The enzymic degradation of porphyran.
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ABSTRACT:
SUBMITTER: Turvey JR
PROVIDER: S-EPMC1198303 | biostudies-other | 1967 Oct
REPOSITORIES: biostudies-other
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Project description:1. A multienzyme system capable of degrading keratosulphates to yield galactose, N-acetylglucosamine and sulphate was found in the liver extract of a marine gastropod, Charonia lampas. 2. During the degradation, neither oligosaccharides nor sulphated sugars were produced. 3. It is suggested that the degradation could be attributed to the concerted action of beta-galactosidase, beta-N-acetylglucosaminidase and a sulphatase (sulphohydrolase), tentatively designated keratosulphatase. 4. Two forms of keratosulphatase (I and II) were separated by DEAE-Sephadex column chromatography. Both forms could release all the sulphate from keratosulphates and neither appeared to be identical with glycosulphatase or chondrosulphatase, both of which are also present in Charonia lampas. 5. beta-Galactosidase and beta-N-acetylglucosaminidase could degrade keratopolysulphate to a greater extent in the presence of keratosulphatase than in its absence. 6. It is suggested that keratosulphate was first desulphated by the action of keratosulphatase, and the desulphated polymer was then degraded to galactose and N-acetylglucosamine by the action of beta-galactosidase and beta-N-acetylglucosaminidase. 7. beta-Galactosidase alone released a small amount of galactose from shark cartilage keratopolysulphate, but beta-N-acetylglucosaminidase alone did not release N-acetylglucosamine. This indicates that unsulphated galactose residues occupy all the non-reducing terminal positions in keratopolysulphate chains.
| S-EPMC1179316 | biostudies-other
Project description:1. Ovalbumin glycopeptides, freed from all amino acids other than aspartic acid and a small proportion of leucine by repeated digestion with Pronase, were hydrolysed by 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (glycoaspartamidase) to the corresponding oligosaccharides. The glycoaspartamidase did not attack ovalbumin itself. 2. Ovalbumin, with mannose/hexosamine ratio 5:4, lost 1.5moles of N-acetylglucosamine and more than 2moles of mannose after incubation with alpha-mannosidase and beta-N-acetylglucosaminidase respectively. 3. In ovalbumin glycopeptides with approximate mannose/hexosamine ratios 5:3 and 5:4, one and two N-acetylglucosamine residues respectively were accessible to the action of beta-N-acetylglucosaminidase. 4. A mixture of alpha-mannosidase and beta-N-acetylglucosaminidase, acting on an ovalbumin glycopeptide with mannose/hexosamine ratio 5:3.7, removed nearly 4moles of mannose and 1.5moles of N-acetylglucosamine. 5. alpha-Mannosidase removed about 1.5moles of mannose from the ovalbumin oligosaccharide with mannose/hexosamine ratio approx. 5:3. The subsequent action of beta-N-acetylglucosaminidase liberated less than 1mole of N-acetylglucosamine and made at least 1mole further of mannose accessible to alpha-mannosidase action. 6. It is concluded that the carbohydrate moiety of ovalbumin is linked through a glycosyl group to asparagine. In a molecule with mannose/hexosamine ratio 5:4, there are two beta-N-acetylglucosamine residues linked together in a terminal position, followed by alpha-mannose. There is also present a side chain containing two alpha-mannose units.
| S-EPMC1185198 | biostudies-other
Project description:The equilibrium model, which describes the influence of temperature on enzyme activity, has been established as a valid and useful tool for characterizing enzyme eurythermalism and thermophily. By introducing K(eq), a temperature-dependent equilibrium constant for the interconversion between E(act), the active form of enzyme, and E(inact), a reversibly inactive form of enzyme, the equilibrium model currently provides the most complete description of the enzyme-temperature relationship; its derived parameters are intrinsic and apparently universal and, being derived under reaction conditions, potentially have physiological significance. One of these parameters, T(eq), correlates with host growth temperature better than enzyme stability does. The vent-dwelling annelid Alvinella pompejana has been reported as an extremely eurythermal organism, and the symbiotic complex microbial community associated with its dorsal surface is likely to experience similar environmental thermal conditions. The A. pompejana episymbiont community, predominantly composed of epsilonproteobacteria, has been analyzed metagenomically, enabling direct retrieval of genes coding for enzymes suitable for equilibrium model applications. Two such genes, coding for isopropylmalate dehydrogenase and glutamate dehydrogenase, have been isolated from the A. pompejana episymbionts, heterologously expressed, and shown by reverse transcription-quantitative PCR to be actively expressed. The equilibrium model parameters of characterized expression products suggested that enzyme eurythermalism constitutes part of the thermal adaptation strategy employed by the episymbionts. Moreover, the enzymes' thermal characteristics correspond to their predicted physiological roles and the abundance and expression of the corresponding genes. This paper demonstrates the use of the equilibrium model as part of a top-down metagenomic approach to studying temperature adaptation of uncultured organisms.
| S-EPMC2227733 | biostudies-literature
Project description:The digestion of the Fc fragment of rabbit immunoglobulin IgG by several proteolytic enzymes was investigated by using gel filtration and starch-gel electrophoresis in 8m-urea-formate as criteria of the extent of degradation. Though fragment Fc and mildly reduced fragment Fc proved resistant to tryptic hydrolysis, papain and pepsin cleaved the fragment at acidic pH values and appeared to give rise to a similar spectrum of products. A (limit) peptide comprising the C-terminal 113 residues of the heavy chain was isolated and identified from the pepsin-digest products of fragment Fc. The products of proteolytic digestion of fragment Fc were no longer able to inhibit passive cutaneous anaphylaxis by rabbit anti-(bovine serum albumin) or demonstrate reversed passive cutaneous anaphylaxis in the guinea pig. Nor were they able to inhibit the intestinal absorption of heterologous immunoglobulin IgG in the young mouse. These studies imply that the site or sites responsible for these biological properties of intact fragment Fc reside in the N-terminal 30-40% of the fragment.
| S-EPMC1270632 | biostudies-other
Project description:1. Cell-free extracts, prepared from a non-fluorescent Pseudomonas grown on m-cresol, oxidized gentisate and certain alkyl-substituted gentisates with the consumption of 1 mol of oxygen and the formation of 1 mol of pyruvate from 1 mol of substrate. 2. In addition to pyruvate, malate was formed from gentisate; citramalate was formed from 3-methylgentisate and 4-methylgentisate; 2,3-dimethylmalate was formed from 3,4-dimethylgentisate. 3. One enantiomer, d-(-)-citramalate, was formed enzymically from 3-methylgentisate, 4-methylgentisate and citraconate. l-(+)-Citramalate was formed from mesaconate by the same extracts. When examined as its dimethyl ester by gas-liquid chromatography, enzymically formed 2,3-dimethylmalate showed the same behaviour as one of the two racemates prepared from the synthetic compound. 4. Maleate, citraconate and 2,3-dimethylmaleate were rapidly hydrated by cell extracts, but ethylfumarate and 2,3-dimethylfumarate were not attacked. 5. Cell extracts oxidized 1,4-dihydroxy-2-naphthoate to give pyruvate and phthalate. 6. Alkylgentisates were oxidized by a gentisate oxygenase (EC 1.13.1.4) present in Pseudomonas 2,5. The ring-fission products were attacked by maleylpyruvase, but not by fumarylpyruvase, and their u.v.-absorption spectra were those expected for alkyl-substituted maleylpyruvates. 7. When supplemented with ATP, CoA, succinate and Mg(2+) ions, an enzyme system from cells grown with 2,5-xylenol formed pyruvate from d- but not from l-citramalate. Extracts from cells grown with dl-citramalate or with itaconate attacked both d- and l-citramalate; other alkylmalates were cleaved in similar fashion to give pyruvate or 2-oxobutyrate. 8. These results accord with a general sequence of reactions in which the benzene nucleus of an alkylgentisate is cleaved to give an alkyl-substituted maleylpyruvate. The ring-fission products are hydrolysed to give pyruvate, plus alkylmalic acids which then undergo aldol fissions, probably as their CoA esters. In Pseudomonas 2,5 several homologous sequences of this general type appear to be catalysed by a single battery of enzymes with broad substrate specificities, whereas the metabolic capabilities of the fluorescent Pseudomonas 3,5 are more restricted. 9. Intact cells of both organisms metabolize d-malic acid by reactions that have not been elucidated, but are different from those which degrade alkylmalates.
| S-EPMC1176684 | biostudies-other
Project description:How genetic information gained its exquisite control over chemical processes needed to build living cells remains an enigma. Today, the aminoacyl-tRNA synthetases (AARS) execute the genetic codes in all living systems. But how did the AARS that emerged over three billion years ago as low-specificity, protozymic forms then spawn the full range of highly-specific enzymes that distinguish between 22 diverse amino acids? A phylogenetic reconstruction of extant AARS genes, enhanced by analysing modular acquisitions, reveals six AARS with distinct bacterial, archaeal, eukaryotic, or organellar clades, resulting in a total of 36 families of AARS catalytic domains. Small structural modules that differentiate one AARS family from another played pivotal roles in discriminating between amino acid side chains, thereby expanding the genetic code and refining its precision. The resulting model shows a tendency for less elaborate enzymes, with simpler catalytic domains, to activate amino acids that were not synthesised until later in the evolution of the code. The most probable evolutionary route for an emergent amino acid type to establish a place in the code was by recruiting older, less specific AARS, rather than adapting contemporary lineages. This process, retrofunctionalisation, differs from previously described mechanisms through which amino acids would enter the code.
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Project description:1. During the enzyme-catalysed degradation of l-serine O-sulphate no exchange occurs between the hydrogen atom attached to the alpha-carbon atom of the substrate and the tritiated water of the incubation medium. 2. The participation of an intermediate carbanion has been demonstrated in the degradation by performing the reaction in the presence of tetranitromethane. 3. Photo-oxidation of the enzyme in the presence of Rose Bengal led to a rapid inactivation of enzyme with the concomitant loss of four histidine residues/molecule. 4. Rose Bengal was also a non-competitive inhibitor of the enzyme.
| S-EPMC1173567 | biostudies-other
Project description:A sulphamidase and a sulphoesterase were isolated from adapted cells of Flavobacterium heparinum. These enzymes were partially purified from the ;heparinases' present in the bacterial extracts and characterized. The sulphamidase has a high specificity for glucosamine N-sulphate and glucosamine 2,6-disulphate. The activity decreases sharply with increasing molecular weight of the substrates tested. The sulphamidase and the sulphoesterase activities were distinguished from each other by their different sensitivities to concentration of phosphate ion and to temperature. The importance of these enzymes in the study of the structure of heparin is discussed.
| S-EPMC1187498 | biostudies-other
Project description:XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure-function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561-->Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.
| S-EPMC1134786 | biostudies-literature
Project description:Aminoglycoside 2''-phosphotransferases are clinically important enzymes that cause high levels of resistance to aminoglycoside antibiotics by the organisms that harbor them. These enzymes phosphorylate aminoglycosides, and the modified antibiotics show significant reduction in the binding ability to target the bacterial ribosome. This report presents a detailed characterization of the antibiotic resistance profile and the aminoglycoside and nucleotide triphosphate substrate profiles of four common aminoglycoside 2''-phosphotransferases widely distributed in clinically important Gram-positive microorganisms. Although the antibiotic resistance phenotypes exhibited by these enzymes are similar, their aminoglycoside and nucleotide triphosphate substrate profiles are distinctive. Contrary to the dogma that these enzymes use ATP as the source of phosphate in their reactions, two of the four aminoglycoside 2'-phosphotransferases utilize GTP as the phosphate donor. Of the other two enzymes, one exhibits preference for ATP, and the other can utilize either ATP or GTP as nucleotide triphosphate substrate. A new nomenclature for these enzymes is put forth that takes into account the differences among these enzymes based on their respective substrate preferences. These nucleotide triphosphate preferences should have ramifications for understanding of the evolution, selection, and dissemination of the genes for these important resistance enzymes.
| S-EPMC2652286 | biostudies-literature