Hyaluronic Acid Biosynthesis Research Articles

Biosynthesis of hyaluronic acid by Streptococcus.

Synthesis of hyaluronic acid was investigated in a cell-free system derived from a strain of Group A streptococci. Preparative procedures were improved so that an enzyme system 70 times more active than that previously reported was obtained. The hyaluronic acid synthesized could be separated into trichloroacetic acid-soluble and -insoluble fractions. On the basis of pulse-chase experiments, it was shown that the trichloroacetic acid-insoluble fraction is a precursor of the soluble fraction. The release of the trichloroacetic acid-insoluble hyaluronic acid is specifically blocked with p-chloromercuribenzoate, without inhibition of chain elongation. The addition of butanol to trichloroacetic acid resulted in solubilization of all of the hyaluronic acid. No detectable difference in molecular size was observed between the two hyaluronic acid fractions, both of which were estimated to be more than one million daltons in size. Testicular hyaluronidase digestion of either one of the two types of hyaluronic acid yielded no high molecular weight fragments, indicating that hyaluronic acid is not bound covalently to protein. However, following incubation of enzyme assay mixtures with UDP-[14C]GlcUA, even in the absence of UDP-GlcNAc, radioactive high molecular weight hyaluronic acid was obtained which suggests that the enzyme system elongates rather than initiates hyaluronic acid chains. Tunicamycin did not inhibit hyaluronic acid synthesis, indicating lack of participation of an intermediate of pyrophosphorylpolyisoprenol type. The results obtained are consistent with the hypothesis that chain elongation of hyaluronic acid proceeds by alternate addition of monosaccharides from UDP-sugars by a membrane-bound synthesizing system followed by release of completed hyaluronic acid chains.

Biosynthesis of hyaluronic acid in the vitreous V. Studies on a particulate hyalocyte glycosyl transferase

Previous studies on the in vitro, enzymatic biosynthesis of hyaluronic acid by extracts of calf vitreous hyalocytes have concentrated on the properties of a soluble glycosyl transferase. The present report describes the properties of a particulate glycosyl transferase from hyalocytes and its role in hyaluronic acid biosynthesis. The enzyme catalyzes the incorporation of radioactivity from UDP-[ 14C]glucuronic acid (UDP-[ 14C]GlcUA) and UDP-[ 3H] N-acetylglucosamine (UDP-[ 3H]GlcNAc) into high molecular weight soluble glycosaminoglycan which appears in the reaction medium. In addition, radioactive material is removed from the denatured enzyme by treatment with 1 m-NaCl, 7 m-urea, and leech hyaluronidase. Pronase treatment also solubilizes bound radioactive material. It is proposed that the tightly bound material, solubilized by Pronase or hyaluronidase, may represent the initial formation of enzyme-bound hyaluronic acid, which is subsequently released into the medium following further polymerization.

Biosynthesis of hyaluronic acid in the vitreous VI. Isolation of a complex containing hyaluronic acid and glycosyl transferase activity and studies on the activity of a soluble glycosyl transferase

Addition of protamine sulfate to calf vitreous, after removal of hyalocytes, results in the formation of an insoluble complex of hyaluronic acid and protamine. The hyaluronic acid-protamine (HA-P) complex was found to possess glycosyl transferase activity, as shown by the incorporation of radioactivity from UDP-[ 14C]glucuronic acid and UDP-[ 14C] N-acetyl-glucosamine into the hyaluronic acid present in the complex. The incorporation of radio-activity was stimulated by the addition of Mg 2+, and for optimal incorporation both nucleotide sugar precursors of hyaluronic acid were required. When the HA-P complex is dissolved in 0·5-1 m-NaCl solutions, the glycosyl transferase can be separated from protamine, thereby regenerating a soluble system for the study of hyaluronic acid biosynthesis. The present report also describes the results of experiments designed to determine which agents affect the activity and heat stability of a soluble glycosyl transferase, extracted from hyalocytes, which similarly catalyzes hyaluronic acid synthesis. Treatment of the enzyme with N-acetylimidazole results in an inhibition of enzyme activity. When the treatment was carried out in the presence of substrates, no protection against the inhibition was observed. The enzyme was also inhibited by UTP and UDP, suggesting the role of this portion of the nucleotide sugar substrates in binding to the enzyme. Treatment with urea caused the enzyme to be inactivated by heat either in the presence or absence of endogeneous hyaluronic acid.

Biosynthesis of acid mucopolysaccharides by the surviving new born rat skin: I. — Kinetics of the biosynthesis at the polymer level

1) The amounts of individual mucopolysaccharides in the new born rat skin have been estimated and their specific rates of labelling assessed in vitro. Total and percentage amounts of these polymers agree satisfactorily with previously published data. 2) Relative rates of labelling from [U14C]-glucose have been estimated by combining column chromatography separation and electrophoresis on cellulose acetate strips. Specific radioactivities have been measured either with respect to the total uronic acid content of the fractions or with respect to their quantitative staining with Alcian Blue. The two methods agreed satisfactorily. 3) Average biosynthetic rates almost identical for hyaluronic acid and the total sulfated mucopolysaccharides. However, within the latter fraction, heparin + heparan sulfate incorporate [U14C]-glucose about 4 to 5 times more rapidly than the chondroitin sulfates. This result could not be expected from previous data obtained in vivo and is discussed with reference to a possible heterogeneity of the cell material whence the various mucopolysaccharides originate. 4) In the presence of puromycin, labelling of the sulfated mucopolysaccharides stops almost immediately, indicating a stringent requirement for protein primers. Biosynthesis of hyaluronic acid is affected only after preincubation of tissue with puromycin (one hour) and subsequent incubation of two hours with [U14C]-glucose.

The Biosynthesis of Hyaluronic Acid by Streptococcus

Abstract The mechanism of hyaluronic acid chain growth in a particulate enzyme preparation obtained from Group A Streptococcus has been examined and additional characteristics of the enzyme detailed. Hyaluronic acid chain elongation occurs by the transfer of monosaccharide units from uridine nucleoside diphosphate derivatives to the nonreducing ends of endogenous oligosaccharide. This oligosaccharide which appeared to be chemically identical with hyaluronic acid was released from the enzyme by either acid hydrolysis or digestion with streptococcal hyaluronidase. The only detectable uridine nucleotide product of hyaluronic acid synthesis was UDP, and synthesis was not inhibited by bacitracin. There was no evidence for the participation of lipid-extractable intermediates during hyaluronic acid formation. The pH optimum for polysaccharide formation is 7.1. The enzyme exhibits an absolute dependence upon divalent metal ion and is maximally activated by 10 mm Mg++ or 1 mm Mn++. Michaelis constants for UDP-GlcUA and UDP-GlcNAc are 5 x 10-5 m and 5 x 10-4 m, respectively. The enzyme is inactivated by treatment with 4% 1-butanol. Addition of Mg++ and UDP, UDP-GlcUA, UDP-GlcNAc, or other uridine nucleoside diphosphate sugars prior to butanol treatment prevented inactivation. Fifty per cent stabilization of the enzyme during butanol treatment occurred at 7 x 10-5 m UDP.

The Biosynthesis of Hyaluronic Acid by Group A Streptococcus: VI. BIOSYNTHESIS FROM URIDINE NUCLEOTIDES IN CELL-FREE EXTRACTS

The Biosynthesis of Hyaluronic Acid by Group A Streptococcus: VI. BIOSYNTHESIS FROM URIDINE NUCLEOTIDES IN CELL-FREE EXTRACTS

Biosynthesis of hyaluronic acid in tissue cultures of human synovial membrane.

Production of acid mucopolysaccharides in tissue cultures from human non-inflammatory synovial membranes was studied. The results indicate that hyaluronic acid was the chief acid mucopolysaccharide produced. Hyaluronic acid was synthesized by the synovial as well as by the periarticular tissues. The results did not indicate an appreciable production of (dialysable) hexosamine containing oligosaccharides. Intrinsic viscosity of the acid mucopolysaccharides produced varied from one case to another but kept rather constant from week to week.

Biosynthesis of hyaluronic acid by cell-free extracts of group-A streptococci

Biosynthesis of hyaluronic acid by cell-free extracts of group-A streptococci