Partial purification of somatomedin from human plasma.

Fractional precipitation of human plasma using ethanol, followed by chromatography on S.P. Sephadex, yielded a somatomedin-enriched fraction freed from substantial amounts of inhibitory substances. Heat coagulation of the proteins present in this fraction allowed the recovery of appreciable amounts of active components which were then chromatographed on Sephadex G-75 and S.P. Sephadex. A major part of the activity was associated with components less than 4,000 daltons, suggesting that somatomedin, or an active fragment thereof, had been dissociated from a carrier protein by the heat treatment. The range of pH employed throughout was 5.3-9.8. Recoveries of about 30% of biological activity with fold-purification up to 38, as measured by radioactive sulphate uptake in the chick pelvic cartilage assay, were higher than those obtained using acid-ethanol extraction.     

Partial purification and characterization of a recombinase from human cells

We describe the partial purification and characterization of a human recombinase activity from RPMI 1788 B lymphoblasts. Stoichiometric amounts of recombinase carry out a strand transfer reaction between linear duplex DNA and homologous circular single-strand DNA. The product of strand transfer by the recombinase is a joint molecule composed of a single-strand circle joined to one end of the linear duplex molecule by a region of DNA heteroduplex at least 150 bp long. Formation of DNA heteroduplexes is accompanied by strand displacement. Strand invasion initiates at the ends of the linear duplex. Finally, strand displacement by human recombinase exhibits polarity and proceeds in a 3' to 5' direction. This is the first demonstration of a strand transfer activity from a high eukaryote. We discuss similarities between our recombinase and the RecA and rec1 recombination proteins from E. coli and Ustilago maydis, respectively.     

Partial purification and characterization of a serine endopeptidase from rat liver plasma membranes

A serine endopeptidase was partially purified from rat liver plasma membranes by using a four-step procedure: solubilization with N-lauroylsarcosine; Ultrogel AcA-34 chromatography; CM Affi-Gel blue chromatography; agarose-soybean trypsin inhibitor chromatography. This enzyme was found to hydrolyze casein and various chromogenic peptide substrates; highest activity occurred with H-D-Val-Leu-Arg-p-nitroanilide, reported to be a specific substrate for human glandular kallikreins. The enzyme was heat-sensitive, showed a pH optimum between 8.0 and 9.0 and was inhibited by D-Phe-L-Phe-L-Arg-CH2Cl, aprotinin, diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, phenylmethylsulphonyl fluoride, leupeptin, antipain and dithiothreitol. This liver plasma membrane proteinase has an apparent molecular weight of about 30 000 as determined by Ultrogel AcA-34 chromatography and by autoradiography of [3H]DFP-labelled protein electrophoresis.     

Partial purification and characterization of protein tyrosine kinases from normal tissues

Three membranous protein tyrosine kinases (PTKs) have been partially purified from human placenta and pig brain. The two placental enzymes (PTK-1 and -2) are distinct with respect to solubility in detergents, molecular weight, and enzymatic properties. The brain protein tyrosine kinase resembles placental PTK-1 with respect to molecular weight and some kinetic properties. However, stimulation of brain PTK is greater with Mn2+ than with Mg2+ whereas placental PTK-1 gives higher rates with Mg2+ than with Mn2+. All three enzymes are inhibited about 50% by 0.1 M NaCl. A monoclonal antibody raised in vitro against the brain enzyme inhibits brain PTK as well as placental PTK-2, but has no effect against PTK-1 or pp60src. It thus appears that these three enzymes are distinct entities that differ from each other both kinetically and immunologically. With synthetic tyrosine-glutamic acid polymers as a substrate, protein tyrosine kinase activity can be detected in crude extracts of membranes.     

Partial purification and characterization of a peroxidase activity from human placenta.

The aim of this work was to use preparations from germinating seeds of Pisum sativum to determine the apparent equilibrium constant of the reaction catalysed by sucrose-phosphate synthase (EC 2.4.1.14) and to compare this with the mass-action ratio of the reaction in the seeds. The apparent equilibrium constant ranged from 5.3 at 0.25 mM-MgCl2, pH 7.0, to 62 at 10 mM-MgCl2, pH 7.5. The sucrose phosphate content of the seeds, 23 nmol/g fresh wt., was determined by separating sucrose phosphate from sucrose by ion-exchange chromatography and then measuring the sucrose released by alkaline phosphatase. Comparison of equilibrium constants and mass-action ratios in the cotyledons of 38 h-germinated seeds showed that the reactions catalysed by glucose-6-phosphate isomerase, phosphoglucomutase and UDP-glucose pyrophosphorylase are close to equilibrium, and those catalysed by sucrose-phosphate synthase and sucrose phosphatase are considerably displaced from equilibrium in vivo.     

Partial purification and characterization of a folatebinding protein from human choroid plexus

A folate-binding protein (binder) from human choroid plexus was solubilized with Triton X-100 and partially purified in three steps: (1) affinity chromatography, (2) Sephadex G-200 column chromatography, and (3) polyacrylamide gel electrophoresis. When the partially purified binder was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the binding activity was located in the region of the gel with a molecular weight between 45,000 and 60,000. The specific activity of the binder after the three purification steps was 1.2 g folic acid/mg protein, a 316-fold purification. Binding activity of the partially purified binder decreased below pH 6.0 and above pH 8.0 was unaffected by treatment with ribonuclease or deoxyribonuclease, but was abolished with trypsin, chymotrypsin, or protease (Streptomyces griesus). The binding of folic acid to the human binder was inhibited by folate > H₄-folate > methyl-H₄-folate dihydrofolate pteroic acid methotrexate aminopterin.     

Partial purification and characterization of a double-stranded RNA-specific nuclease from human placenta

A double-stranded RNA-specific nuclease (ds RNase) has been isolated and partially purified from human placenta by DEAE-cellulose and DNA-cellulose column chromatography. Denatured DNA-cellulose retained most of the single-stranded RNA-specific nuclease (ss RNase) activity, whereas the ds RNase came out in the void volume. N-ethylmaleimide at a concentration of 5 mM, selectively inhibited ds RNase activity by 60% under the conditions in which the ss RNase activity was inhibited to an extent of 7%. The ds RNase was specifically inhibited by Penicillium chrysogenum viral ds RNA and by ethidium bromide. The partially purified ds RNase showed requirements for Mg+ whereas Mn2+ and NH4+ ions were inhibitory. The DEAE-enzyme cleaved 32P-labelled 45S ribosomal precursor RNAs from Yoshida ascites sarcoma cells into species that had similar electrophoretic mobilities as the mature rRNAs.     

Partial purification and characterization of cysteine proteinase inhibitor from chicken plasma

A high-molecular-weight cysteine proteinase inhibitor (CPI) was purified from chicken (Gallus gallus) plasma using polyethylene glycol (PEG) fractionation and affinity chromatography on carboxymethyl–papain–Sepharose-4B. The CPI was purified 96.8-fold with a yield of 28.9%. Based on inhibitory activity staining for papain, CPI was shown to have an apparent molecular mass of 122 kDa. No inhibitory activity was obtained under reducing condition, indicating that CPI from chicken plasma was stabilized by disulfide bonds. CPI was stable in temperature ranges from 40 to 70 °C for 10 min; however, more than 50% of the inhibitory activity towards papain was lost within 30 min of heating at 90 °C. CPI was stable in the presence of salt up to 3%. The purified CPI exhibited the inhibitory activity toward autolysis of arrowtooth flounder (Atheresthes stomias) and Pacific whiting (Merluccius productus) natural actomyosin (NAM) in a concentration-dependent manner.     

Kinin-converting aminopeptidase from human urine partial purification and properties

• 1.1. An aminopeptidase from human urine, which hydrolyses dipeptides and β-naphthylamides of neutral and basic amino acids and which converts the peptides lysylbradykinin and methionyllysyl-bradykinin into bradykinin, was highly purified by a four-step procedure.
• 2.2. The enzyme (mol. wt 100,000) has several similarities with kinin-converting aminopeptidases found in human serum and liver, and is inhibited by 1,10-phenanthroline and puromycin.

     

Acid sphingomyelinase from human urine: purification and characterization

Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine in the presence of 0.1% Nonidet P-40. The activity could be enriched 23,000-fold by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue Sepharose and DEAE-cellulose. The last purification step yielded an enzyme preparation with a specific activity of about 2.5 mmol sphingomyelin cleaved/h per mg protein and with a yield of about 3%. Purified sphingomyelinase appeared to be homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 70 kDa. In the presence of 0.08% (w/v) sodium taurodeoxycholate the preparation showed phosphodiesterase activity toward sphingomyelin, phosphatidylcholine and phosphatidylglycerol. These activities co-purified during the entire purification procedure, indicating that the acid sphingomyelinase hydrolyses not only sphingomyelin but also the other two phospholipids, phosphatidylcholine and phosphatidylglycerol. Addition of 100 microM tripalmitoylglycerol to the assay system (which contains 100 microM sphingomyelin) instead of detergent, stimulated the reaction about 20-fold compared to an assay which did not contain detergents, thus offering a very sensitive and efficient system for the assay of sphingomyelinase in a system free of detergents. Sphingomyelin degradation was strongly inhibited by phosphatidylinositol 4',5'-bisphosphate, adenosine 3',5'-diphosphate and adenine-9-beta-D-arabinofuranoside 5'-monophosphate (50% inhibition at inhibitor concentrations of 1-5 microM).