Purification of clathrin assembly protein from rat liver

Recently, the gene encoding clathrin assembly protein of lymphoid myeloid leukemia (CALM), which is homologous to the AP180, was cloned from rat brain, and its expression differential to AP180 was reported (Kim and Lee, 1999). This gene product promotes the polymerization of clathrin into clathrin cage and found to be a regulator in membrane trafficking between intracellular compartments in eukaryotic cells (Kim et al., 2000). In this study, we have purified the CALM protein from clathrin-coated vesicles of rat liver using the monoclonal antibody against the recombinant N-terminal region of the CALM. The coated proteins extracted from the coated vesicle fraction was further purified by multi-step procedures involving gel-filtration and ion-exchange chromatography and SDS-PAGE. The purified protein with an apparent molecular weight of 100 kD promoted the assembly of clathrin triskelia into clathrin cage. In this respect the CALM protein bears a functional resemblance to the AP180 that has been previously described.     

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 tryptophan-5-monooxygenase from rabbit hindbrain

An affinity chromatography procedure for the rapid purification of tryptophan-5-monooxygenase from rabbit hindbrains was developed using e-aminocaproyl-D-tryptophan methyl ester-Sepharose-4B gels. The precise requirements for the optimal biospecific interaction between the affinity ligand and the ligate (enzyme) was established from a study of the effects of the variation in the length of the "spacer’’ on the affinity properties of the gel. The enzyme preparation isolated by this procedure was found to be essentially homogeneous and was characterized by a molecular weight of 200,000 ±20,000. SDS-polyacrylamide gel electrophoresis of the enzyme revealed it to be a dimer, the molecular weight of each subunit being approximately 90,000. The specific activity of the enzyme preparation is approxi-mately 7-10 times that of the crude homogenate, but a further fivefold enhancement in the specific activity could be obtained by limited proteolysis with trypsin. The extreme lability of the enzyme could be circumvented by its immobilization on activated Sepharose or by cross-linking with dimethyl suberimidate. The kinetic properties as well as the advantages of such stabilized enzyme preparations are presented.     

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 properties of multiple forms of dihydrodiol dehydrogenase from monkey liver

Two major and two minor forms of dihydrodiol dehydrogenase with similar molecular weights of around 36000 were purified from monkey liver cytosol. All the forms oxidized trans-dihydrodiols of benzene and naphthalene and reduced aromatic aldehydes, but showed differences in charge, specificity for other substrates and inhibitor sensitivity. One major (pI 8.7) and one minor (pI 7.9) form of the enzyme exhibited high activity for alicyclic alcohols and sensitivity to o-phenanthroline. The other major form (pI 6.2) oxidized 3 alpha-hydroxysteroids and was inhibited by dexamethasone and indomethacin, whereas the other minor form (pI 5.8) showed high reductase activity for aldehydes including D-glucuronate and sensitivity to barbital and sorbinil, and cross-reacted with human aldehyde reductase. The results indicate that the multiple forms of monkey liver dihydrodiol dehydrogenase are indanol dehydrogenases, 3 alpha-hydroxysteroid dehydrogenase and aldehyde reductase.     

Purification of glutathione reductase from chicken liver and investigation of kinetic properties

Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75 M Tris-HCl buffer including 1 mM EDTA, and 50°C, respectively. K _M and V _max values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme.     

Synthesis and some properties of 2,3-dihydrofuro[2,3-b]quinol-4-one

The reaction of O-ethylbutyrolactonium tetrafluoroborate with ethyl anthranilate gave the corresponding imido ester, which undergoes cyclization to a furo[2,3-b]-quinol-4-one derivative when it is heated in a solution of sodium butoxide. When the latter product is heated in diphenyl oxide, it is converted to a furo[3,2-c]-quinol-2-one derivative.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 1, pp. 65–67, January, 1982.