Action of endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. on amino acid-O-glycans: comparison with the enzyme from Diplococcus pneumoniae

Endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. released the disaccharide, Gal beta 1----3GalNAc, from both dansylated serine-GalNAc-Gal and threonine-GalNAc-Gal, and showed higher activity on the former than the latter. The Km values were 0.17 mM and 1.43 mM with DNS-Ser-GalNAc-Gal and DNS-Thr-GalNAc-Gal, respectively. The optimum pHs were found to be 4.5-7.5 and 4.5-6.0 on DNS-Ser-GalNAc-Gal and DNS-Thr-GalNAc-Gal, respectively. On the contrary, the enzyme from Diplococcus pneumoniae had low activity to release the disaccharide from the amino acid-O-glycans. The possibility that the same O-glycoside but linked to different aglycon amino acids may play a different biological role in glycoproteins is discussed.     

Purification and characterization of an endo-beta-galactosidase produced by Diplococcus pneumoniae.

An endo-beta-galactosidase acting on blood group A and B substances was found in the culture fluid of Diplococcus pneumoniae. The enzyme was purified 1000-fold, and its properties were studied in detail. The enzyme preparation, thus obtained, was practically free from various exoglycosidases, endo-beta-N-acetylglucosaminidase and proteases. The enzyme releases trisaccharides from blood group A and B active mucins purified from ovarian cyst fluid. The structures of the trisaccharides liberated from A and B active mucins were elucidated to be GalNAcalpha1 leads to 3(Fucalpha1 leads to 2)Gal and Galalpha1 leads to 3(Fucalpha1 leads to 2)Gal, respectively. The enzyme also hydrolyzes blood group A and B active oligosaccharides composed of type 2 chains, yielding the same products as in the case of ovarian cyst blood group substances. An H active mucin from ovarian cyst fluid, H active oligosaccharides, and A and B active oligosaccharides with type 1 chains were not hydrolyzed by the enzyme. Consequently, the enzyme catalyzes the following reaction, resulting in the degradation of blood type A and B determinants. (see article).     

Characterization of an endo-alpha-N-acetyl galactosaminidase from Diplococcus pneumoniae.

Evidence is presented for the presence in filtrates of Diplococcus pneumoniae of an endo-glycosidase capable of acting on the O-glycosidic linkage between N-acetyl galactosamine and serine or threonine residues. The glycosidase was partially purified by chromatography on Affi-Gel 202. The resulting preparation acted on glycopeptides from mouse melanoma, fetuin and pig submaxillary mucin to release a disaccharide characterized as galactosyl-N-acetyl galactosamine. The enzyme had no action on phenyl α-N-acetyl-D-galactosaminide, asialo ovine submaxillary mucin or monosialoganglioside. A similar activity was detected in a commercial preparation of Clostridium perfringens neuraminidase.     

Purification and properties of an endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae.

An enzyme that hydrolyzes the O-glycosidic linkage between alpha-N-acetyl-D-galactosamine and serine or threonine in mucins and mucin-type glycoproteins was purified by chromatography on an Affi-Gel 202 column or isoelectric focusing from filtrates of Diplococcus pneumoniae cultures. The final preparations were free of protease and a wide range of other glycosidase activities. The preparation obtained by isoelectric focusing was shown to consist of a single protein by gel filtration and sodium dodecyl sulfate-gel electrophoresis. This preparation had an apparent molecular weight of about 160,000, determined by gel filtration, an optimum pH of 7.6, and an isoelectric point in the range pH 8 to 9. The enzyme releases the disaccharide Gal-GalNAc from a variety of glycopeptide and glycoprotein substrates and appears to have a specific requirement for an unsubstituted galactose in the nonreducing terminus and an alpha linkage between N-acetylgalactosamine and the aglycone. This is the only endoenzyme known capable of cleaving the linkage between a carbohydrate and serine or threonine residues in glycoproteins. The ability of this enzyme to act on macromolecular substrates and its pH optimum makes it ideally suited to explore the distribution and function of mucin-type glycoproteins on normal and cancer cell surfaces.     

Isolation and characterization of a new endo-beta-galactosidase from Diplococcus pneumoniae

An endo-beta-galactosidase, which hydrolyzes the internal beta-galactosidic linkages of R----GlcNAc (or GalNAc) beta 1----3Gal beta 1----4GlcNAc (or Glc), was isolated from the culture supernatant of Diplococcus pneumoniae. The enzyme, named endo-beta-galactosidase DII, hydrolyzed linear N-acetyllactosamine repeating structures in glycolipids and glycopeptides to release oligosaccharides. The specificity of endo-beta-galactosidase DII is the same as that of Escherichia freundii endo-beta-galactosidase as far as described above, but the following differences between these two enzymes were found: Branched lactosaminyl glycolipids and H-antigenic glycolipids were resistant to endo-beta-galactosidase DII, even when linear structure was present at the inner part. Throughout the enzymic hydrolysis, endo-beta-galactosidase DII released mostly small oligosaccharides (tetra-, tri-, and disaccharides) from substrates, suggesting that the enzyme split off the oligosaccharides stepwise from the nonreducing terminal. Lactosaminoglycans were partially hydrolyzed by endo-beta-galactosidase DII to produce small oligosaccharides as the major product and residual glycopeptides. The residual glycopeptides were readily hydrolyzed by E. freundii endo-beta-galactosidase to produce various sizes of oligosaccharides. Keratan sulfate was not degraded by endo-beta-galactosidase DII. These properties of endo-beta-galactosidase DII characterize it as a new endo-beta-galactosidase with a unique specificity.     

Partial purification and characterization of an exonuclease from Xanthomonas oryzae.

An exonuclease with a strong preference for single-stranded deoxyribonucleic acid over double-stranded deoxyribonucleic acid has been purified 500-fold from Xanthomonas oryzae. This enzyme liberates 5'-mononucleotides in a reaction which requires Mg2+.     

Soluble adenylate cyclase from the culture medium of Bordetella pertussis: purification and characterization.

Culture medium of exponentially growing Bordetella pertussis (strain 114) contains significant quantities of soluble (100,000 X g for 1 h) adenylate cyclase. The enzyme was purified by chromatography on diethylaminoethyl-cellulose and Sephadex G-200. The purest material yielded a single band on sodium dodecyl sulfate-disc gel electrophoresis. It is heat labile, has a temperature optimum of 30 degrees C, a pH optimum of pH 7 to 8, and a Km for adenosine 5'-triphosphate of 0.4 mM, and requires Mg2+ for maximum activity. The molecular weight, by sodium dodecyl sulfate-disc gel electrophoresis and sucrose density gradient, is approximately 70,000. The enzyme is markedly inhibited by fluoride and weakly inhibited by monovalent salts, but its activity is not altered by alpha-keto acids of nonsubstrate nucleoside triphosphates. Thus, but its presence in the culture supernatant, its smaller molecular weight, and its insensitivity to alpha-keto acids and nucleotides, this enzyme differs from the bacterial adenylate cyclases previously described.     

Partial purification and characterization of a BCGFII from EL4 culture supernatants

Two B cell growth factor activities have been previously described. One activity, present in the culture supernatants of PMA-induced EL4 is active in a co-stimulator assay with normal B cells and anti-immunoglobulin. The other activity is present in the culture supernatants of the alloreactive T cell line C.C3.11.75 and can be assayed in a co-stimulator assay with normal B cells and dextran sulfate or with BCL1 in vivo line B cell tumor. We have termed the first activity BCGFI and the second BCGFII. We have now shown that a very similar BCGFII activity can be obtained from EL4 culture supernatants induced by PMA. This (EL4)BCGFII has an apparent m.w. of 55,000, is eluted from DEAE Sephacel at 0.05 M NaCl, and has a pI of 5.5, which is clearly distinct from the properties of (EL4)BCGFI activity. (EL4)BCGFII activity is similar to but not identical to (DL)BCGFII. It differs from (DL)BCGFII in chromatographic behavior and in the kinetics of the response of BCL1 to the two factors. (EL4)BCGFII activity can be detected in 18 to 24 hr by virtue of its ability to cause in vitro proliferation of in vivo BCL1 tumor B cells.     

Partial purification and characterization of an alkaline proteinase from the rabbit testis

A proteolytic enzyme capable of cleaving intact proteins and synthetic substrates α?N?benzoyl?DL?arginine β?naphthylamide (Bz-Arg-NNap), α-N-benzoyl-L-arginine p-nitroanilde (Bz-Arg-NPhNO₂), and α-N-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) was purified 92– fold from the rabbit testes. The enzyme exhibited optimal activity at pH 9.0 and 50°C. The polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme demonstrated multiple forms; the major band in the SDS-polyacrylamide gel electrophoresis corresponded to a M_t 48,000. The same value was established by the gel filtration over Sephadex G-75. The rabbit testicular alkaline proteinase (TAP) resembled acrosin in the hydrolysis of Bz-Arg-OEt. However, CaCl₂, a potential stimulator of acrosin activity, inhibited the alkaline proteinase. The strong inhibitors of acrosin, eg pheny methyl sulphonyl fluoride (PMSF), tosyl lysine chloromethyl ketone (TLCK), and benzamidine did not inhibit the alkaline proteinase. TAP was activated by an acrosin inhibitor isolated from the rabbit testes. Since 0.5 M KCl was necessary for complete extraction of the enzyme and the bulk of the activity was present in 9,000g pellet of the testicular homogenate. The alkaline proteinase appeared to be associated with the membranous structures.     

Partial purification and characterization of an anion-activated ATPase from radish microsomes

Previous investigation showed two distinct ATP-dependent proton-transporting systems in microsomal vesicle from radish seedlings, one inhibited by vanadate and one inhibited by NO-3. On the bases of the effects of these inhibitors we could discriminate two distinct ATPase activities in the same material. The NO-3 sensitive activity was separated from the vanadate-sensitive activity and partially purified by a single-step chromatographic method, which lead to approx 35-fold purification from the microsomes and to a specific activity of 2.3 mumol Pi X min-1 X mg protein-1, at 30 degrees C. The partially purified activity was specific for ATP, some activity being observed toward GTP, and even less toward CTP, UTP and ITP. No significant Pi hydrolysis was found with ADP, AMP, p-nitrophenylphosphate and glucose 6-phosphate. ADP but not AMP was inhibiting in the presence of ATP. The activity was dependent on divalent cations in the order of preference: Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+ greater than Zn2+. The activity was unaffected by monovalent cations, strongly activated by Cl-, inhibited by 90% by 50 mM NO-3, virtually unaffected by oligomycin and NaN3. At least 90% of the activity was abolished in the presence of each: 10 microM N,N'-dicyclohexylcarbodiimide, 10 microM erythrosin B, 10 mu mersalyl, 100 microM trimethyltin, 100 microM diethylstilbestrol, 100 microM N-ethylmaleimide. No inhibition has been found in the presence of Ca2+, at a concentration blocking the vanadate-sensitive activity. Nigericin, gramicidin and carbonylcyanide p-trifluoromethoxyphenylhydrazone stimulated the activity of this preparation after it was incubated in the presence of sonicated phospholipids, suggesting the capacity of the ATPase to function as a H+-transporting system. All characteristics mentioned were closely similar to those described in the vacuolar ATPases.     

Partial purification and characterization of an NAD-dependent 3 beta-hydroxysteroid dehydrogenase from Clostridium innocuum.

In nine strains of Clostridium innocuum, 3 beta-hydroxysteroid-dehydrogenating activities were detected. 3 beta, 7 alpha, 12 alpha-Trihydroxy- and 3 beta-hydroxy-12-keto-5 beta-cholanoic acids were identified as reduction products of the respective 3-keto bile acids by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. One strain was shown to contain a NAD-dependent 3 beta-hydroxysteroid dehydrogenase. Enzyme production was constitutive in the absence of added bile acids. The specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids, with trisubstituted acids being more effective than disubstituted ones. A pH optimum of 10.0 to 10.2 was found after partial purification by DEAE-cellulose chromatography. A molecular weight of about 56,000 was established. 3 beta-hydroxysteroid dehydrogenase activity was also found in the membrane fraction after solubilization with Triton X-100, suggesting that the enzyme was originally membrane bound. The enzyme reduced a 3-keto group in unconjugated and conjugated bile acids, lower Km values being demonstrated with disubstituted than with trisubstituted bile acids. Keto functions at C-7 and C-12 further reduced the Km value. The enzyme was found to be partially heat labile (86% inactivation at 50 degrees C for 10 min).