Purification of alkaline protease of Rhizopus oryzae by foam fractionation

Studies on the cost effective purification method is very much essential for the industrially important enzyme like protease. As foam fractionation is an important technique in downstream processing of the biologicals, purification of the crude proteolytic enzyme produced by Rhizopus oryzae has been attempted using converging-diverging foam fractionator. The effects of different parameters studied on the purification efficiency by foam fractionation were pH of the broth, air flow rate, initial liquid height. The results obtained were found quite encouraging at pH 10 with 1 LPM of air flow rate at low initial liquid height.     

Isolation, purification and antitumor activity of lipopolysaccharide from cow placenta

Lipopolysaccharide from cow placenta (LPS-CP-2) has been isolated and purified by hot phenol-water extraction, enzyme hydrolysis, chloroform-petroleum ether method, ion-exchange and gel-filtration chromatography. Also, LPS-PS-2 was evaluated for antitumor activity against Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. LPS-PS-2 caused significant (P<0.05) decrease in tumor volume, and viable cell count; and it prolonged the life span of EAC-tumor-bearing mice. Hematological profile indicates that LPS-CP-2 possessed protective action on the haemopoietic system. Further, administration of LPS-CP-2 reduced the tumor volume of both DLA and EAC cell lines in a dose-dependent way. The LPS-PS was found to be devoid of pyrogenic response in the rabbits. These results indicate that LPS-PS exhibited significant antitumor activity without pyrogenic response, suggesting its potential as antitumor agent.     

The purification and identification of calmodulin from human placenta

A protein which showed similarity to bovine brain calmodulin in electrophoretic mobilities on polyacrylamide gels in the presence of 40% glycerol (pH 8.6) and 0.1% sodium dodecyl sulfate (pH 7.2) was isolated from human placenta. Its final yield was approx. 4 mg per kg human placenta. The placenta protein was similar to bovine brain calmodulin in stimulating bovine brain calmodulin-deficient cyclic nucleotide phosphodiesterase in the presence of calcium. However, its stimulating activity was eliminated by ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) or trifluoperazine. In addition, there is a close resemblance in amino acid composition between the placental protein and bovine brain calmodulin. These results indicate that calmodulin is present in human placenta.     

Isolation and characterization of thrombomodulin from human placenta

Protein C, a plasma protein, is activated by thrombin to a protease (protein Ca) that functions as a physiological anticoagulant. We have isolated thrombomodulin, a cofactor required for the rapid activation of protein C, from human placenta. The purification to near homogeneity was achieved using a crude Triton-solubilized protein fraction from a placental particulate fraction as starting material. Chromatography on DEAE-Sepharose removed 95% of the protein and achieved a 3-fold purification. Thrombomodulin was then isolated by affinity chromatography on a column of thrombin-Sepharose wherein the thrombin had been previously inactivated with diisopropyl fluorophosphate. The final preparation was purified 7,900-fold over the membrane extract with a yield of 7%. We obtained 0.88 mg of thrombomodulin from 100 g of membrane extract derived from 5 kg of placenta. The protein was nearly homogeneous as judged by electrophoresis on 10% acrylamide sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol with an apparent Mr = 105,000. Western blot analysis without 2-mercaptoethanol gave an apparent Mr = 75,000. The protein stimulated the rate of protein C activation by thrombin 800-fold to 10 mol of Ca formed/min/mol of thrombin. Thrombin and thrombomodulin appear to form a 1:1 stoichiometric complex as judged from experiments where we measured the effect of varying the concentration of thrombomodulin with respect to thrombin and the converse, on rates of protein C activation. An antibody directed against rabbit lung thrombomodulin inhibited the human placenta protein by 66%, and the amino acid composition of the proteins from the two species was similar indicating that the proteins are closely related. The apparent Michaelis constant of the thrombin-thrombomodulin complex for protein C is 9.8 microM. The protein C activation reaction requires calcium ions and is maximal at 1 mM Ca2+; higher concentrations inhibited the reaction. Coagulation factor Va and factor Va light chain both stimulate the activity of human thrombomodulin 2- to 3-fold.     

Rapid purification of annexin V from human placenta by affinity chromatography

Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues.     

Immunoaffinity purification and characterization of nucleotide pyrophosphatase from human placenta

Nucleotide pyrophosphatase [EC 3.6.1.9] was purified to homogeneity from human placenta using a monoclonal antibody affinity column. By sodium dodecylsulfate--polyacrylamide gel electrophoresis, the purified enzyme showed a major band at a molecular size of 130 K. The enzyme was a glycoprotein with N-linked oligosaccharides consisting of both complex- and oligomannoside-types. Substrate specificity to hydrolyze phosphodiester and phosphosulfate linkages as well as other properties were similar to those of nucleotide pyrophosphatase and phosphodiesterase from other sources.     

Isolation and characterization of methylmalonyl-CoA mutase from human placenta

Methylmalonyl-CoA mutase, one of two known cobalamin-dependent enzymes present in mammalian tissues, has been isolated from 2.5 kg of human placenta utilizing affinity chromatography on 5'-deoxyadenosylcobalamin-Sepharose as the major purification step. The enzyme gives a single band on polyacrylamide disc gel electrophoresis. The Mr of the enzyme is 145,000 and it has two subunits of Mr = 72,000. Amino acid analysis reveals major differences from other human cobalamin-binding proteins. Based on x-ray fluorescence, the enzyme has 2 mol of cobalamin bound/mol of enzyme. In contrast to purified cobalamin transport proteins, most of the cobalamin bound to the enzyme is not released by boiling at low pH in the presence of KCN, or dialysis against 7.5 M guanidine containing 0.2 M dithiothreitol, or both, suggesting the possibility that cobalamin may be covalently attached to the purified enzyme. Both precipitating antibodies and antibodies that inhibit enzyme activity have been raised in a chicken.     

Isolation and characterization of methionine synthetase from human placenta

The cobalamin-dependent enzyme, methionine synthetase, has been purified approximately 1000-fold to apparent homogeneity from human placenta with a 19% recovery. The final two steps of the purification utilized two different affinity columns. The first was a N5-methyltetrahydrofolate-cystamine-agarose column, and the second was a S-adenosylhomocysteine-agarose column. The enzyme was eluted from the first affinity column by buffer containing reducing agent which released the folate and the enzyme while elution from the second affinity column was accomplished with buffer containing 0.5 M sodium chloride. Criteria for purity were the observations that single peaks of enzyme activity, protein, and cobalamin with an apparent molecular weight of 160,000 were obtained by gel filtration and that holomethionine synthetase contained 1 mol of cobalamin/mol of protein. Furthermore, analysis by high performance liquid chromatography using a molecular weight sizing column demonstrated a single peak of protein with a corresponding cobalamin peak. This single peak of protein was progressively converted to a second protein peak that was enzymatically inactive, and this conversion was associated with a directly proportional loss of enzyme activity and cobalamin from the first peak. Methionine synthetase appeared to have a molecular weight of 160,000 on unreduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and subunits of Mr 90,000, 45,000, and 35,000 on reduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.     

Isolation of villous microvessels from the human placenta

A method for isolating the microvessels of the human placental villi has been developed in order to culture perivascular cells. It consists of an initial selection of the villi by serial sieving. The villi retained by the 75 μm sieve were digested by collagenase-dispase. A Percoll gradient permitted the isolation of microvessels still surrounded by stromal fibres and cells. Another digestion by collagenase-dispase eliminated the contaminant elements and allowed, after a new Percoll gradient, microvessels with endothelium, basement membrane and a few perivascular cells to be obtained. Each step of the isolation of microvessels was monitored by light or electron microscopy. Our study confirms the isolation of microvessels embedded in their basement membrane and the preservation of endothelial and perivascular cells after digestion. This method, which has permitted the culture of placental endothelial cells and pericytes, appears of interest for studying microvascular angiogenesis and permeability.     

Isolation of the hemopexin receptor from human placenta

A hemopexin receptor detected in detergent-solubilized placental membranes was purified from the human placenta, using hemopexin-Sepharose affinity chromatography. The solubilized membranes exhibited binding sites of 2.77 pmol of hemopexin/mg of protein with a dissociation constant (Kd) of 6.6 X 10(-8) M. The purified receptor has a molecular weight of 80,000, determined on sodium dodecyl sulfate-gel electrophoresis. Immunoinhibition experiments using the antibody against the placental receptor revealed inhibition of binding of 125I-hemopexin to human leukemia K562 and HL 60 cells, thereby strongly supporting that the polypeptide isolated from the human placenta was the hemopexin receptor.     

Isolation of inhibin like peptides from human placenta

Two moieties of inhibin could be obtained by chromatography of partially purified preparations of inhibin from human placenta on Sephadex G-100, G-25 and ion exchange chromatography on diethylaminoethyl Sephadex A-50. The higher molecular weight moiety (14,000) designated as HPI-H appears to be similar to inhibin from human seminal plasma. While the lower molecular weight moiety (1500) designated as HPI-L appears to be similar to that of sheep testicular inhibin. The preparations from both human term placenta and human seminal plasma inhibited the binding of [¹²⁵I] human follicle stimulating hormone to rat testicular receptors. This effect of inhibins could be neutralized by antisera raised against corresponding polypeptide. Further these antibodies could neutralize endogenous inhibin resulting in 2 to 3 fold increase in serum follicle stimulating harmone levels, which could then be reversed by exogenous administration of the isolated inhibin preparations.     

Partial purification and characterization of cytochrome P-450 from human placenta

Two isozymes of cytochrome P-450 were partially purified to specific contents of 7.0 and 0.5 nmol/mg of protein, respectively, from placenta of non-smoking women by chromatography on octyl Sepharose, hydroxylapatite, DEAE-cellulose and CM-cellulose. NADPH-cytochrome P-450 reductase was purified from phenobarbital-induced mouse liver and from human placenta and was combined with cytochrome P-450 and dilauroylphosphatidylcholine to reconstitute the cytochrome P-450 monooxygenase system. Substrates investigated were benzo[a]pyrene, 7-ethoxycoumarin and delta 4-androstene-3,17-dione.     

The purification and preliminary characterization of UMP synthase from human placenta

Orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23) are the final two of six enzymatic steps required in the de novo biosynthesis of uridine 5'-monophosphate (UMP). Earlier work of this laboratory showed that, in the mouse Ehrlich ascites carcinoma, both of these enzymatic activities were contained on the single multifunctional polypeptide chain, UMP synthase. We report here that the placenta provided an available human source for UMP synthase with 40-fold higher orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase specific activities than erythrocytes, a human source previously used by others. By using the placenta as a source of UMP synthase and by developing a novel purification procedure different from that used in the purification of UMP synthase from the Ehrlich ascites carcinoma (the only other homogeneous preparation of a mammalian UMP synthase), we achieved the purification of human UMP synthase to apparent homogeneity. This represents the first publication to homogeneity of UMP synthase from a human source as well as from a source other than malignant cell lines. Contrary to earlier reports human placental UMP synthase was found to be a multifunctional protein containing both enzymatic activities on a single polypeptide of 51,000 molecular weight. Preliminary characterization of the human placental UMP synthase showed it to be similar to UMP synthase from the Ehrlich ascites carcinoma in subunit molecular weight, native molecular weight, isozyme pattern (although not absolute pI values), pH optima of enzymatic activities, and kinetic constants for orotidine 5'-monophosphate (Km) and 6-azauridine 5'-monophosphate (Ki) at the decarboxylase site.