Isolation and certain properties of human pituitary prolactin]

A simple method of isolation of highly purified prolactin from acetonated preparations of anterior hypophysial lobes is described. The new method permits to obtain higher (about 10-fold) yields of the hormone, as compared to those obtained using previously described methods. Prolactin was extracted by acid aqueous acetone and was subsequently purified of extract by fractionation with acetone and NaCl and by isoelectric precipitation. The final stage of the hormone purification involved gel-filtration through Sephadex G-200; prolactin yield was 400 microgram per 1 hypophysis. The lactogenic activity of the hormone is 14 MU/mg; the sequence of N-terminal amino acid residues of prolactin is as follows: NH2-Leu-Pro-Ile-x-Pro-Leu(?)-Gly-Ala-.     

Isolation and physico-chemical properties of oligosaccharides of human milk]

From a non-dialysable fraction of human milk the authors have isolated 30 oligosaccharides by combining anion exchange and paper chromatography. These oligosaccharides (MW 998 to 3113) contain D (+)-galactose, L (-)-fucose, N-acetyl-D (+)-glucosamine, N-acetyl-D (-)-neuraminic acid in variable proportions and one single D (+)-glucose residue in terminal reducing position. The characteristics of the oligosaccharides are those of the so-called Polonovski and Lespagnol gynolactose. These sugars do not originate from glycoproteins nor glycolipids. The authors suggest that the biosynthesis of human milk oligosaccharides is due to an activation of glycosyltransferases related to blood group substances, induced by lactose. They base this hypothesis on the fact that, as demonstrated by Strecker and Montreuil, spontaneous or glucose, galactose and lactose induced meliturias are accompagnied by an important urinary excretion of blood group substance related oligosaccharides.     

Isolation and properties of superoxide dismutase from human blood plasma]

A procedure for purification of superoxide dismutase (SOD) from human blood plasma has been developed, which includes gel filtration on Ultrogels AcA-34 and AcA-44 (LKB, Sweden). The protein purified from blood plasma is a glycoprotein which is thermostable at 70-80 degrees C. The molecular mass of the protein determined immediately after gel filtration is approximately 147,000 daltons. A comparative analysis of effects on the SOD activity of plasma and erythrocytes of compounds capable of forming chelating complexes with metals within the enzyme active center has been carried out. The purified enzyme differs by its physico-chemical characteristics from cytosolic Cu,Zn-SOD and pertains to a new class of SOD, the so-called extracellular SOD, detected in some biological fluids.     

Isolation and properties of human kappa-casein

Human kappa-casein was isolated from human whole casein by gel filtration with Sephadex G-200 and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate (SDS). The kappa-casein was calcium-insensitive and did stabilize human beta-casein and bovine alpha s1-casein against precipitation by calcium ions. Formation of micelles from human beta- and kappa-caseins, and calcium ions was confirmed by electron microscopic observation. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a single band was obtained. The formation of para-kappa-caseins by chymosin was confirmed by SDS-PAGE. Two para-kappa-caseins with apparent molecular weights of 13,000 and 11,000 appeared. The molecular weight of intact human kappa-casein was estimated to be approximately 33,000. The human kappa-casein contained about 40% carbohydrate (15% galactose, 3% fucose, 15% hexosamines, and 5% sialic acid) and 0.10% (1 mol/mol) phosphorus. Its amino acid composition was similar to that of bovine kappa-casein except for serine, glutamic acid, and lysine contents.     

Isolation and properties of human transketolase

Recombinant human (His)₆-transketolase (hTK) was obtained in preparative amounts by heterologous expression of the gene encoding human transketolase in Escherichia coli cells. The enzyme, isolated in the form of a holoenzyme, was homogeneous by SDS-PAGE; a method for obtaining the apoenzyme was also developed. The amount of active transketolase in the isolated protein preparation was correlated with the content of thiamine diphosphate (ThDP) determined in the same preparation. Induced optical activity, facilitating studies of ThDP binding by the apoenzyme and measurement of the transketolase reaction at each stage, was detected by circular dichroism spectroscopy. A single-substrate reaction was characterized, catalyzed by hTK in the presence of the donor substrate and in the absence of the acceptor substrate. The values of the Michaelis constant were determined for ThDP and a pair of physiological substrates of the enzyme (xylulose 5-phosphate and ribose 5-phosphate).     

Isolation and properties of glucose-6-phosphate dehydrogenase from human cultivated cells]

A new procedure for purification of glucose-6-phosphate dehydrogenase resulting in an electrophoretically homogenous preparation made up of 5.10(8) cells (390 mg of protein) is proposed. The enzyme yield is more than 20%. The molecular weights of a subunit and a native enzyme are 55000 and 220000, respectively. The isoelectric point for the protein lies at 4,8. The kinetics of the enzyme thermal inactivation obey the first order equation with the inactivation rate constant of 6.10(-3) min-1.     

Physarum tropomyosin-troponin complex. Isolation and properties.

The relaxing protein (TM-TN complex) was isolated from plasmodia of Physarum. SDS-gel electrophoresis revealed that the relaxing protein consists of tropomyosin subunits with a molecular weight of 35,000 troponin subunits with molecular weights of 38,000 (T) and 24,000 (I) and several other components. No component corresponding to muscle troponinC (MW-18,000) was detected in the plasmodium relaxing protein. The relaxing protein combined with muscle F-actin, and inhibited the ATPase [EC 3.6.1.3] activity and superprecipitation of reconstituted muscle actomysin in the absence of Ca2+ ions. The inhibition was reversed by adding 1 muM Ca2+ ions.     

Isolation and physico-chemical properties of a complex kinetoplast DNA associate from the cells of Trypanosoma lewisi]

An associate of kinetoplast DNA (k-DNA) was isolated from the cells of Trypanosoma lewisi and characterized in terms of its sedimentation properties, melting parameters and reassociation kinetics. Electron microscopy studies showed that k-DNA isolated is a complex associate of circular molecules. The contour length of minicircular molecules is 0.77 mkm. k-DNA contains sites enriched by AT-pairs; melting of native associate and k-DNA fragments in the presence of 6.5 M sodium perchlorate results in the appearance of six zones within the temperature range of 47-64 degrees C. Data from k-DNA reassociation studies suggest that k-DNA associate constitutent molecules differ in sizes and nucleotide sequences. k-DNA is found to consist of two components with molecular weights of 1.7 . 10(6) and 17.5 . 10(6), respectively.     

Isolation and properties of extracellular cellulase-hemicellulase complex of Phoma hibernica

A cellulase-hemicellulase complex was obtained from the culture supernatant of Phoma hibernica. It was purified by ammonium sulfate precipitation, column chromatography on diethylaminoethyl-Sephadex A-50 and Sephadex G-100. The preparation was capable of degrading carboxymethyl-cellulose, insoluble cellulose, xylan, galacto-, gluco-, and galactogluco-mannan. The distinct protein band obtained after isoelectrofocusing also showed activities towards these substrates. Optimum pH for cellulase and galactomannase activities was 4.5 and for xylanase activity 4.5–5.5. Tetranitromethane, urea and Fe³⁺ inhibited all the enzymatic activities of the complex. The preparation attacked carbohydrate polymers in different manners depending on the substrate. Cellulose was attacked in an exo-wise, xylan in an endowise manner. Nitrophenyl derivatives of carbohydrates were hydrolyzed slowly. It is suggested that the purified enzyme preparation is a complex most probably composed of subunits of different enzymatic activities.Abbreviations Used CM carboxymethyl - DEAE diethylaminoethyl - CMC carboxymethylcellulose     

Isolation and properties of noncovalent complex of transketolase with RNA

A method for isolation of homogenous transketolase from baker's yeast using immunoaffinity chromatography was significantly simplified. It was demonstrated that transketolase could be isolated from fresh yeast in the form of a complex with a high molecular weight RNA. Storage of yeast led to the dissociation of the complex to a low molecular weight complex and then to the free enzyme. Conditions were chosen for complex dissociation and free enzyme isolation. In comparison to the free enzyme, the specific activities of the high and low molecular weight complexes were decreased 20-25- and 3-5.5-fold, respectively. The affinity to the cofactor thiamine diphosphate and to xylulose-5-phosphate (donor substrate) did not change for the low molecular weight complex, while the time of binding to calcium increased. The latter was necessary for the complete manifestation of the enzymatic activity. Changes in the circular dichroism spectrum between 300 and 360 nm after the addition of thiamine diphosphate, which characterize the formation of the catalytically active holoenzyme, were significantly lower for the low molecular weight complex than for the free enzyme.     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Isolation and physical chemical properties of rat hemopexin]

Rat hemopexin was purified by a procedure involving three different steps : ammonium sulfate precipitation, rivanol precipitation and DEAE-cellulose chromatography with concave gradient of molarity. Purity of the preparation was checked by three different methods : analytical ultracentrifugation, immunoelectrophoresis and acrylamide gel electrophoresis. The principal physical properties were studied. The amino acid and carbohydrate composition was determined and compared with that of human and rabbit hemopexin.     

Isolation and properties of bovine kidney aminopeptidase A]

Aminopeptidase A, which specifically hydrolyses N-terminal dicarbonic amino acid residues containing free alpha-amino groups, is isolated from bovine kidney. The enzyme is 500-fold purified and is homogenous under electrophoresis and ultracentrifugation. Aminopeptidase A has pH optimum of 7.5, it is activated with Ca2+ and inactivated with EDTA. Its molecular weight is 53000. The enzyme hydrolyses alpha-L-aspartyl-beta-naphtylamide and splits peptides having N-terminal glycine, lysine, arginine and alanine are hydrolyzed by the enzyme much slower. Aminopeptidase A does not attack alpha-L-alanyl-beta-naphtylamide, leucineamide, insulin, peptides with blocked N-terminal amino acid and peptides which have proline to be the second N-terminal amino acid.     

Isolation and properties of tripolyphosphatase from Neurospora crassa]

Homogenous preparation of tripolyphosphatase from Neurospora crassa is obtained. The enzyme is found to consist of two equal subunits with molecular weight of 40 000 and to have pH optimum 7.0 and temperature optimum 50 degrees C. Bivalent metal ions are required for its catalytical activity, the hest activators being Co2+, Mg2+ and Mn2+. Strict specificity of the enzyme to tripolyphosphate is demonstrated, Km being 5.9-10(-4) M. The enzyme hydrolyses tripolyphosphate to equimolar mixture of ortho- and pyrophosphate. The enzyme activity depends on orthophosphate and pyrophosphate concentrations in the incubation medium.     

Isolation and properties of intracellular peptidase from Brevibacterium]

The intracellular peptidase of Brevibacterium E531, a lysine-producing bacterial species, was purified 6500-fold by chromatography on DEAE-cellulose and the affinity adsorbent H-Thr(But)-Phe-Pro-hexamethylene-diamine-Sepharose 4B and by gel filtration on Sephadex G-200. The enzyme displayed the maximum activity towards proline p-nitroanilide at pH 7.7-7.9 and readily split glycine, alanine and proline from di-, tri- and tetrapeptides but did not practically hydrolyze oligopeptides of a greater chain length. The enzyme was not inhibited by complexons (EDTA, 8-oxiquinoline and 1.10-phenanthroline). The peptidase was not activated by divalent metal ions and was inhibited by Zn2+; Cd2+, Hg2+ and Cu2+. Data brom gel filtration on Sephadex G-200 suggest that the molecular mass of the enzyme is no less than 250 kDa. In the presence of sodium dodecyl sulfate the molecular mass of the enzyme is 43 kDa, which is suggestive of the presence of a quaternary structure. One peculiarity of the enzyme is its activation by alkaline metal halogenides and sodium nitrate which reaches a maximum at the 0.05-0.1 M concentration of the salts.     

Isolation and several properties of Streptomyces griseus carboxypeptidase]

Enzymatic and physico-chemical properties of homogenous preparation of carboxypeptidase from Streptomyces griseus are studied. pH-Optimum is found to be 7.9 and 8.2 under the hydrolysis of cbs-Gly-Leu and hyppuryl-arg respectively, temperature optimum --60 degrees C. The enzyme splits more efficiently basic amino acids and leucine from N-terminal-protected dipeptides. Str. griseus carboxypeptidase is activated by reducting agents (NaCN, cisteine, ascorbic acid), it is inhibited by KMnO4 and it does not belong to "serine" type enzymes. SH-groups are essential for the enzyme activity. No significant effect of metal ions on the enzyme activity is observed. The inhibitory effect of EDTA developed only after the prolonged treatment. The enzyme has one N-terminal group (alanine), which evidences the presence of one polypeptide chain in the enzyme molecule.