Tokyo
K. Miura, Mishima
S. Nishimura, Tokyo
E. Ohtsuka, Osaka
M. Tsuboi, Tokyo
T. Uchida, Tokyo
1.1 Nucleic Acid Components
Yasuo Iwanami oxycytidine and thymidine) in deoxyribonucleic
Sasaki Institute, Kanda-Surugadai, Chiyoda-ku, Tokyo, acid (DNA). Phosphodiester linkages connect
101 Japan the nucleosides from 3' to 5' so as to construct
the biopolymers.
Nucleic acids contain four bases as their main This sugar phosphate backbone of the macro-
components, while proteins are constituted by molecules inlaid with the above four bases in a
more than twenty amino acids. On the basis of certain sequence is essentially the same in all
these facts, the concept of "genetic code" (tri­ living organisms.
plet of the bases) was developed by pioneers The DNA of several viruses does not contain
in molecular biology. During the last fifteen cytosine, but rather some modified base instead.
years, however, many bases other than the four The base always present in the DNA of the T-
constituents have been found in nucleic acids. even phages of Escherichia coli is 5-hydroxy-
They are called "minor components" or "modi­ methylcytosine 20. Furthermore, one or more D-
fied bases". Hence, the view as to what compose glucose molecules are attached to some of the
nucleic acids has markedly changed. hydroxymethyl groups by glycosidic linkage 21.
Since these unusual bases can base-pair with
1 Major Components of Nucleic Acids guanine like cytosine, the viral DNA can main­
The four common bases of nucleic acids are tain double-helical structure. The linked glucose
adenine, guanine, cytosine and uracil occurring residues probably protrude from the double
as their D-ribosides (adenosine, guanosine, cy- helix. Although the biological significance of
tidine and uridine) in ribonucleic acid (RNA) or the complicated bases remains obscure, one
the former three bases and thymine as D-deoxyri- speculative hypothesis asserts that they may
bosides (deoxyadenosine, deoxyguanosine, de- protect the viral DNA from a virus-specific
0
HN
Η Ν^ΝΓΝ*
2
—0-CH
2 NH
2
cxN>
:H H:
H H
HO 0
-P-0-CH
: 0
H H
HO
■l-o- CH
2 NH
I l/°\ 2
ki s>
o- N
Hη— f H
HO 0 O^N
PII -0-CH
2
0.
o
. H H:
H
HO
M0: -
Fig. 1. A Section of RNA (-GpApUpCp·■·)·
2
Methylated Bases in RNA 1.1:2.1
enzyme which only breaks down unmodified nucleic acid either right after the completion
DNA of the host cells1. The search on this sub­ or during its biosynthesis. Because the nucleic
ject is in progress2. acid is not constructed from any pieces of
The glucoses of T-even DNA are present either methyl-containing precursors, except thymidines
in the a- or ^-configuration or the a-glucosyl- for DNA, but methylated later, the reaction
/3-glucoside structure. In most cases, more than products of methylation are generically named
70 per cent of the hydroxymethyl groups are "methylated bases".
linked to glucose3. No free 5-hydroxymethyl-
cytosine residues are found in T4 DNA. 2.1 Methylated Bases in RNA
Likewise, thymine is replaced with 5-hydroxy- Some methylated bases are labile. Alkaline
methyluracil 6 in the DNA of some phage of hydrolysis causes rearrangement of the 1-methyl-
Bacillus subtilis*. After infection by the phage, adenine 29 residue to ATe-methyladenine 31 and
the normal DNA precursor, 2'-deoxycytidine also cleavage at the imidazole ring of 7-methyl-
or 2'-deoxyuridine triphosphate, is converted guanine 43 residue in RNA. This often gave
by the viral enzymes into the corresponding 5- confusing results to those who were working with
hydroxymethyl derivative, which is then incor­ the methylated base analysis of RNA. Some
porated in the phage DNA serving as a sub­ years ago, a new general systematic analysis for
strate for DNA polymerase. In the DNA of bac- methylated components, selectively 14C-labeled
teriophage SP-15, the thymine residues are re­ in the methyl group, has been reported7. In
placed with (5)-(+)-5-(4,5-dihydroxypentyl)- this method the unstable bases are protected
uracil 10\ while in that of phage 0W-14 5-(4- from structural change by maintaining acidic
aminobutylaminomethyl)uracil T is in replace­ or netural conditions during the necessary
ment of about half the thymine bases. steps of the determination. The techniques of
paper chromatography for separating 1-methyl-
2 Methylated Bases as Minor Constituents guanine 40, iV2-methylguanine 41, AP-dimethyl-
Although minor in content, 5-methylcytosine 19 guanine 42 and 7-methylguanine7, which have
has long been known as a definite constituent of similar properties, and of safe hydrolysis of N4*
DNA. This was followed by discovery of 5- methylcytidine (cf. 18) are also described in a
hydroxymethylcytosine in the phage DNA. Oc­ related paper8. Relative amounts of methylated
currence of methyladenines in RNA was first components in transfer (i)RNA [4S RNA]7 and
reported in 1958. Since then, a variety of methyl- ribosomal (r)RNA [18S and 28S RNA species]8
purines, methylpyrimidines, and other deriva­ of HeLa cells, obtained by the systematic analy­
tives of the nitrogenous bases have been found sis, are summarized in Table 1. It is of interest
in RNA, which has now continued to be a that with increasing molecular weight, the rate
"treasure-house" of these unusual bases. Re­ of methylation decreases within these three kinds
cently, DNA became a source of new findings of RNA species in addition to DNA which con­
for them again. tains one methylated base per 115 nucleotide
In contrast to the formation of 5-hydroxy- units9.
methylcytosine in the viral DNA, the origin of Three minor bases, iVe-methyladenine, iVe-dim-
these minor components has shown to be that ethylademine 32 and AT4-methylcytosine 18, were
methylation or other modification of the regular
not found in fRNA, whereas rRNA lacked the
bases takes place on the macromolecule of
other three, 1-methylhypoxanthine 45, 5-methyl­
1 J. D. Watson, Molecular Biology of the Gene, p. 353, cytosine and 5-methyluracil (thymine), which
W. A. Benjamin, Inc., New York 1965. were also found only in fRNA in the case of
2 R. A. Fleischman, /. L. Campbell, C. C. Richardson, L cells7. 5-Methylcytosine and thymine exist in
J. Biol. Chem. 251, 1561 (1976).
DNA as their deoxyribosides, but the ribosides
8 B. White, P. Handler, E. L. Smith, Principles of Bio­
of the three bases may be rRNA-specific in
chemistry, p. 185, McGraw-Hill Book Co., New York
1964.
4 H. V. Aposhian, Biochem. Biophys. Res. Commun. 18, 7 Y. Iwanami, G. M. Brown, Arch. Biochem. Biophys.
230 (1965). 124, 472 (1968).
5 H. Hayashi, K. Nakanishi, C. Brandon, J. Marmur, 8 Y. Iwanami, G. M. Brown, Arch. Biochem. Biophys.
J. Am. Chem. Soc. 95, 8749 (1973). 126, 8 (1968).
6 A, M. B. Kropinski, R. J. Bose, R. A. J. Warren, 9 G. M. Brown, G. Attardi, Biochem. Biophys. Res.
Biochemistry 12, 151 (1973). Commun. 20, 298 (1965).
3
1.1:2.1 Methylated Baes in RNA
Table 1. Relative content of methylated components in shown to contain this common partial struc­
/RNA and rRNA of HeLa cells ture14. However, a calculation based upon the
Methylated component Radioactivity of isolated values in Table 1 shows that, if thymine was
component (%*) present included in each /RNA molecule, its content
~4S 18S 28S should be much higher than 7.3 %. It has been
RNA7 RNA8 RNA8 shown that two glycine /RNA from S. epider-
1-Methyladenine 13.9 3.5 1.5 midis and methionine /RNA (initiator /RNA)
JVe-Methyladenine 0 5.2 3.9 from mouse myeloma cells, rabbit liver and
Ne-Dimethyladenine 0 7.3 3.7 sheep mammary gland lack in such a se-
1-Methylguanine 6.6 2.1 0.5 quence18-17. One of the lysine /RNA of rabbit
JV2-Methylguanine 12.1 1.9 0.3 liver contains the 2'-<9-methyl derivative of
7V2-Dimethylguanine 12.3 2.8 0.5
thymine riboside 25.
7-Methylguanine 5.3 4.0 1.6
Occurrence of iV2,/V2,7-trimethylguanine 46 in an
J
1 -Methylhypoxanthine 1.8 0 0 enzymatic hydrolyzate of 2-10S RNA from
3-Methylcytosine 3.3 1.2 2.9 Chinese hamster cells has also been reported.19
iV4-Methylcytosine 0 18.0 18.6 3-Methyluracil 2 seems to be widely distribut­
5-Methylcytosine 21.5 0 0 ed7'8·20.
3-Methyluracil 1.4 7.3 8.7 Another point of interest is the higher level of
5-Methyluracil(thymine) 7.3 0 0 activity of the RNA methylating enzyme
Unkown methylated uracil ** 1.5 6.0 (methylase) in neoplastic cells21"23 or the amount
2-O-Methylribose 7.2 42.8 50.0 of methylated base, especially 7-methylguanine,
arising during the course of chemical carcino-
Approximately one
methylated component/ 1/12 1/48 1/71 genesis by methylating agents24. Studies of these
nucleotide residues7 altered methylations25 including "aberrant" or
"hyper" methylation by such carcinogens may
HeLa DNA contains one methylated base per 115 nu-
cleotide residues. shed light on the cancer problem.
* The recovery was close to 100 per cent. The weak point in general assay systems of
** From the corresponding fraction of L cells 5-hydroxy- iRNA methylases from tumors and normal
methyluracil was detected and determined.
cells is to use E. coli /RNA as a substrate; the
heterologous substrate has already been methyl­
these cells. In iRNA of E. coli, however, pre­
ated by its own enzymes23. However, transcrip­
sence of JVe-methyladenine and 2-methylade-
tion of /RNA genes in vitro by purified RNA
nine 30 has been demonstrated10. Thus minor
polymerase may have the advantage of produc­
components of mammalian RNA may be dif­
ing completely unmodified /RNA precursors
ferent from those of microorganisms11. After
which provide a substrate for the study of the
the first report on the primary sequence of
processes and modifications leading to the for-
/RNA and its clover leaf structure presented by
Holley et al.12 in 1965, the tetranucleotide se­
14 S. Takemura, Seikagaku 43, 135 (1971).
quence, -GpTp^pCp- (thymine riboside 24
15 R. J. Roberts, Nature 237, 44 (1972).
abbreviated to T and pseudouridine to ψ) was 18 P. W. Piper, B. F. C. Clark, Nature 247, 516 (1974).
thought to be common to all kinds of /RNA 17 M. Simsek, U. L. RajBhandary, M. Boisnard, G.
beyond the species of their origins13. Even in Petrissant, Nature 247, 518 (1974).
the beginning of 1971, there seemed to be no 18 H. /. Gross, M. Simsek, M. Raba, K. Limburg, J.
Heckman, U. L. RajBhandary, Nucl. Acid. Res. 1, 35
exception to this rule. Actually, all twenty /RNA
(1974).
of known sequences at that time, had been 19 A. G. Saponara, M. D. Enger, Nature 223,1365 (1969).
20R.H Hall, Biochem. Biophys. Res. Commun. 12,
361 (1963).
10 M. Saneyoshi, F. Harada, S. Nishimura, Biochim. 21 E. Tsutsui, P. R. Srinivasan, E. Borek, Proc. Nat.
Biophys. Acta, 190, 264 (1969). Acad. Sci. U.S. 56, 1003 (1966).
11 M. Staehelin et ah, Nature 219, 1363 (1968). 22 V. M. Craddock, Biochim. Biophys. Acta 195, 351
12 R. W. Holley, /. Apgar, G. A, Everett, /. T. Madison, (1969).
M. Marquisee, S. H Merrill, /. R. Penswick, A. Zamir, 28 Y. Iwanami, M. Inose, Gann 65, 481 (1974).
Science 147, 1462 (1965). 24 E. D. Whittle, Biochim. Biophys. Acta 195, 381 (1969).
18 A. Zamir, R. W, Holley, M. Marquisee, J. Biol. 26 M. Inose, S. Miyata, Y. Iwanami, Biochim. Biophys.
Chem. 240, 1267 (1965). Acta 259, 96 (1972).
4
Methylated Bases in DNA 1.1:2.2
mation of mature iRNA molecules26,27. led 'bizarre' 5'-termini. The methylation seems
S-Adenosyl-L-methionine serves in enzymatic to be coupled with the initiation of wRNA
reactions as methyl donor2829 for all four groups synthesis34. Moreover, m7Gp5' prevents the as­
of bases methylated at nitrogen, oxygen, carbon sociation of wRNA with ribosomal subunits to
and even sulfur atoms. Several species of RNA form an initiation complex for protein syn­
and DNA methylases are known18,19,30·31. In thesis38.
the case of mammalian cells, 45S and 35S pre­
cursors of rRNA are present in nucleoli. In the 2.2 Methylated Bases in DNA
maturation steps, the 45S RNA first splits into Some 5-methylcytosine is contained in DNA
18S RNA and the 35S precursor, and then the from various sources. jye-Methyladenine is also
latter precursor in turn changes into 28S RNA. a minor component of the DNA of bacteria
Evidence suggests that these precursors liberate and phages. Since they can form hydrogen-
non-ribosomal fragments during this course32. bonded pairs, the double-helical structure of
The hypothetical methanism of maturation is the DNA containing them is essentially the same.
supported by our unpublished results of methyl­ Rather most, if not all, native DNA double
ation pattern analyses, which can be interpreted helices possess methylated base residues as a
to mean that non-ribosomal fragments, less matter of principle.
methylated and enriched with uracil residues, In addition to these minor components, the
are liberated from the precursors. On the other existence of 3-methylcytosine 17 and methylated
hand, these results would not provide definite guanines in animal DNA has been reported39.
evidence for the secondary methylation mech­ Sensitive radioisotope techniques showed that
anism in rRNA maturation33. 3-methylcytosine was found, besides 5-methyl­
Messenger (/w)RNA is also found to be methyl- cytosine which was present in the largest quan­
ated34"36. The wRNA from mouse myeloma cells tities, only in the DNA from human sources
contains JVe-methyladenosine and a novel 5'- among the four lines of HeLa, L, human em­
termini having 7-methylguanosine connected to bryonic lung and chick embryo cells used in
2'-0-methylated nucleosides 23 with 5',5'-tri- this study. Very small amounts of 1-methyl-
phosphate linkage:* m7Gp6'pp5' CmpUp-, m7- guanine, JV2-methylguanine, jy2-dimethylguanine
Gp*'pp5'GmpCp-, m7Gp8'pp5'AmpGp-, m7- and 7-methylguanine were detected only in
Gp5'pp5'GmpAp-, m7Gp5'pp6'GmpGp-, m7- HeLa DNA. However, the possibility of their
Gp6 'pp6 'CmpCp-, m7Gp5 'pp5 'CmpUmpCp-, occurrence in lung cell DNA has not been ruled
m7Gp6'pp6'GmpUp-, m7Gp6'pp5'AmpCp- and out. These newly found components might,
m7Gp5'pp5'AmpAp-37. They are sometimes cal- therefore, be specific to human DNA. This is
a very important finding because 3-methyl­
* Abbreviations: m7Gp5/: 7-methylguanosine 5'-phos­ cytosine and 1-methylguanine probably hinder
phate; p8'pp5': triphosphate connecting from 5' to 5'; base pairing where they occur. Even though
Cmp: 2'-0-methylcytidine 3'-phosphate, etc.
they base-pair, their hydrogen bonding can not
™N.S. Petersen, C. S. McLaughlin, D.P. Nierlich,
be normal because the methyl blockage inter­
Nature 260, 72 (1976).
27 C. Ilgen, L. L. Kirk, J. Carbon, J. Biol. Chem. 251, feres with the hydrogen bond. If this is the case,
922 (1976). an interest is lent as to how frequent are the
28 E. Borek, P. R. Srinivasan, Ann. Rev. Biochem. 35, base-pair hindrances in the DNA double helix
275 (1966). (see also Ref.82).
29 R. Michelot, M. Legraverend, G. Farrugia, E. Lederer,
The Opposition of guanine in DNA is a critical
Biochemie 58, 201 (1976).
80 /. Hurwitz et al, J. Biol. Chem. 239, 3462, 3474 (1964); reaction site for the initiation of malignant
240. 1256 (1965). transformation by monofunctional alkylating
81 Y. Kuchino, S. Nishimura, Biochem. Biophys. Res. agents40. A new class of enzymes acting on and
Commun. 40, 306 (1970).
reparing damaged DNA containing 3-methyl-
82 P. Jeanteur, F. Amaldi, G. Attardi, J. Mol. Biol. 33,
adenine and Oe-methylguanine residues has
757 (1968).
88 E. F. Zimmerman, Biochemistry 7, 3156 (1968).
84 Y. Furuichi, K. Miura, Nature 253, 374 (1975). 38 E. D. Hickey, L. A. Weber, C. Baglioni, Proc. Nat.
85 Y. Furuichi, 5. Muthukrishnan, A. J. Shatkin, Proc. Acad. Sci. U.S. 73, 19 (1976).
Nat. Acad. Sci. U.S. 72, 742 (1975). 39 L. Culp, E. Dore, G. M. Brown, Arch. Biochem.
88 R. P. Perry, D. E. Kelley, Cell 1, 37 (1974). Biophys. 136, 73 (1970).
87 /. M. Adams, S. Cory, Nature 255, 28 (1975). 40 P. Kleihues, G. P. Margison, Nature 259, 153 (1976).
5