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Solubilization of a mannose-polymerizing enzyme from Phaseolus aureus.

ABSTRACT: A soluble enzyme preparation, which catalyses the polymerization of mannose, was obtained by Triton X-100 extraction of a particulate fraction derived from Phaseolus aureus hypocotyls. The product that resulted when GDP-alpha-d-mannose was used as a substrate was a beta-(1-->4)-linked mannan, about three-quarters of which was alkali-insoluble. The mannose-polymerizing enzyme activity was at least as great in the soluble preparation as in the particulate preparation, and the specific activity of the solubilized enzyme was greater by a factor of at least 3.5. Kinetic studies of the soluble enzyme indicate that the apparent K(m) is 55-62mum, and a disproportionate increase in rate is observed at high concentrations. GDP-alpha-d-glucose is a strong competitive inhibitor of the mannose-polymerizing reaction, with an apparent K(i) of 6.2mum. The soluble enzyme is relatively unstable, losing about two-thirds of its original activity in 5h at 0 degrees C or in 24h at -20 degrees C. A solvent (acetone, butanol, diethyl ether)-extracted particulate preparation, which also exhibits the same enzyme activity, is more stable, retaining full activity for at least 5 days at -20 degrees C. There was no polymerizing-enzyme activity in the soluble enzyme preparation when UDP-d-glucose, UDP-d-galactose, UDP-d-xylose, UDP-l-arabinose or UDP-d-glucuronic acid were used as substrates. However, the soluble enzyme preparation would catalyse the polymerization of glucose, with GDP-d-glucose as substrate.

SUBMITTER: Heller JS

PROVIDER: S-EPMC1173760 | biostudies-other | 1972 Jun

REPOSITORIES: biostudies-other

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Project description:With a particulate enzyme preparation from Phaseolus aureus hypocotyls, UDP-alpha-d-[U-(14)C]galactose served as a precursor for a number of products. One of these products was characterized as a beta-(1-->4)-linked galactan. The ADP-, GDP-, TDP- and CDP- derivatives of alpha-d-galactose did not serve as biosynthetic precursors for any products insoluble in 70% ethanol, nor as substrates for a sugar nucleotide 4-epimerase which is present in the particulate enzyme preparation. The (14)C-labelled beta-(1-->4)-galactan is alkali-insoluble and was characterized by analysis of partial acetolysis products. The labelling pattern of the [(14)C]oligosaccharides derived from acetolysis indicates that (1) only slightly more than two [(14)C]galactose moieties are added to the growing polysaccharide chain on average, and (2) these additions take place at the reducing end of the polysaccharide chain. The radioactive beta-(1-->4)-linked galactan chain represented 8.5% of the radioactivity initially added, and 20% of the water- and butanol-insoluble products derived from UDP-alpha-d-[(14)C]galactose. Total hydrolysis of the alkali-insoluble fraction of Phaseolus aureus hypocotyl yielded d-glucose and d-mannose in a 5:1 ratio but no detectable quantities of d-galactose. A trace quantity of a radioactive disaccharide, identified as (1-->3)-linked galactobiose, was isolated from the partial acetolysate of the alkali-insoluble [(14)C]polysaccharide material. Also isolated from this partial acetolysate was a C-1 derivative of [(14)C]galactose, which could not be identified. An alkali-soluble galactose-containing polysaccharide was also synthesized in this enzymic reaction, and represented 20% of the water- and butanol-insoluble products derived from UDP-alpha-d-[(14)C]galactose. The spectrum of radioactive oligosaccharides produced by partial acetolysis of this alkali-soluble polysaccharide material was different from that obtained from the alkali-insoluble polysaccharide, indicating a different structure.

Project description:1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.

Project description:ObjectivesMannose-binding lectin (MBL) is an important component of innate immunity. Structural and promoter polymorphisms in the MBL2 gene that are responsible for low MBL levels are associated with susceptibility to infectious diseases. The objective of this study was to investigate the association of serum MBL levels and MBL2 polymorphisms with persistent Staphylococcus aureus bacteremia (SAB) in adult Korean patients.MethodsWe conducted a case-control study nested in a prospective cohort of patients with SAB. The study compared 41 patients with persistent bacteremia (?7 days) and 46 patients with resolving bacteremia (<3 days). In each subject, we genotyped six single-nucleotide polymorphisms in the promoter region (alleles H/L, X/Y, and P/Q) and exon 1 (alleles A/B, A/C, and A/D) of the MBL2 gene and measured serum MBL concentrations. We also compared MBL2 genotypes between SAB patients and healthy people.ResultsPatients with persistent bacteremia were significantly more likely to have low/deficient MBL-producing genotypes and resultant low serum MBL levels, than were patients with resolving bacteremia (P?=?0.019 and P?=?0.012, respectively). Independent risk factors for persistent bacteremia were metastatic infection (adjusted odds ratio [aOR], 34.7; 95% confidence interval [CI], 12.83-196.37; P?=?0.003), methicillin resistance (aOR, 4.10; 95% CI, 3.19-29.57; P?=?0.025), and low/deficient MBL-producing genotypes (aOR, 7.64; 95% CI, 4.12-63.39; P?=?0.003). Such genotypes were significantly more common in patients with persistent bacteremia than in healthy people (OR, 2.09; 95% CI, 1.03-4.26; P?=?0.040).ConclusionsThis is the first demonstration of an association of low MBL levels and MBL2 polymorphisms responsible for low or deficient MBL levels with persistent SAB. A combination of factors, including clinical and microbiological characteristics and host defense factors such as MBL levels, may together contribute to the development of persistent SAB.

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Solubilization of a mannose-polymerizing enzyme from Phaseolus aureus.

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