Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase.
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ABSTRACT:
SUBMITTER: Zecher R
PROVIDER: S-EPMC1162188 | biostudies-other | 1980 Oct
REPOSITORIES: biostudies-other
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Project description:Spermidine synthase was purified to apparent homogeneity from human spleens (8700-fold) by affinity chromatography. The native enzyme was composed of two subunits of identical Mr (35,000) and showed an apparent Mr of 62,000 in pore-gradient gel electrophoresis. Its pI was 5.1, Spermine synthase was purified to apparent homogeneity from placenta (5300-fold) and from kidney (4600-fold). The native enzyme was composed of two subunits of identical Mr (45,000) and showed an apparent Mr of 78,000 in pore-gradient gel electrophoresis. In isoelectric focusing it revealed two bands, with pI values of 4.9 and 5.0. Both synthases were present in all human tissues studied, but revealed a clear tissue-specific pattern. Mouse antisera against spermidine synthase revealed only one band, of Mr 35,000, in all purified enzyme preparations and in crude human tissue extracts in immunoblotting. Antisera against spermine synthase showed an immunoreactive band corresponding to the Mr of the subunit of spermine synthase. These antisera did not indicate any cross-reactivity in immunoblotting. Thus spermine synthase and spermidine synthase do not share homologous antigenic sites and are totally different proteins.
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Project description:?-Glucosidase (EC 3.2.1.21) is a prominent member of the GH1 family of glycoside hydrolases. The properties of this ?-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. ?-Glucosidase from chayote (Sechium edule) was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other ?-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus ?-glucosidase (NCBI reference sequence XP_004154617.1), in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of ?-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results). One hundred ?g/mL ?-D-glucose inhibited ?-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min(-1). The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.
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Project description:1. The partial purification of adenylate kinase, types 1 and 2, from human erythrocytes is described. 2. Gel chromatography of both forms of the enzyme gave estimates of the molecular weights in the range 20000-23000. 3. Studies on crude haemolysates at various pH values indicated that the type 2 enzyme was less stable than the type 1. Heat denaturation studies on the partially purified enzymes confirmed these findings. 4. Measurements of rates of inhibition by iodoacetate and iodoacetamide showed that the type 2 enzyme reacts more readily than the type 1 enzyme with both reagents. 5. The effect of temperature on the initial velocity of ADP formation was measured at a single concentration of both AMP and MgATP(2-). The two forms of the enzyme responded differently to increasing temperature.
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Project description:1. Intact erythrocytes were incubated with 100 microM-4,4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS), a concentration sufficient to inhibit lactate transport irreversibly by 65%. DIDS-labelled proteins were detected by immunoblotting of erythrocyte membrane proteins with an anti-DIDS antibody. Labelled polypeptides of 35-45 kDa in rat erythrocytes, and of 40-50 kDa in rabbit and guinea pig erythrocytes, were detected by this technique. In human erythrocytes, which have 10-fold less transport activity, no labelled polypeptide in this molecular mass range was detected. 2. Labelling of these 35-50 kDa polypeptides was decreased markedly in the presence of the specific inhibitors of lactate transport alpha-cyano-4-hydroxycinnamate and 4,4'-dibenzamidostilbene-2,2'-disulphonate (DBDS), which compete with DIDS for binding to the transporter. However, the weakly bound inhibitor 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS) afforded little protection against labelling by DIDS. 3. The lactate transporter from rat erythrocytes was solubilized with decanoyl-N-methyl glucamide (MEGA-10) and partially purified by Mono-Q anion-exchange chromatography, with transport activity eluting at 0.1-0.15 M-NaCl. The 35-45 kDa DIDS-labelled polypeptide from rat erythrocytes was eluted in the same peak of protein as lactate transporter activity during Mono-Q chromatography. 4. These observations provide strong evidence that the lactate transporter is a polypeptide of 35-45 kDa in rat erythrocytes and of 40-50 kDa in rabbit and guinea pig erythrocytes.
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Project description:Human angiotensinogen has been purified 390-fold from serum by a rapid high-yielding procedure that involved chromatography on Blue Sepharose, phenyl-Sepharose, hydroxyapatite and immobilized 5-hydroxytryptamine (5-HT). Angiotensinogen was specifically bound to immobilized 5-HT, which effected a partial resolution into multiple forms, which were also evident when analysed by SDS/polyacrylamide-gel electrophoresis (Mr 59,400, 60,600, 62,600 and 63,800). This heterogeneity was confirmed by resolution into six main bands on isoelectric focusing, ranging from pI 4.40 to 4.82. N-terminal analysis, digestion with human renal renin and deglycosylation studies implied that the preparation comprised several forms of angiotensinogen, varying in their degree of glycosylation. The presence of sialic acid was shown to be a major factor in determining the heterogeneity.
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Project description:Phytase is a phosphatase enzyme widely used as feed additive to release inorganic phosphorus from plant phytate and enhance its uptake in monogastric animals. Although engineered fungal phytases are used most, a natural enzyme gives opportunity to understand novel properties, if any. In the current study, a novel fungal strain, Aspergillus foetidus MTCC 11682 was immobilized on poly urethane cubes and used for phytase production, purification and molecular characterization. Phytase produced by this method was partially purified by ammonium sulphate precipitation and Sephacryl S-200HR gel filtration to 23.4-fold (compared to crude extract) with recovery of 13% protein. Electrophoresis analysis revealed that phytase has molecular weight of 90.5 kDa on non-reducing and 129.6 kDa on reducing SDS-PAGE. The purified phytase exhibited a wider pH and temperature stability. Analysis of the cloned sequence showed that the gene has 1176 bp that encodes for a peptide of 391 amino acids of the core catalytic region. It was also found that phytase from A. foetidus has a sequence identity of 99% with the phytase gene of other Aspergillus species at nucleotide level and 100% at protein level in A. niger, A. awamori, A. oryzae. In silico analysis of sequence identified the presence of two consecutive and one non-consecutive intra chain disulfide bonds in the phytase. This probably contributed to the differential migration of phytase on reducing and non-reducing SDS-PAGE. There are predicted 11 O-glycosylation sites and 8 N-glycosylation sites, possibly contributed to an enhanced stability of enzyme produced by this organism. This study opened up a new horizon for exploring the novel properties of phytase for other applications.
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Project description:Seven xylanolytic bacterial strains were isolated from saw-dust dump soil. The bacterial strain X6 was selected on the basis of the highest xylanase activity with no cellulase contamination. It was identified as Stenotrophomonas maltophilia by biochemical tests and 16S rRNA gene sequencing approach. Xylanase production studies by S. maltophilia on different commercial xylans and agro-industrial residues suggested that wheat bran was the best carbon source for xylanase production (26.4 ± 0.6 IU/mL). The studies with inorganic and organic nitrogen sources suggested yeast extract as the best support for xylanase production (25 ± 0.6 IU/mL). Maximum xylanase production was observed at initial medium pH = 8.0 (23.8 ± 0.4 IU/mL) with production at pH = 7.0 and pH = 9.0 being almost comparable. Xylanase produced by S. maltophilia was purified to homogeneity using ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The final purification was 5.43-fold with recovery of 19.18%. The molecular weight of the purified xylanase protein was ~142 kDa. Both crude and purified xylanase had good stability at pH = 9.0 and 80°C with activity retention greater than 90% after 30 min incubation. The enzyme stability at high temperature and alkaline pH make it potentially effective for industrial applications.
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