Purification and some properties of phospholipase C from Achromobacter xylosoxidans.

A non-haemolytic phospholipase C (EC 3.1.4.3) was purified from the culture medium of Achromobacter xylosoxidans with a 5% yield and a purification factor of 330. A combination of ultrafiltration, acetone precipitation and two subsequent affinity chromatographic steps was used. The affinity chromatography is a new application of 2-(4-aminophenylsulphonyl)ethyl-cellulose, a sorbent that has previously been used for the purification of phospholipase C from Bacillus cereus. The purified enzyme gave four distinct bands on polyacrylamide gel electrophoresis, and each band was catalytically active. Under our experimental conditions, the phospholipids examined were hydrolysed in the following order: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin. Neither the synthetic substrate p-nitrophenylphosphorylcholine nor phosphatidylinositol was hydrolysed under different experimental conditions. For maximal hydrolytic activity toward phosphatidylcholine, the enzyme required Triton X-100 and Ca2+ ions. EDTA was inhibitory, but the enzyme activity was almost completely restored by Zn2+. The molecular mass of the phospholipase C, estimated by gel permeation, was 34,000 daltons.     

Purification and some properties of three serine carboxypeptidases from Aspergillus niger

Three enzymes exhibiting peptidyl-L-amino acid hydrolase and esterase activities have been purified by immobilized metal-ion affinity chromatography and ion-exchange chromatography. The three enzymes were entirely free of the acid protease activity that normally exists along with them in the crude culture filtrates of Aspergillus niger. Although all three exo-peptidases possessed nearly identical molecular weights (ca. 140,000), isoelectric points (ca. 5.0) and other properties, their affinities for the two substrates tested, carbobenzoxy-L-Glu-L-Tyr and benzoyl L-arginine ethyl ester, differed. All three peptidases were inhibited by phenylmethanesulphonyl fluoride, indicating that they are serine carboxypeptidases. They were also inhibited by tosyl phenylalanine chloromethyl ketone, suggesting the presence of a histidyl residue in their active sites. The differences in the number of accessible histidyl residues on the enzyme surfaces could explain the differences in their retentions on Cu2+-iminodiacetate-Sepharose 6B.     

Purification and some properties of inducible lysine decarboxylase from Vibrio parahaemolyticus.

Inducible lysine decarboxylase from Vibrio parahaemolyticus AQ 3627 was purified to apparent homogeneity and characterized. The enzyme displayed a molecular weight of 531000 by gel filtration and 79000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required pyridoxal phosphate as a cofactor, and the pH optimum was 5.5. The Km value for L-lysine was 3.2 mM, and the enzyme was inhibited by 6-aminocaproic acid and alpha-fluoromethyl analogs of cadaverine. delta-Hydroxylysine and S-aminoethyl-L-cysteine was active as substrates to a lesser extent than L-lysine. The amino-terminal amino acid sequence was determined to be Met-Asn-Ile-Phe-Ala-Ile-Leu. These properties were compared with those of other bacterial lysine decarboxylases.     

Purification and some properties of chitinase fromAspergillus carneus

An extracellular chitinase fromAspergillus cerneus was purified by ammonium sulphate precipitation, gel filtration through Sephadex G-100, preparative HPLC chromatography and large slabs of polyacry-lamide gel electrophoresis.The mol wt of the enzyme was estimated to be 25000 by SDS gel electrophoresis, and it contained 9.37% (w/w) carbohydrate residue, as glucose. The pattern of its amino acid composition showed high contents of asparagine, serine, and threonine. The enzyme was active at pH 5.2 and 50°C. The K_m value of the enzyme was 4.37 mM (expressed asN-acetylglucosamine). The enzyme was stable at pH 3–9, whereas it was unstable at 70°C or more. Calcium and Mg ions slightly activated the enzyme, whereas Hg²⁺, I₂, andp-chloromercuribenzoate inhibited the enzyme activity. The enzyme hydrolyzed chitin, colloidal chitin, glycol chitin, and chitooligsac-charides, but did not hydrolyze chitosan, starch, xylan, inulin, and cellulose. The lysis ofA. niger and Micorcoccus lysodeikticus cell walls by the action of the enzyme was also investigated.     

Purification and some properties of squalene-2,3-epoxide: lanosterol cyclase from rat liver

Squalene-2,3-epoxide: lanosterol cyclase was purified from rat liver in five steps as a soluble and homogeneous protein. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis with a molecular weight of 75 kD. In its native state it behaved as a homo-dimer. The isoelectric point of 5.5 and the apparent Km value for (3S)-squalene-epoxide of 55 microM were estimated for the cyclase.     

Purification and some properties of a protein inhibitor of sucrose synthetase from ripening wheat grain

A heat-stable protein with a molecular mass of 30 kDa inhibiting the reverse reaction of sucrose synthetase has been isolated from ripening wheat grain and purified to the homogeneous state by the methods of gel filtration on Sephacryl S-200, ion-exchange chromatography on DEAE-cellulose 52, and chromatofocusing in a pH 6–4 gradient. The influence of this protein on K_M for sucrose has been shown which is expressed in a decrease in the affinity of the substrate for the enzyme. An inverse correlation has been found between the activity of sucrose synthetase and the inhibitor in the process of ripening of the wheat grain. On the basis of the results obtained, a hypothesis has been put forward of the participation of the inhibitor protein in the regulation of sucrose synthetase in the ripening of wheat grain.M. A. Aitkhozhin Institute of Molecular Biology and Biochemistry, Kazakh SSR Academy of Sciences, Alma-Ata. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 381–383, May–June, 1989.     

Purification and characterization of befunolol reductase from rabbit liver

An enzyme (befunolol reductase) which catalyzes the reduction of befunolol to dihydrobefunolol was purified from the cytosolic fraction of rabbit liver to homogeneity by various chromatographic techniques. Befunolol reductase had molecular weights of 29000 on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and 34000 on gel filtration. The enzyme required reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor and showed an optimal pH of 6.5. The apparent Km and Vmax values of the enzyme for the reduction of befunolol were 1.7 mM and 4.4 units/mg, respectively. Flavonoids, sulfhydryl reagents, heavy metals and coumarins strongly inhibited the enzyme. The enzyme catalyzed the reduction of a variety of aromatic ketones. In addition to befunolol, some ketone-containing drugs such as daunorubicin and levobunolol were efficiently reduced by the enzyme. On the basis of substrate specificities for steroids, befunolol reductase purified from the cytosolic fraction of rabbit liver appeared to be a 3 alpha-hydroxysteroid dehydrogenase.     

Purification of the M-type phosphoglyceromutase from rabbit muscle

A new method for the purification of M-type phosphoglyceromutase from rabbit muscle involving affinity chromatography on dye ligand media in the presence of 2,3-diphosphoglycerate is described. The method is rapid and technically simple. The purity of the enzyme was verified by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate and by Cellogel electrophoresis. Immunological techniques showed that the anti-M antibodies are specific and do not cross-react with the B isozyme.     

Purification and properties ofClostridium thermocellum endoglucanase 5 produced inEscherichia coli

Endoglucanase 5 (EG5) has been isolated from the strain ofE. coli TGI harboring recombinant plasmid pCU108, which contains thecel5 gene ofC. thermocellum. The enzyme has been produced with 98-fold purification and a final yield of 27% by using subsequent twofold high performance ion-exchange chromatography on Mono Q and high performance chromatofocusing on Mono P. The protein has a mol mass of 35 kDa and includes 3 multiple forms with pI 4.4–4.8 as evidenced by analytical gel isoelectrofocusing. EG5 cleaves CMC (K_m = 0.097 g/L, V_max = 8.2 mg/min·mg of protein), amorphous cellulose, xylan, lichenan as a substrate with an optimum temperature of 80?C and pH 6.0 and Avicel (K_m = 18.2 g/L, V_max = 0.035 mg/min·mg of protein) with an optimum temperature of 60?C and pH 6.O. Cellobiose in concentrations up to 200 Μg/mL do not inhibit the hydrolysis of CMC by EG5, but 10–30 Μg/mL of glucose significantly decrease the activity of this enzyme. The stimulating role of calcium chloride and concentration of protein in the system has been demonstrated for Avicel hydrolysis by EG5.     

Transketolase from human erythrocytes. Purification and properties

Human erythrocyte transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde-transferase) was purified 8200-fold by adsorption onto hydroxylapatite, DEAE-cellulose treatment, acetone fractionation, and chromatography on Sephadex G-100. The purified transketolase could not be separated from glyceraldehyde-3-phosphate dehydrogenase, whereas the latter enzyme could be isolated in a pure state. Its homogeneity is suggested by sedimentation velocity, sedimentation equilibrium, and acrylamide electrophoresis. A molecular weight of 136000 was found. The physicochemical properties of glyceraldehyde-3-phosphate dehydrogenase and transketolase are very similar. A molecular weight of 136000 is suggested for transketolase, although gel filtration with Sephadex G-100 gave only 104000 ± 10%. This discrepancy is a reflection of an interaction of transketolase with the gel filtration medium. The isoelectric point for transketolase as well as for glyceraldehyde-3-phosphate dehydrogenase, as determined by isoelectric focussing, was found to be around 8.5. The activity of the enzyme is close to the maximum for pH 7.5 to pH 8.6. Additions of thiamine pyrophosphate or other cofactors do not influence the activity. Several divalent cations were tested. Sulfate and phosphate inhibit transketolase approximately to 50% between 50 and 100 mM concentration. Thiamine was present in transketolase, as shown by a microbiological assay and by the thiochrome reaction. The activation energy for the formation of sedoheptulose-7-phosphate from xylulose-5-phosphate was estimated from rate measurements to be 11.2 kcal/mole in the temperature range from 5° to 55°.     

Synthesis and some properties of 1, 3, 5-trinitrocyclohexane

Conclusions A method was developed for the synthesis of 1,3,5-trinitrocyclohexane and some of its properties were studied.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 11, pp. 2639–2641, November, 1970.