Docsity
Docsity

Prepare for your exams
Prepare for your exams

Study with the several resources on Docsity


Earn points to download
Earn points to download

Earn points by helping other students or get them with a premium plan


Guidelines and tips
Guidelines and tips

Polysaccharide Synthesis: Determining Factors and Mechanisms, Summaries of Biochemistry

The synthesis of polysaccharides from the perspective of the sources of biological information that determine their structures and control their rates of synthesis. The authors explore various aspects of polysaccharide synthesis, including the role of enzyme specificity, membrane components, and lipid intermediates. They also touch upon the synthesis of different types of polysaccharides, such as intracellular storage polysaccharides, cell wall structural polysaccharides, and extracellular or capsular polysaccharides.

What you will learn

  • How does the cytoplasmic membrane function in cell wall polysaccharide synthesis?
  • What is the significance of lipid intermediates in the formation of cell wall components?
  • What are the different ways to approach the study of polysaccharide synthesis?
  • How do membrane components contribute to complex polysaccharide synthesis?
  • What role does enzyme specificity play in polysaccharide synthesis?

Typology: Summaries

2020/2021

Uploaded on 05/24/2021

judyth
judyth 🇺🇸

4.6

(27)

321 documents

1 / 16

Toggle sidebar

This page cannot be seen from the preview

Don't miss anything!

bg1
Polysaccharide
Biosynthesis
P.
W.
ROBBINS,
A.
WRIGHT,
and M.
DANKERT
From
the
Division
of
Biochemistry,
Massachusetts
Institute
of
Technology,
Cambridge
AB
ST
R
ACT
Polysaccharide
synthesis
is
discussed
from
the
point
of
view
of
the
sources
of
biological
information
that
determine
the
structures
and
control
the
rates
of
synthesis
of complex polysaccharides.
It
is
concluded
that
three
types
of
information
contribute
in
important
and
different
ways,
namely
enzyme
specificity,
primer
substances,
and
the
structure
of
the
cytoplasmic
membrane.
Each
of
these factors
is
discussed
in
a general way
with
examples of
its
contri-
bution
to
the
structure and
organization
of
specific
polysaccharides.
The
general
problem
of
polysaccharide
synthesis
may be
considered
from
a
number
of
points
of view.
It
is
possible, for
instance, to divide polysaccharides
into
groups on
the
basis
of
chemical complexity
and
it
would be
feasible
to
discuss
homopolysaccharides, polysaccharides
with
simple
repeating
units,
and
complex
heteropolysaccharides
as
three distinct
problems of
biosynthesis.
Alternatively,
it
would
be
possible
to
separate
polysaccharides
into functional
groups
and
consider
as
separate
topics
the
synthesis
of
intracellular
storage
polysaccharides,
cell
wall
structural
polysaccharides,
and
extracellular
or
capsular
polysaccharides. Both
approaches are
reasonable
and
it
is
possible
to develop
the
themes
that
the
biochemical mechanisms
of biosynthesis
be-
come
more
complex
as
one
considers
polysaccharides
with
increasing struc-
tural
complexity
or
increasing functional
complexity.
It
is
obvious
that
more
enzymes
will
be
involved
in the formation
of
the variety
of linkages of
a
complex
heteropolysaccharide
than
will
be
required
to
form
the
one
or
two
types
of
linkage in
a homopolysaccharide.
It
is
also
obvious
that
a
complex
function may
require
the
intervention
of enzymes,
cofactors,
and
control
systems
to
ensure
the coordination
of
polysaccharide
synthesis
with
the
forma-
tion
of
other
substances,
as
in
the
synthesis of
the
bacterial
cell
wall,
or
to
regulate
synthesis
and
degradation,
as
in
the
case
of
glycogen
formation
and
breakdown.
A
third
way
of discussing
polysaccharide
synthesis
is
to consider
the
sources
of
biological
information
that
determine
the
structure
and
control
the
syn-
thesis
of
polysaccharides.
This
is
the
point
of
view
that
will
be
taken
in
the
present
paper
and
it
will
be
assumed
that
there
are
primarily
three
sources
of
information, namely
enzyme
specificity,
primer
substances,
and
the struc-
ture
of
the
cytoplasmic
membrane.
These
sources
of
information
may
well
be
33'
The Journal of General Physiology
Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021
pf3
pf4
pf5
pf8
pf9
pfa
pfd
pfe
pff

Partial preview of the text

Download Polysaccharide Synthesis: Determining Factors and Mechanisms and more Summaries Biochemistry in PDF only on Docsity!

Polysaccharide Biosynthesis

P. W. ROBBINS, A. WRIGHT, and M. DANKERT From the Division of Biochemistry, Massachusetts Institute of Technology, Cambridge

AB ST R ACT Polysaccharide synthesis is discussed from the point of view of the sources of biological information that determine the structures and control the rates of synthesis of complex polysaccharides. It is concluded that three types of information contribute in important and different ways, namely enzyme specificity, primer substances, and the structure of the cytoplasmic membrane. Each of these factors is discussed in a general way with examples of its contri- bution to the structure and organization of specific polysaccharides.

The general problem of polysaccharide synthesis may be considered from a number of points of view. It is possible, for instance, to divide polysaccharides into groups on the basis of chemical complexity and^ it^ would be^ feasible^ to discuss homopolysaccharides, polysaccharides with simple repeating units, and complex heteropolysaccharides as three distinct problems of biosynthesis. Alternatively, it would be possible to separate polysaccharides into functional groups and consider as separate topics the synthesis of intracellular storage polysaccharides, cell wall structural polysaccharides, and extracellular or capsular polysaccharides. Both approaches are reasonable and it is possible to develop the themes that the biochemical mechanisms of biosynthesis be- come more complex as one considers polysaccharides with increasing struc- tural complexity or increasing functional complexity. It is obvious that more enzymes will be involved in the formation of the variety of linkages of a complex heteropolysaccharide than will be required to form the one or two types of linkage in a homopolysaccharide. It is also obvious that a complex function may require the intervention of enzymes, cofactors, and control systems to ensure the coordination of polysaccharide synthesis with the forma- tion of other substances, as in the synthesis of the bacterial cell wall, or to regulate synthesis and degradation, as in the case of glycogen formation and breakdown. A third way of discussing polysaccharide synthesis is to consider the sources of biological information that determine the structure and control the syn- thesis of polysaccharides. This is the point of view that will be taken in the present paper and it will be assumed that there are primarily three sources of information, namely enzyme specificity, primer substances, and the struc- ture of the cytoplasmic membrane. These sources of information may well be

33'

The Journal of General Physiology

Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021

APPROACHES TO THE BIOSYNTHESIS OF MACROMOLECULES

related to one another. At the present stage of understanding in molecular

biology, however, each of these components may be considered as an inde-

pendent source of information and, in each case, the source of a different

type of information. Enzyme specificity derives from protein structure (ulti-

mately from nucleic acid nucleotide sequences) and specific enzymes are

responsible for the synthesis of activated monosaccharides, the nucleoside

diphosphate sugars, and for the specific transfer of glycosyl residues from these

nucleoside diphosphate sugars to acceptor molecules,^ usually^ other^ saccharide

residues. Primers may be defined as^ substances^ that^ cannot^ be^ derived^ readily

de novo from enzymes and metabolites; they serve as enzyme substrates^ and

probably also direct enzyme action by^ allosteric^ interactions^ and^ structural

effects. The cytoplasmic membrane has recently come to^ the foreground^ as

the site of cell wall polysaccharide synthesis and, as^ will^ be^ mentioned^ below,^ a

phospholipid that is probably present^ in^ the^ cytoplasmic^ membrane^ serves

as a coeznyme in the synthesis of at least some cell wall polysaccharides.

What determines the structure of the cytoplasmic membrane is at present

almost purely a matter of speculation and, in fact, Sonneborn (1) has pointed

out that the relationship between the cell genome and the structure and func-

tion of the cytoplasmic membrane remains one of the most important un-

explored areas of biology. To summarize, therefore, enzyme specificity,

primer materials, and the structure of the cytoplasmic membrane may be

considered as the three basic sources that determine and control the syn-

thesis of polysaccharides, although all three factors may not operate directly

in every case.

The possibility that other sources of information may function in poly-

saccharide synthesis has been proposed. McMullen (2) has suggested that

"nucleoside diphosphate saccharides... are analogous to the activated

amino acid in^ protein^ template^ biogenesis^ and^ correspondingly^ enter^ into

the same kind of ... coding mechanisms involving polynucleotide struc-

tures .... " Such speculation is interesting but, as far as we are aware, has

no experimental basis.

Enzyme Specificity

Little comment is required on the major role played by enzyme specificity

in determining and controlling polysaccharide synthesis. The synthesis of

most polysaccharides begins with the formation of a nucleoside diphosphate

glycosyl derivative from a nucleotide triphosphate and a glycosyl phosphate

ester. Fig. 1 illustrates the formation of uridine diphosphate glucose (^ =^ UDP-

glucose) from^ uridine^ triphosphate^ and^ glucose-l-phosphate.^ Even^ at^ this

initial stage the reaction shows a high degree of specificity and control.

Beinstein and Robbins (3), for example, studied the formation of the struc-

turally similar nucleotides UDP-glucose and thymidine diphosphate glucose

332

Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021

APPROACHES TO THE BIOSYNTHESIS OF MACROMOLECULES

( =TDP-glucose) (^) by extracts of enteric bacteria. It was shown that the nucleo- tides are formed by separate (^) soluble enzymes, each with its own pattern of feed-back control. (^) Approximately sixty nucleoside diphosphate sugars are known at the present (^) time. Some of these are formed directly from an a-D- glycosyl-l-phosphate (^) and a nucleoside triphosphate, but many are products of oxidation, reduction, and epimerization (^) reactions that are catalyzed by specific soluble enzymes. For example, (^) such reactions bring about the trans- formation of TDP-glucose (^) to TDP-rhamnose, the direct precursor of poly- saccharide rhamnose. The known (^) systems are summarized in the reviews by Leloir (4) and Ginsburg (^) (5) and will not be enumerated here. The (^) enzymes that catalyze the transfer of glycosyl residues from nucleoside diphosphate (^) sugars to the oxygen residues of other saccharide moieties in general show (^) absolute specificity with respect to the linkage position and anomeric configuration. (^) Since, as far as is known, all nucleoside diphosphate sugars (^) have the same absolute configuration (=a-D) at the phosphate- glycosyl linkage (^) some transfer reactions clearly take place with retention of this (^) configuration at the glycosyl position while other transfer reactions lead to inversion of configuration. In either case the energy of the system is pre- served, presumably by way of covalent intermediates; thus, it is possible (^) that transfer (^) reactions that take place with retention of the glycosyl carbon atom configuration are actually double inversion reactions. According (^) to this concept (^) (6) the enzyme that will catalyze transfer with retention of configura- tion reacts with the nucleoside diphosphate sugar with displacement of the nucleoside diphosphate and the formation of an enzyme-sugar covalent intermediate. This intermediate has the inverted glycosyl configuration (^) and reaction with the final acceptor in a second displacement reaction leads to restoration of the original glycosidic configuration. Reactions that involve over-all inversion, on the other hand, would be visualized (^) as simple dis- placement reactions on the nucleoside diphosphate sugar glycosyl (^) linkage by the acceptor group oxygen moiety. While these (^) simple schemes would account adequately for the occurrence of both types of glycosidic linkages (^) in polysaccharides, little serious work hasyet been carried out on the mechanisms of transfer reactions.

The Role of Primer in PolysaccharideBiosynthesis

Although enzymatic (^) specificity adequately accounts for the sequential ad- dition of subunits into correct linkage (^) with growing polysaccharide chains, it appears that the entire assembly of the molecule (^) cannot be achieved by enzymes, coenzymes, and metabolites alone. (^) An interesting example of such an instance is the biosynthesis of glycogen. (^) Glycogen is composed of several hundred to several thousand chains of a-1 ,4-linked D-glucose units, the interchain linkage being of the a-1,6-glucosidic type (Fig. 2). It is synthe-

334

Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021

P. W. ROBBINS, A. WRIGHT, AND M. DANKERT Polysaccharide Biosynthesis 335

sized by the transfer of glucose units from UDP-glucose to the nonreducing end groups of primer glycogen, catalyzed by the enzyme glycogen synthetase (7). The action of the enzyme is to lengthen the exterior chains of the molecule (Fig. 3). Branching of these newly created chains is catalyzed by amylo- 1,4 - 1,6-transglucosidase (branching enzyme), the over-all effect of the two enzymes being to cause the outward growth of the molecule. Glycogen itself is the best acceptor or primer in the glycogen synthetase reaction, al- though oligosaccharides as small as maltose may also act as acceptors, but at a very much reduced rate (8). The question of how the first glycogen molecules appear in the cell raises two alternatives. Either glycogen is synthesized de novo from precursors present in the cell or preformed glycogen is present at the birth of each new cell. De novo synthesis by glycogen synthetase does not seem likely at present since

6 G G ~G (^) G ,G G G GG +UDP UDPG' + 'IN FIGURE 3. Action of glycogen synthetase. The figure shows transfer of glucose from UDP-glucose to the nonreducing end group of a glycogen molecule resulting in chain elongation.

glucose and other common metabolites do not act as acceptors in the reaction. As mentioned previously, this enzyme is able to use maltose as an acceptor at a slow rate, but since maltose and its homologues were shown by Ola- varria (9) to be degradation products of glycogen rather than its precursors, this pathway seems unlikely. Brown (10), in fact, has reported that no de novo glycogen synthesis can be detected when purified glycogen synthetase is incubated with UDP-glucose in the absence of primer glycogen. The de novo synthesis of glycogen from glucose-l-phosphate by a mixture of highly purified, primer-free phosphorylase and branching enzyme has been reported by Brown et al. (11). It is suggested that the process involves the formation of a glucosyl enzyme which becomes the acceptor for chain growth. However, such a reaction in vivo would require a favorable glucose- - phosphate to inorganic phosphate ratio which would necessitate compart- mentalization of the cell, since this ratio normally favors the phosphorolysis reaction. It has also been observed that the appearance of glycogen in chick embryo precedes by a significant period the appearance of phosphorylase (12). Furthermore, it has been shown that in McArdle's disease, which is characterized by an accumulation of glycogen in muscle, there is a complete absence of phosphorylase (13). Thus from the standpoint of present knowledge the question of de novo synthesis of glycogen must be left open. Considering the

Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021

P. W. RoBBINS, A. WRIGHT, AND M. DANKERT PolysaccharideBiosynthesis

or other components on or near the cytoplasmic membrane. It has also been

postulated that in the absence of a cell wall the various component macro-

molecules that would make up the cell wall diffuse away from the exterior

surface of the membrane too rapidly to undergo "self-organizing" reactions

such as covalent cross-linking and complex hydrogen bond interactions. This

"diffusion barrier" postulate for the priming function of cell wall in its own

synthesis is attractive, but is not compatible with the reversion of stable L-forms

on hard agar since agar has little effect on the diffusion rate of even rather

large molecules. It is possible that hard agar and hard gelatin change the

conformation of the cytoplasmic membrane and that this conformational

change leads in some as yet undefined way to the organization of the cell

wall macromolecules into a fixed pattern. Certainly it would be useful to

know whether the initiation of cell wall synthesis in the hard agar system

bears any relationship at all to the resumption of cell wall synthesis in re-

verting L-forms, since in one case it is logical to assume that priming is a

physical effect of the medium while in the other case a chemical priming by

wall fragments would seem more reasonable.

The Role of the Cytoplasmic Membrane in PolysaccharideSynthesis

Work with cell-free systems that catalyze the synthesis of heteropolysaccharides

by transfer from nucleoside diphosphate sugar derivatives has suggested that

elements of the cytoplasmic membrane are involved in the synthetic process.

For example, studies by Glaser and Brown (17) with a preparation from

Rous sarcoma cells and the work of Dorfman and coworkers (14) with en-

zymes from Group A Streptococci showed that hyaluronic acid synthesis is

associated with cell particulate fractions. Smith et al. (18) purified the system

that catalyzes the synthesis of Type III Pneumococcus polysaccharide by am-

monium sulfate fractionation and found that although the final preparation

is essentially free of nucleic acid it can be precipitated by ultracentrifugation

and exhibits typical "microsomal" properties. Recent work on the biosyn-

thesis of the lipopolysaccharide component of the cell wall of Gram-negative

bacteria has provided direct evidence for the involvement of membrane

components in complex polysaccharide synthesis. Lipopolysaccharide has a

highly complex structure which, as indicated in Fig. 4, can be subdivided into

three main components: the backbone and the R-specific side chains, which

together constitute the "core," and the smooth O-antigen. The backbone

(19, 20) contains heptose, phosphate, 0-phosphorylethanolamine, and 2-keto-

3-deoxy-octonate (21), which links it to the lipid A of Westphal (20). The

R-specific chains (19) contain glucose, galactose, and N-acetylglucosamine

and are probably joined to the backbone by a glucose-heptose linkage (22).

The O-specific side chains determine the serological groups of enteric bac-

teria and may be linked to the N-acetylglucosamine units of the R-chains

Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021

APPROACHES TO THE BIOSYNTHESIS OF MACROMOLECULES

(22). Unlike the core material the smooth (^) O-antigen varies widely in mono- saccharide composition. Salmonella groups B and (^) E, which will be discussed below, have repeating sequences containing galactose, mannose, and rham- nose. Mutants that lack the capacity to synthesize UDP-glucose or UDP- galactose (^) form an incomplete lipopolysaccharide that is deficient in both the R-specific and O-specific side chains (23, 24). Such mutants have been used by Rothfield, Osborn, and Horecker (25) to study the incorporation of specific glucosyl or galactosyl residues into the incomplete lipopolysaccharide core.

Heptose Heptose KDO

H-C CH 2 I

o 0

I I 0- 0 CH 2 Glu,Gal, AcGm CH - NH (^3)

' Lipid A

Gal Rh Man

FIGURE 4. Structural components of lipopolysaccharide. The core substance is en- closed by the square brackets and contains 2-keto-3-deoxy-octonate (KDO), heptose, O-phosphorylethanolamine, phosphate, glucose (Glu), galactose (Gal), and N-acetyl- glucosamine (AcGm). The core is linked to lipid A through KDO. The round brackets enclose the repeating sequence of the O-antigen of Salmonella newington which contains galactose, rhamnose (Rh), and mannose (Man). The values of n and m are unknown.

The reactions have been (^) formulated as follows:-

(a) (^) Glucose-deficient lipopolysaccharide + UDP-glucose

- glucosyl-lipopolysaccharide (+ UDP) (b) Glucosyl-lipopolysaccharide + UDP-galactose -- > galactosyl-glucosyl-lipopolysaccharide (+ UDP)

In each case enzyme activity is found in both the cell wall membrane frac- tion and (^) "soluble" fraction derived from sonicated cells. The substrate speci- ficity and the binding of (^) enzyme to its macromolecular substrate have been

338

Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021

APPROACHES TO THE BIOSYNTHESIS OF MACROMOLECULES

galactose (Fig. 5), and the former also contains the dideoxyhexose abequose. In each of these cases the presence of membrane material is necessary for synthesis. Weiner et al. (29) and Wright et al. (30) have also demonstrated that the O-antigen chains are not directly derived from the nucleotide sugars but are assembled from trisaccharide repeating units preformed on a lipid intermediate. The sequence of reactions involved in the biosynthesis of the

TDP-Rh [TDP]

UDF

(Man-Rh-Gal)- "Core'^ "Core" FIGURE 6. Scheme for the involvement of lipid intermediates in the^ synthesis^ of^ Sal- monella O-antigen. The system is described in the text. Postulated intermediates and products are shown in square brackets. The following abbreviations are^ used: UDP-Gal, uridine diphosphate-D-galactose TDP-Rh, thymidine diphosphate-L-rhamnose GDP-Man, guanosine diphosphate-D-mannose.

Salmonella O-antigen is^ shown^ in^ Fig.^ 6.^ The^ first^ transfer^ to^ the lipid^ ac- ceptor is that of^ galactose-l-phosphate^ from^ the donor^ UDP-galactose^ with the concurrent release of UMP. UMP inhibits the reaction and can also cause its reversal. The second transfer to the lipid acceptor is that of rhamnose, added to every available galactosyl residue to^ form^ the^ disaccharide^ inter- mediate. In the presence of GDP-mannose the^ trisaccharide^ repeating^ unit^ is formed and immediately converted^ into^ polysaccharide.^ Direct^ evidence^ for the participation of^ the^ trisaccharide^ phosphate^ lipid^ intermediate^ at^ this stage has been provided^ by^ Weiner^ et^ al.^ (29).^ Working^ with^ S.^ typhimurium

340

Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021

P. (^) W. ROBBINS, A. WRIGHT, AND M. DANKERT (^) Polysaccharide Biosynthesis

they isolated and characterized the mannosylrhamnosylgalactose- l-phosphate

lipid. These workers also isolated long O-antigen chains terminated by ga-

lactose-l-phosphate, a finding which is indicative of the formation of a lipid-

linked polysaccharide by a chain-elongating mechanism based on the poly-

CH 2 0H

H NH CO CH 3

L-Ala

D -Glu

L-Lys

D-Ala [

CH20H

I CH$ NH H-C -CH (^3) CO NH 10 HC -C-NH (^2) (CH 2 ) 2 CO NH l 0O HC-(CH 2 ) 3 -CH-NH-C (^) hvVv\V CO HN HC-C O VVVVVVVV CH 3 FIGURE 7. Repeating unit of the mucopeptide (^) of Gram-positive bacteria. The poly- saccharide backbone has alternating (^) units of N-acetylglucosamine and N-acetylmuramic acid. The N-acetylmuramic acid is substituted (^) by a tetrapeptide containing L-alanine, D-isoglutamine, L-lysine, and D-alanine. (^) The zigzag lines indicate the points of attach- ment of interchain bridges.

merization of repeating units provided (^) by the trisaccharide intermediate. The formation of lipopolysaccharide, (^) the final reaction in the sequence, is achieved by a transfer of polysaccharide (^) to the appropriate acceptor in the lipopolysaccharide (^) core. Such a mechanism has important implications (^) since it illustrates how sub- units of the O-antigen, (^) which is exterior to the cell membrane, (^) might be as- sembled from cytoplasmic precursors in the form of lipid-linked (^) intermediates

341

n

Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021

P. W. ROBBINS, A. WRIGHT, AND M. DANKERT PolysaccharideBiosynthesis

there are in fact a series of related lipids which^ have^ such^ a^ function;^ struc- tural variation of the lipids could provide the specificity necessary for the concurrent formation of the several complex components of the bacterial cell wall. Studies of other membrane-controlled systems, not only in bacteria but in plant and animal cells, should resolve this interesting and important question.

REFERENCES

  1. SONNEBORN, T. M., The evolutionary integration of the genetic material into genetic systems, Presented at the Mendel Centennial Celebration at the Gene- tics Society of America at Fort Collins, Colorado, September, 1965, Madison, University of Wisconsin Press.
  2. McMuLLEN, A. I., A template mechanism for the biogenesis of specific poly- saccharides, Excerpta Medica, International Congress Series No. 77, XIth In- ternational Congress of Cell Biology, New York, Excerpta Medica Founda- tion, 1964, 32.
  3. BERNSTEIN, R. L., and ROBBINS, P. W., Control aspects of uridine 5'-diphosphate glucose and thymidine 5'-diphosphate glucose synthesis by microbial en- zymes, J.^ Biol.^ Chem.,^ 1965,^ 240,^ 391.
  4. LELOIR, L. F., Nucleoside diphosphate sugars and saccharide synthesis, Bio- chem. J., 1964, 91, 1.
  5. GINSBURG, V., Sugar nucleotides and the synthesis of carbohydrates, Advances Enzymol., 1964, 26, 35.
  6. KOSHLAND, D. E., JR., Group transfer as an enzymatic substitution mechanism, in A Symposium on the Mechanism of Enzyme Action, (W. D. McElroy and B. Glass, editors), Baltimore, The Johns Hopkins University Press, 1954, 608.
  7. LELOIR, L. F., The biosynthesis of polysaccharides, Sixth International Congress of Biochemistry, New York City, Proceedings of the Plenary Sessions, 1964, 15.
  8. GOLDEMBERG, S. H., Specificity of uridine diphosphate glucose-glycogen glucosyl- transferase, Biochim. et Biophysica Acta, 1962, 56, 357.
  9. OLAVARRIA, J. M., The metabolism of oligosaccharides, J. Biol. Chem., 1960, 235, 3058.
  10. BROWN, D. H., in discussion of the paper by L. F. Leloir, Role of uridine diphos- phate glucose in the synthesis of glycogen, in Ciba Foundation Symposium on Control of Glycogen Metabolism, (W. J. Whelan and M. P. Cameron, editors), Boston, Little, Brown and Company, 1964, 85.
  11. ILLINGWORTH, B., BROWN, D. H., and CORI, C. F., The de novo synthesis of poly- saccharide by phosphorylase, Proc. Nat. Acad. Sc., 1961, 47, 469. BROWN, D. H., ILLINGWORTH, B., and CORI, C. F., The mechanism of the de novo synthesis of polysaccharide by phosphorylase, Proc. Nat. Acad. Sc., 1961, 47, 479.
  12. GRILLO, T. A. I., and OZONE, K., Uridine diphosphate glucose-glycogen synthe- tase activity in the chick embryo, Nature, 1962, 195, 902.
  13. SCHMID, R., ROBBINS, P. W., and TRAUT, R. R., Glycogen synthesis in muscle lacking phosphorylase, Proc. Nat. Acad. Sc., 1959, 45, 1236.

Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021

APPROACHES TO THE BIOSYNTHESIS OF MACROMOLECULES

  1. MARKOVITZ, A., and DORFMAN, A., Synthesis of capsular polysaccharide (hya- luronic acid) by protoplast membrane preparations of group (^) A Streptococcus, J. Biol. Chem., 1962, 237, 273.
  2. MARECHAL, L. R., and GOLDEMBERG, S. H., Uridine diphosphate glucose-A3-1,3- glucan -3-glucosyltransferase from Euglena gracilis, J. Biol. Chem., 1964, 239,
  3. LANDMAN, O. E., and HALLE, S., Enzymically and physically induced inheritance changes in Bacillus subtilis, J. Mol. Biol., 1963, 7, 721.
  4. GLASER, L., and BROWN, D. H., The enzymatic synthesis in vitro of hyaluronic acid chains, Proc. Nat. Acad. Sc., 1955, 41, 253.
  5. SMITH, E. E. B., MILLS, G. T., BERNHEIMER, H. P., and AUSTRIAN, R., The syn- thesis of type III pneumococcal capsular polysaccharide from uridine nucleotides by a cell-free extract of Diplococcus pneumoniae Type III, J. Biol. Chem., 1960, 235, 1876.
  6. OSBORN, M. J., ROSEN, S. M., ROTHFIELD, (^) L., ZELEZNICK, L. D., and HORECKER, B. L., Lipopolysaccharide of the Gram negative cell wall: biosynthesis of (^) a complex heteropolysaccharide occurs by successive (^) addition of specific sugar residues, Science, 1964, 145, 783.
  7. LUDERITZ, O., STAUB, A. M., and WESTPHAL, O., Immunochemistry of O and R antigens of Salmonella and related Enterobacteriaceae, (^) Bact. Rev., in press.
  8. HEATH, (^) E. C., and GHALAMBOR, M. A., 2-keto-3-deoxy-octonate, a constituent of cell wall lipopolysaccharide preparations obtained (^) from E. coli, Biochem. and Biophysic. Research Comm., 1963, 10, 340.
  9. NIKAIDo, H., Biosynthesis of cell wall polysaccharide in mutant strains of Sal- monella. III. Transfer of L-rhamnose and (^) D-galactose, Biochemistry, 1965, 4,
  10. NIKAIDO, M., Studies on the biosynthesis of cell wall polysaccharide in mutant strains of Salmonella. (^) I and II, Proc. Nat. Acad. Sc., 1962, 48, 1337, 1542.
  11. OSBORN, M. J., ROSEN, S. M., ROTHFIELD, L., and HORECKER, B. L., Biosynthe- sis of bacterial (^) lipopolysaccharide. I. Enzymatic incorporation of galactose in a mutant (^) strain of Salmonella. Proc. Nat. Acad. Sc., 1962, 48, 1831.
  12. ROTHFIELD, L., OSBORN, M. J., and HORECKER, B. L., Biosynthesis of bacterial lipopolysaccharide. (^) II. Incorporation of glucose and galactose catalyzed by particulate and soluble (^) enzymes in Salmonella, J. Biol. Chem., 1964, 239, 2788: ROTHFIELD, L., and HORECKER, B. L, The role of cell-wall lipid in the biosynthe- sis of bacterial lipopolysaccharide, Proc. Nat. Acad. Sc., 1964, 52, 939. ROTHFIELD, L., and TAKESHITA, M., The role of cell envelope phospholipid in the enzymatic synthesis (^) of bacterial lipopolysaccharide: binding of transferase enzymes to a lipopolysaccharide complex, Biochem. Biophysic. (^) Research Comm., 1965, 20, 521.
  13. ZELEZNICK, L. D., (^) ROSEN, S. M., SALTMARsH-ANDREW, M., OSBORN, M. J., and HORECKER, B. L., Biosynthesis of bacterial lipopolysaccharide. IV. Enzymatic incorporation of mannose, rhamnose, and galactose in a mutant strain of Salmonella typhimurium, Proc. Nat. Acad. Sc., 1965, 53, 207.
  14. NIKAIDO, H., and NIKAIDO, K., Biosynthesis of cell wall polysaccharide in mutant

Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021

APPROACHES TO THE BIOSYNTHESIS OF MACROMOLECULES

looked and yet they may play a very important role. An example of that has been, I think, provided in the last 2 or 4 years by the work of Roden in Dorfman's labora- tory who has shown that the acid mucopolysaccharides-I don't remember whether hyaluronic acid^ also,^ but^ certainly^ chondroitin^ sulfuric^ acid^ and^ heparin-are^ at- tached to small protein peptides by a handle consisting of one molecule of xylose and two molecules of galactose. So you can have a molecule of about 300,000 which is at- tached to protein by one single molecule of xylose and this xylose is absolutely neces- sary to combine these polysaccharides to the protein. I think that such minute trace constituents in polysaccharides will probably be found to be of much greater im- portance than we at present realize, and that they are prone to be overlooked due to the complex architecture of such polysaccharides as the Salmonella polysaccharides. But I think this problem is one of the most important if, out of the chemistry of the polysaccharides, results of the kind Dr. Luria envisions should eventuate. Dr. Luria: If there are no other questions, we proceed to the last paper in the pro- gram. This really brings us to what I think is the essential problem of building the functional organization of the cell. The problem is how to keep things together in such a way that they are going to work at the right time and at the right place, and only into the kind of relations in which they are going to be useful for the specific purpose of the cell. This leads us to what I would call the biochemistry of the future, that is, the biochemistry of cellular membranes. It is fit that the biochemistry of the future should come last in a symposium of the past. Dr. Kennedy has very kindly agreed to open the future for us in closing the symposium.

Downloaded from http://rupress.org/jgp/article-pdf/49/6/331/1243879/331.pdf by Pakistan user on 29 April 2021