Microcin 7: purification and properties

Microcin 7 is an antibiotic peptide, produced and excreted to the culture medium by E. coli strains harboring the plasmid pRYC7. This peptide was extracted from the culture media by adsorbing it on octadecyl silica. It was purified by gel filtration on Sephadex G-25 and reverse phase high performance liquid chromatography. Its amino acid composition is the following: Ala (0.8), Arg (1.9), Asx (1.9), Gly (1.5), Met (0.8) and Thr (0.9). The purified peptide dose not react with ninhydrin and it is resistant to carboxypeptidase degradation, indicating that the molecule may be a cyclic or end-blocked oligopeptide.     

N-acetyl-aspartate amidohydrolase: purification and properties.

Abstract— The enzyme in rat brain responsible for the de-acetylation of N-acetyl-aspartic acid has been partially purified. In contrast to the enzyme from hog kidney which is stable at 70°C, it rapidly denatures above 57°C. The rat brain enzyme has the same K_m for its substrate and the same solubility in ammonium sulphate solution as the hog kidney enzyme. Results of migration on starch gel electro-phoreses and isoelectric focusing indicate a pI for the amidohydrolase of 5.1. A variety of potential substrates, modulators, and inhibitors have been examined.     

Corn phosphoglycolate phosphatase: purification and properties

Phosphoglycolate phosphatase (EC 3.1.3.18), isolated from maize leaf bundle sheath, was purified 200-fold to a specific activity of about 99 mol mg^-1 protein · min^-1. The purification procedure included Sephadex G-75 filtration, and diethylaminoethyl-cellulose and Phospho-Ultrogel A6R chromatography. This partially purified enzyme exhibited optimum activity over a broad pH range, from pH 6.3 to pH 8.0. It displayed a very high degree of specificity for phosphoglycolate and required a divalent cation to be active; Mg²⁺ was the most effective activator. Saturation curves of the Michaelis-Menten type were observed both with phosphoglycolate (K_m=0.57 mmol·l^-1) and with Mg²⁺ (K_m=0.015 mmol·l^-1). The activation constant for Mg²⁺ was unchanged when the pH was raised from 7.0 to 8.0. These results indicate that variations of stromal pH and Mg²⁺ during the darklight transition could not directly modifity the activity of the phosphoglycolate phosphatase in maize bundle-sheath chloroplasts. The undissociated protein showed a pI of 4.95, as determined by isoelectric focusing. For the native phosphatase a molecular mass of about 61 500 Da was estimated by polyacrylamide gradient gel electrophoresis. The subunit was found to have a relative molecular mass of 31 500 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is concluded that maize phosphoglycolate phosphatase is a dimer.Abbreviations DEAE diethylaminoethyl - P-glycolate phosphatase phosphoglycolate phosphatase - P-glycolate phosphoglycolate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl[glycine - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Sweet corn phosphorylase: purification and properties

Sweet corn 1,4-α-glucan phosphorylase was purified 190-fold to a near homogeneous state. The enzyme had a molecular weight of about 315,000 on Sephadex G-200 chromatography. The pyridoxal 5′-phosphate content was found to 1 mole per 140,000 g protein, suggesting that the enzyme is dimeric. On sucrose density gradient ultracentrifugation the sweet corn phosphorylase was dissociated to an active monomeric species with a molecular weight of 150,000 and a sedimentation coefficient of 8 S. The priming specificity of the sweet corn phosphorylase was investigated; maltose was not a primer and maltotriose was the smallest apparent primer. The Michaelis constants for the maltosaccharide series from maltopentaose to maltooctaose were determined. The effect of d-enzyme on the apparent priming specificity of the enzyme was investigated. Adenosine diphosphoglucose and 2,3-diphosphoglycerate were found to inhibit the enzyme activity.     

Human interleukin 1: purification and properties

Human interleukin 1 (IL 1) has been purified to homogeneity by a procedure of molecular weight fractionation, isoelectric focusing, and preparative polyacrylamide gel electrophoresis. The homogeneity of the purified material has been demonstrated by silver staining of analytical polyacrylamide gels. The homogeneous IL 1 retains only a trace of its original biological activity because of the denturing effects of the sodium dodecyl sulfate used in the final step of purification. Very highly purified IL 1, retaining strong biological activity, has been eluted from nondenaturing polyacrylamide gels. This IL 1 has been demonstrated to stimulate human and mouse T and B lymphocytes, fibroblasts, and synovial cells. In addition, in vivo treatment of animals with IL 1 resulted in the immunologically relevant symptoms of fever, increased plasma levels of acute phase proteins, and increased numbers of circulating neutrophils.     

Peroxidase from Astragalus maritimus: purification and properties

A peroxidase has been purified to homogeneity from Astragalus maritimus seeds using ammonium sulfate precipitation and chromatography on DEAE-cellulose and hydroxylapatite. The purification obtained was 255 fold. The enzyme preparations were homogenous by the criteria of SDS-PAGE and analytical gel electrofocusing. The protein contained 0.11% of iron that corresponds to a minimum molecular size of 50,700. Determinations of molecular size by SDS-PAGE gave values of 48,000 +/- 1,000 while the one obtained by Sephadex gel filtration was 49,000. The pH optimum of the enzyme was 6.0. The activation energy was estimated to be 6 Kcal/mol. The prosthetic group was shown to be ferriprotoporphyrin IX. The presence of 13% neutral sugars was found. The spectrophotometric analysis showed the presence, in the visible region, of absorption maxima at 403, 490 and 633 nm. The Rz value (A403/A275) was 2.7.     

Chicken ornithine transcarbamylase: purification and some properties

Ornithine transcarbamylase [EC 2.1.3.3] has been purified from chick kidney to homogeneity. The molecular weight is 110,000 as determined by gel filtration. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the enzyme showed that the enzyme exists as a trimer of identical subunits of 36,000 daltons like other mammalian species ornithine transcarbamylases. In 0.1 M triethanolamine/HCl, the apparent optimum pH of the purified enzyme was 7.5 in the presence of 5 mM ornithine. The curve shifted toward a more alkaline region with a decrease in ornithine concentration. The specific activity of the purified enzyme as 77 units at pH 7.5. The Km for carbamyl phosphate was 0.11 mM and the Km for ornithine was 1.21 mM. With an increase in pH, a decrease in Km values for ornithine and an increase in the extent of inhibition by ornithine were observed. On using antibody against bovine liver ornithine transcarbamylase, the precipitin lines for the chick and bovine enzymes showed a spur pattern. Even when excess amounts of the antibody were added, the chick enzyme did not lose the activity while the bovine enzyme activity was inhibited completely.     

Rabbit liver alcohol dehydrogenase: purification and properties

Alcohol dehydrogenase [EC 1.1.1.1] was purified to homogeneity from rabbit liver by water extraction, DEAE-cellulose treatment, affinity chromatography on 5'-AMP-Sepharose and gel filtration on Sephadex G-150 using dithiothreitol as a stabilizer. The purified enzyme has an estimated molecular weight of 72,000 and consists of two subunits with a molecular weight of about 36,000 each. The enzyme contains 4 g-atoms of zinc and 18 sulfhydryl groups per mol of protein and exhibits maximal activity at pH 10.8, with a second maximum at pH 7.5. The apparent Km values for ethanol and NAD+ are 0.45 mM and 53.19 microM, respectively, at pH 10.8 and 3.33 mM and 6.94 microM, respectively, at pH 7.5. The enzyme oxidizes ethanol most readily among the aliphatic alcohols studied and has very low substrate specificity for methanol. Among steroid alcohols, 5 beta-androstan-3 beta-ol-17-one serves as a substrate for the enzyme. Pyrazole and 4-methylpyrazole (which are well known alcohol dehydrogenase inhibitors), sulfhydryl reagents, heavy metal ions and metal-chelating agents inactivate the enzyme.     

Human T-lymphoblast deoxycytidine kinase: purification and properties

Previous observations present tremendous variations in the properties of deoxycytidine kinase. To clarify the properties and physiologic role of deoxycytidine kinase, we have undertaken its purification. Deoxycytidine kinase was purified from cultured human T-lymphoblasts (MOLT-4) to 90% purity with an estimated specific activity of 8 mumol min-1 (mg of protein)-1. The purification procedure included ammonium sulfate precipitation, Superose-12 HPLC gel filtration chromatography, DE-52 ion-exchange chromatography, AMP-Sepharose 4B affinity chromatography, and dCTP-Sepharose-4B affinity chromatography. Deoxyguanosine, deoxyadenosine, and cytidine phosphorylating activities copurified with deoxycytidine kinase to final specific activities of 7.2, 13.5, and 4 mumol min-1 (mg of protein)-1, respectively. The enzyme is very unstable at low protein concentration and is stabilized by storage at -85 degrees C with 1 mg/mL bovine serum albumin, 20% glycerol (v/v), 200 mM potassium chloride, and 25 mM dithiothreitol. The molecular weight was 60,000, and the Stokes radius was 32 A by gel filtration chromatography. The subunit molecular weight was 30,500. This enzyme had apparent Km values of 1.5, 430, 500, 450, and 40 microM for deoxycytidine, deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside, respectively. The pH optimum ranged from 6.5 to 9.0. Mg2+ and Mn2+ were the preferred divalent cations. ATP, GTP, dGTP, ITP, dITP, TTP, and XTP were substrates for the enzymes. Our study indicates that deoxycytidine kinase is a dimer with two subunits and has phosphorylating activity for deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside. This highly purified enzyme will facilitate the study of its regulation and phosphorylation of anticancer or antiviral nucleoside analogues.