Bovine Submaxillary Glycoprotein Research Articles

Purification and characterization of .BETA.-N-acetylhexosaminidase from the liver of red sea bream.

β-N-Acetylhexosaminidase was purified to an electrophoretically homogeneous form from the liver of red sea bream Pagrus major and the properties of the purified enzyme were studied. The molecular weight of the enzyme was estimated to be 150, 000 by gel filtration. The enzyme exhibited its maximum activity at pH 4.0, 4.5 and 5.0 toward the substrates p-nitro-phenyl β-N-acetylglucosaminide, chitobiose and bovine submaxillary glycoprotein, respectively. The Km and Vmax of the enzyme for p-nitropheny β-N-acetylglucosaminide were 0.42 mM and 235μmol/min.mg. The enzyme liberated approximately 100% of the N-acetylglucosamine occurring in bovine submaxillary glycoprotein as a minor carbohydrate component, revealing that the N-acetylglucosamine existed at the nonreducing end of the carbohydrate moieties.

Isolation and characterization of a blood group A substance-degrading .ALPHA.-N-acetylgalactosaminidase from an Acremonium sp.

A fungus isolated from soil was found to produce α-N-acetylgalactosaminidase in culture, this enzyme being the dominant glycosidase. The fungus was identified as an Acremonium sp., based on various taxonomical characteristics. The enzyme produced by the fungus was purified to electrophoretic homogeneity from the culture broth by procedures including ammonium sulfate fractionation, and chromatography on DEAE-Sephadex A-50, hydroxylapatite, Sephadex G-150 and Concanavalin A-Sepharose 4B. The molecular weight of the enzyme was estimated to be 55, 000 and 57, 000 by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, and the enzyme appeared to have a monomer structure. The enzyme was most active at pH 4.0-4.5, and was stable at pH 6.0-7.5 and below 40°C. The Michaelis constant for p-nitrophenyl α-N-acetylgalactosaminide was 1.3 mM. The enzyme liberated the N-acetylgalactosamine from the bovine submaxillary glycoprotein, which had been desialyzed with neuraminidase. The enzyme also liberated the N-acetylgalactosamine from various blood type A active substances, including porcine gastric mucin, horse gastric mucin and human salivary mucin, which was ascertained by a hemagglutination inhibition test. The enzyme could change blood type A erythrocytes into blood type O erythrocytes, which was confirmed by a hemagglutination test.

Chemical signals of fish skin for the attachment response of Acanthostomum brauni cercariae.

The chemical signals of the skin surface of fish, which stimulate the attachment responses of Acanthostomum brauni cercariae, were identified by offering chemicals and fish-skin extracts in agarose substrates to the cercariae. Smaller molecules such as amino acids, fatty acids, monosaccharides, electrolytes, urea, and carbonate solutions did not stimulate attachments, but hyaluronic acid had some effects. Bovine submaxillary glycoproteins had a strong stimulating activity that disappeared after neuraminidase digestion. The stimulating components of the skin surface of fish were hydrophilic substances with molecular weights of more than 10,000. They were sensitive to neuraminidase digestion but not to hyaluronidase digestion and thus can be identified as glycoproteins. A. brauni cercariae respond only to the complete glycoprotein molecules and not to their monosaccharide components. The known attachment triggers of other cercariae are small molecules. Large glycoproteins as host signals for A. brauni cercariae may be an adaptation to muddy habitats, where various substances with low molecular weights may interfere with the host identification.

β-elimination and reduction reactions and some applications of dimethylsulfoxide on submaxillary glycoproteins

It has been shown that dimethylsulfoxide is useful in separating protein contaminants and minor mucins from the submaxillary glycoproteins prepared by the method of Tettamanti, G. and Pigman, W. (1968) Arch. Biochem. Biophys., 124, 41–50. In addition, alkaline dimethylsulfoxide was found to greatly accelerate the β-elimination reaction of glycoproteins. As this procedure does not produce diffusible protein fragments, it should be of value in studies of the protein core and for the investigation of those protein-carbohydrate linkages that are resistant to treatment by alkali. Optimal conditions for carrying out the β-elimination and reduction reactions of bovine submaxillary glycoprotein in aqueous alkaline NaBH 4 systems have been described. Under such conditions, the conversion of 2-aminopropenoic and 2-amino-2-butenoic acid residues into alanine and α-aminobutyric acid, respectively, is complete. This reduction procedure is of a broad application and can be used for the reduction of the phenylhydrazone derivatives of α-keto acids to the corresponding amino acids. A comparison of our procedure with that described by Simpson, L. D., Hranisavljevic, J. and Davidson, E. A. (1972) Biochemistry, 11, 1849–1856, shows that the method outlined here is superior both in efficiency and for quantitative recovery of unsaturated hydroxyamino acids.

The occurrence of repetitive glycopeptide sequences in bovine submaxillary glycoprotein.

Bovine submaxillary mucin, after removal of its sialic acid component, is cleaved by trypsin into two principal glycopeptide fractions; their final yield corresponds to 70% by weight of the original mucin and 85% of the original amount of hexosamine in the mucin. These glycopeptides have amino acid and carbohydrate compositions that are very similar to those of the original and desialyzed mucin. The main glycopeptide contains about 28 amino acids; the second one seems to be a covalently linked trimer of the former and is resistant to further action by trypsin. The protein core of bovine submaxillary mucin is composed mainly of several hundred covalently bound sequences of these glycopeptides. These results are compatible with those reported by Downs and Pigman (1969) for the products obtained by chemical cleavage of this mucin.

The acid and enzymic hydrolysis of O-acetylated sialic acid residues from rabbit Tamm-Horsfall glycoprotein.

Rabbit Tamm-Horsfall glycoprotein and bovine submaxillary glycoprotein were both found to contain sialic acid residues which are released at a slow rate by the standard conditions of acid hydrolysis. These residues are also resistant to neuraminidases from Vibrio cholerae and Clostridium perfringens. This behaviour was attributed to the presence of O-acetylated sialic acid, since the removal of O-acetyl groups by mild alkaline treatment normalized the subsequent release of sialic acid from rabbit Tamm-Horsfall glycoprotein by acid and by enzymic hydrolysis. Determination of the O-acetyl residues in rabbit Tamm-Horsfall glycoprotein indicated that on average two hydroxyl groups of sialic acid are O-acetylated, and these were located on the polyhydroxy side-chain of sialic acid or on C-4 and C-8. These findings confirm the assumption that certain O-acetylated forms of sialic acid are not substrates for bacterial neuraminidases. Several explanations have been suggested to explain the effect of O-acetylation of the side-chain on the rate of acidcatalysed hydrolysis of sialic acid residues.

Glycoprotein biosynthesis in the developing rat brain II. Microsomal galactosaminyltransferase utilizing endogenous and exogenous protein acceptors

1. 1. In rat brain, the enzyme UDP- N-acetylgalactosamine: protein N-acetylgalactosaminyltransferase, which transfers N-acetylgalactosamine from UDP- N-acetylgalactosamine to both endogenous protein acceptors and defined exogenous acceptors, is localized almost exclusively in the microsomes. The enzyme requires Mn 2+ and Triton X-100 for maximal activity and has a pH optimum of 6.8. The sulfhydryl nature of the enzyme is indicated by the observations that the activity is inhibited by p-chloromercuriphenylsulfonate and that the inhibition can be prevented by dithiothreitol. The transfer activity is higher in smooth than in rough microsomes and is present in all the brain regions (cortex, cerebellum, brain stem, hypothalamus and hippocampus) studied. Basic myelin protein fractions from bovine brain and spinal cord and bovine submaxillary glycoprotein from which sialic acid and hexosamine have been removed act as efficient acceptors in the transfer reaction catalyzed by the microsomal enzyme: the K m value with respect to the UDP- N-acetylgalactosamine is lowest for the endogenous system. Presumptive evidence is provided which suggests that the transfer of the hexosamine takes place at the hydroxyl groups of serine/threonine residues of the acceptor. 2. 2. The transfer of N-acetylgalactosamine to endogenous acceptors is low in the neonatal rat brain but increases steadily during development. The activity in the 5-week-old animal is about 4 times as that found in the brains of the newborn rats. The enzyme preparation from the neonatal rat brain transfers significant amounts of N-acetylgalactosamine to added protein acceptors and this activity toward the exogenous acceptors increases only slightly during brain development. These results lend support to our earlier suggestion that, in rat brain, the formation of polypeptide chains and the subsequent addition of sugars might be subject to independent alterations during development.

Separation of N-acetyl-alpha-glucosaminidase and N-acetyl-alpha-galactosaminidase from ox spleen. Cleavage of the O-glycosidic linkage between carbohydrate and polypeptide in ovine and bovine submaxillary glycoprotein by N-acetyl-alpha-galactosaminidase.

1 Two specific N-acetyl-α-hexosaminidases were detected in bovine spleen homogenates, purified by ammonium sulphate precipitation, separated by gel filtration and identified as N-acetyl-α-glucosaminidase and N-acetyl-α-galactosaminidase respectively. In contrast to the established N-acetyl-β-hexosaminidase which splits phenyl-β-d-glycosides of both N-acetyl-glucosamine and N-acetylgalactosamine, the two N-acetyl-α-hexosaminidases exhibits an absolute specificity towards the hexosamine moiety of their substrates. N-Acetyl-α-galactosaminidase acts on phenyl-N-acetyl-α-d-galactosaminide and not on the corresponding glucosaminide. The reverse holds for N-acetyl-α-glucosaminidase. 2 In the cell-free supernatant of a bovine spleen homogenate N-acetyl-α-galactosaminidase is present with a specific activity about 50-fold of that of the corresponding glucosaminidase, using the appropriate phenyl-α-d-hexosaminides as substrate. 3 Ox spleen N-acetyl-α-galactosaminidase was found to split both phenyl-N-acetyl-α-d-galactosaminide and the O-glycosidic linkage between terminal N-acetylgalactosamine and peptide-bonded serine (threonine) in ovine and bovine submaxillary glycoproteins. The identity of bovine O-seryl-N-acetylgalactosaminide glycosidase and N-acetyl-α-galactosaminidase is clearly demonstrated by the presence of these enzymatic activities in a single chromatographic peak even after re-chromatography. 4 One substrate of natural origin for N-acetyl-α-glucosaminidase was found in UDP-N-acetylglucosamine.

Glycoprotein biosynthesis: The solubilization of glycosyl transferases from membranes of hela cells and bovine submaxillary glycoproteins

Two glycosyl transferases, the polypeptidyl: N-acetylgalactosaminyl and glycoprotein: galactosyl transferase, which participate in the assembly of the carbohydrate units of glycoproteins, were purified from both HeLa cells and submaxillary glands. In the case of HeLa cells these enzymes are intimately associated with the smooth internal membranes where they form part of the multienzyme group of transferases synthesizing membrane glycoproteins; in submaxillary tissues, however, these enzymes are involved in synthesis of secreted submaxillary glycoproteins and are strongly bound to membranes, possibly plasma membranes, which sediment to the 35–45% sucrose interface. In preparing the respective membranes, the glycosyl transferases were purified from 10–40-fold over the cellular homogenate. It was found that from 80–95% of the total enzyme activities are masked in the isolated membranes; disruption of the membranous structure with nonionic detergents or with phospholipase A liberated the transferases. The largest increase in activity was obtained with the polyoxyethylene octylphenol type. The linear alcohol polyoxy-ethylene types were slightly less effective; less than 50% of the maximum activity was obtained. These results, together with the effect of phospholipase A, emphasize the significance of nonpolar forces in membrane substructure, particularly with regard to the interaction of the transferases with their membranous environment. By contrast, negatively charged detergents, such as sodium deoxycholate and sodium dodecyl sulfate, led to strong inhibition of enzymes. A positively charged detergent, cetylpyridinium bromide, also produced strong inhibition. It was concluded that the influence of detergents on the activity of membrane-bound transferases appears to depend on two factors, solubilization and inhibition.

Glycoprotein biosynthesis: Studies on the receptor specificity of the polypeptidyl: N-acetylgalactosaminyl transferase from bovine submaxillary glands

The receptor requirements of the polypeptidyl: N-acetylgalactosaminyl (galNAc) transferase, purified from bovine submaxillary glands, was investigated. The transfer of galNAc- 14C to the hydroxyamino acid residues of the polypeptide chain of submaxillary glycoprotein denuded of carbohydrate is a highly specific process. All other proteins and glycoproteins studied, intact and partially degraded, were nonfunctional in this reaction except for the submaxillary polypeptide. Receptors were obtained from desialized bovine submaxillary glycoprotein (BSM) by three procedures: (1) removal of the N-acetylhexosamine residues by epididymal hexosaminidase; (2) periodate treatment followed by reduction with NaBH 4 and mild acid hydrolysis; and (3) directly by acid hydrolysis (0.2 n H 2SO 4, 2 hr 100 °). Up to 17–22% of the N-acetylhexosamine previously removed could be replaced by the galNAc- 14C using the purified transferase. The best receptor material was obtained by removal of 50–70% of the N-acetylhexosamine by hexosaminidase or the periodate procedure. A linear relationship was found between the percentage of N-acetylhexosamine removed by hexosaminidase, up to approximately 50%, and the incorporation of galNAc- 14C. When more than 70–75% of the N-acetylhexosamine had been removed, however, no further increase in receptor activity was found; upon removal of 75% or more of the N-acetylhexosamine, the receptor activity was significantly decreased. Materials such as intact BSM or desialized BSM, from which the N-acetylhexosamine had not been removed, were nonfunctional as receptors. The hydroxyl groups of sugar residues would not substitute for those of specific hydroxyamino acids of the submaxillary polypeptide chain. In this respect, N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, and sialic acid were all nonfunctional as receptor sites as demonstrated by the inactivity of bovine and porcine submaxillary glycoproteins, the α 1-glycoprotein, and fetuin, each of which had been partially degraded with specific glycosidic enzymes. The specificity of the reaction for particular hydroxyamino acids of the BSM polypeptide receptor was inferred by the inactivity of the submaxillary glycoprotein after alkali-borohydride treatment. Twenty-five proteins, native and denatured, were tested for receptor activity with negative results. The only material 2 2 After this study had been completed, it was found that the basic protein encephalitogen, a homogeneous protein isolated from bovine spinal cord, had approximately 25% of the receptor activity of the bovine submaxillary polypeptide from which 50% of the N-acetylhexosamine had been removed (1). with receptor activity was found in the extract of the submaxillary glands and most likely represents endogenous BSM material at incomplete stages of synthesis. The receptors prepared from BSM exhibited both conformational and size requirements although they were unusually stable to some conditions known to denature proteins. In spite of treatment with 0.2 n H 2SO 4, 90 °, 2 hr, or with 65% ethanol, receptor activity was not affected. After the use of 8 m urea or heating at 100 °, 5 min, approximately 50% of the receptor activity still remained. The size requirements of the hydroxyamino acid-containing polypeptide chain was illustrated by the complete loss of receptor activity after Pronase digestion. After trypsin treatment, which yields larger peptides than Pronase, 35% of the receptor activity still remained. Small molecules, such as serine, threonine, triserine, trithreouine, and a pentapeptide containing both serine and threonine were also inactive. The results from this work emphasize the high specificity required of the gly cosyl transferases in general for synthesis of the specific structures found in the oligosaccharide units of glycoproteins. The existence of the polypeptidyl: galNAc transferase is consistent with a proposed “one enzyme-one linkage” concept of glycoprotein biosynthesis.

Glycoprotein biosynthesis: The basic protein encephalitogen from bovine spinal cord, a receptor for the polypeptidyl: N-acetylgalactosaminyl transferase from bovine submaxillary glands

A basic protein from bovine myelin, which contains no carbohydrate, was found to serve directly as a natural receptor for the N-acetylgalactosamine transferred from UDP- N-acetylgalactosamine (UDP-galNAc) by the polypeptidyl: N-acetylgalactosamine transferase of bovine submaxillary glands. This receptor, which mimicked in all respects the “artificial” receptor prepared from the bovine submaxillary glycoprotein by removal of the N-acetylhexosamine with hexosaminidase, possessed approximately 25% of the receptor activity (cpm galNAc- 14C incorporated per mg protein) when compared to the latter receptor. Using either receptor, the transfer reaction requires Mn 2+ ion. The product formed in both cases involves a glycosidic linkage between N-acetylgalactosamine- 14C and hydroxyamino acids; the galNAc- 14C was released from the basic protein- 14C product by hexosaminidase and N-acetylgalactos-aminitol- 14C was isolated after alkaline borohydride treatment. The importance of receptor size as a requirement for receptor function was revealed by the loss of activity following Pronase digestion of the A1 protein. The relative stability of both receptors to heating at 100 °, or 8 m urea treatment, suggested that the reactive site of the receptors is primarily dependent on the requirements of a polypeptide sequence rather than tertiary structure. The results of this work support and extend the conclusions derived from study of the specificity of the receptor requirements using the submaxillary protein as a receptor for the submaxillary gland transferase. The basic protein- 14C material was degraded with proteolytic enzymes; two of the radioactive peptides obtained following purification on Dowex 50 resin and high-voltage paper electrophoresis revealed that a threonine was joined in these peptides in an O-glycosidic linkage to galNAc- 14C. It was concluded that a polypeptide sequence exists in the A1 protein, containing a functional threonine residue, that meets the receptor requirements of the polypeptidyl: galNAc transferase as found in the submaxillary glycoprotein.

Physical and chemical studies on glycoproteins: II. Isolation and characterization of oligosaccharides from porcine submaxillary glycoproteins

Treatment of porcine submaxillary glycoproteins with alkaline borohydride (0.1N NaOH, 0.3N NaBH 4 for 120 hours at 28 °) liberated approximately one-third of the carbohydrate in the form of reduced oligosaccharides. The oligosaccharides were isolated by a procedure that included chromatography on Dowex-1 (formate), paper and char-coal-celite. The following compounds were identified by structural determination: α- l-fucopyranosyl-(1 → 2)-β- d-galactopyranosyl-(1 → 4)- d- N-acetylgalactosaminitol; α- l-fuco-pyranosyl-(1 → 2)-β- d-galactopyranosyl-(1 → 4)-[α- N-glycolyhieuraminyl-(2 → 6)]- d- N-acetylgalactosaminitol; α- N-glycolylneuraminyl-(2 → 6)- d- N-acetylgalactosaminitol; α- l-fucopyranosyl-(1 → 2)- d-galactitol; d-galactitol; and d- N-acetylgalactosaminitol. Two additional oligosaccharides were assigned the following structures on the basis of periodate oxidation and their inhibition of the human A-anti A hemagglutination reaction: α- d- N-acetylgalactosaminyl-(1 → 3)-[α- l-fucopyranosyl-(l → 2)]-β- d-galactopyranosyl-(1 → 4)- d- N-acetylgalactosaminitol and α- d- N-acetylgalactosaminyl-(l → 3)-[α- l-fucopyranosyl-(1 → 2)]-β- d-galactopyranosyl-(1 → 4)-[α- N-glycolylneuraminyl-(2 → 6)]- d- N-acetylgalactosaminitol. The carbohydrate structure of the porcine submaxillary glycoproteins is discussed in relation to the above oligosaccharides, ovine and bovine submaxillary glycoproteins, and the blood group substances.