Purification and characterization of a high-molecular-weight protease, ingensin, from human placenta

We purified a high-molecular-weight protease, ingensin, from extract of human placenta by successive DEAE-cellulose, hydroxyapatite, and high performance liquid chromatographies. The activity of ingensin was determined by using a synthetic substrate, succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarinamide (MCA). The purified ingensin, which gave a single band in 6.5% nondenaturing polyacrylamide gel electrophoresis, was activated by linoleic acid and sodium dodecyl sulfate (SDS). Maximum activity was observed at pH 9.5 in the presence of 0.06% SDS, but at pH 8.0 in the presence of linoleic acid. A subcellular fractionation study showed that a large amount of ingensin activity was present in the cytosol or microsome fraction rather than in the precipitate of low-speed centrifugation. The effect of protease inhibitors on the activated ingensin was also investigated.     

A high-molecular weight complex with acid phosphatase activity in human breast cancer

Summary The presence of a high-molecular weight complex with acid phosphatase activity in the cytosol of human mammary tumors is reported. This complex appeared in the cytosol after tissue homogenization in the presence of dithiotreitol, with or without Triton X-100 and at acidic or neutral pH. Upon gel electrophoresis, this fraction showed only one band of enzyme activity which did not enter the fine pore gel. Lubrol or n-butanol had no apparent effect on this complex, and 8 M urea or 2% sodium dodecyl sulfate did not disaggregate this large molecule. After purification by gel filtration, ammonium sulfate precipitation and ion-exchange chromatography an apparent molecular weight or 10⁶ was measured. It hydrolyzed typical acid phosphatase substrates such as p-NPP and -NP, but also ATP and PPi. Only 44% inhibition was observed with L-(+)tartrate and it was still 40% active after 1 hr incubation at 60 °C. Reduction in the presence of SDS yielded several polypeptide bands. It was also detected in some samples of normal mammary tissues, but not in normal human placenta or liver.Abbreviations AP acid phosphatase - C-AP high-molecular weight complex with acid phosphatase activity - SDS sodium dodecyl sulfate - p-NPP p-nitrophenyl phosphate - -NP -naphthyl phosphate - enzyme code number acid phosphatase or ortophosphoric monoester phosphohydrolase (EC 3.1.3.2)     

Effects of water activity on oxygen-binding in high-molecular weight,extracellular invertebrate hemoglobin and hemocyanin

We have investigated the effects of water activity on oxygen-binding properties of giant invertebrate oxygen-binding proteins: hemocyanins from the intertidal crab, Carcinus maenas and the terrestrial snail, Arion ater and hemoglobin from the marine polychaete, Arenicola marina, using the osmotic stress method. We show that in contrast to the water-sensitive dimeric or tetrameric hemoglobins from humans, fish and cyclostomes, changes in water activity exert small effects on the oxygen affinity of these polymeric proteins, indicating that their deoxygenated and oxygenated states are almost similarly hydrated. The small effects of water activity may correlate with small surface to volume ratios in these high-molecular-weight proteins that pose a limit to water-accessible sites, and suggest smaller quaternary structural changes associated with oxygenation compared to those in dimeric and tetrameric hemoglobins.     

Microbial production of a novel trihydroxy unsaturated fatty acid from linoleic acid

A bacterium isolated from a dry soil sample collected from McCalla, AL, USA, converted linoleic acid to a novel compound, 12,13,17-trihydroxy-9 (Z)-octadecenoic acid (THOA). The organism is a Gram-positive, non-motile rod (0.5 μ m × 2 μ m). It was identified as a species of Clavibacter ALA2. The product was purified by high pressure liquid chromatography, and its structure was determined by ¹H and ¹³C nuclear magnetic resonance and Fourier transform infrared spectroscopies, and by mass spectrometer. Maximum production of THOA with 25% conversion of the substrate was reached after 5–6 days of reaction. THOA was not further metabolized by strain ALA2. This is the first report of a 12,13,17-trihydroxy unsaturated fatty acid and its production by microbial transformation. Some dihydroxy intermediates were also detected. THOA has a structure similar to those of known plant self-defense substances. Received 13 January 1997/ Accepted in revised form 05 May 1997     

Cathepsin Q, a novel lysosomal cysteine protease highly expressed in placenta

The complete nucleotide sequence of a novel cathepsin cDNA derived from rat placenta was determined and is termed cathepsin Q. The predicted protein of 343 amino acid is a member of the family C1A protease related to cathepsin L. Rat cathepsin Q and its mouse counterpart were found highly expressed in placenta, whereas no detectable levels were found in lung, spleen, heart, brain, kidney, thymus, testicle, liver, or embryonic tissues. It is predicted that cathepsin Q will differ in catalytic specificity to another placental-specific protease, cathepsin P, indicating that these enzymes will have unique proteolytic functions in extra-embryonic tissues.     

Effects of linoleic acid and mitogenic stimulation on the fatty acid composition of human lymphocytes

Perturbation of the fatty acid composition of human lymphocytes in vitro was investigated by addition of linoleic acid complexed to bovine serum albumin (BSA-LA) and by mitogenic stimulation with phytohaemagglutinin (PHA). BSA-LA resulted in a 45% increase in linoleic acid in phosphatidylethanolamine (PE) and over 100% in phosphatidylcholine (PC) in peripheral blood cells. Supplementation with BSA-LA in PHA-stimulated lymphocytes produced even greater changes: 100% increase in linoleic acid content for PE and over 300% for PC. There was a large decrease in oleic acid: 40% for PE and almost 100% in PC. Significant decreases in arachidonic acid occurred in both phospholipid fractions. PHA alone also altered membrane phospholipid fatty acid composition, with reductions in palmitic, stearic and linoleic acid for PE and increases in oleic acid and arachidonic acid (almost 100%). For PC, there were large decreases in stearic (40%), linoleic (30%) and arachidonic (40%) acids, together with an increase in oleic acid (65%). Cells supplemented with linoleic acid grown in the presence of PHA, compared with those grown in linoleic acid-supplemented medium alone, showed a 40% decrease in palmitic acid and a 55% increase in arachidonic acid in PE. For PC, there were large decreases in stearic acid (40%) and arachidonic acid (57%). Antibody-induced redistribution of surface molecules ('capping') was inhibited by some 14% after incubation with BSA-LA. However, no consistent alterations in PHA-induced cell proliferation were observed. These data suggest that profound alterations of membrane fatty acid composition occur spontaneously during the mitotic cycle, and may be further induced by experimental manipulation, without gross perturbation of cell function.     

Isolation and purification of protease from human placenta by foam fractionation

The effect of operating parameters like pH, protein concentration, column geometry, and gas flow rate on the separation efficiency of proteolytic enzymes from crude human placental homogenate has been studied in a batch foam column. Purification has been found to be optimum at pH 8.0, close to the isoelectric pH, at which the surface adsorption of the protein on the foam bubbles is maximum. Both purification and recovery varied significantly with total protein concentration. Stable bubble formation was hindered at lower protein concentrations, while extraneous proteins rather than the protease were preferentially adsorbed at higher protein concentrations, decreasing the purification efficiency. Column diameter and column height should be optimized for any specific feed protein concentration and gas flow rate. However, the enrichment ratio was found to decrease with the increase in flow rate. The results indicate that foam fractionation is an effective separation process for recovering valuable biochemicals from biological materials.     

Molecular cloning,expression, and anti-tumor activity of a novel serine protease from Arenicola cristata

Arenicola cristata, a marine annelid, is a wellknown and prized traditional Chinese medicine. However, the serine protease gene of A. cristata has not been cloned yet. In this study, a novel protease ofA. cristata was cloned, sequenced, and expressed in Escherichia coli, and the functions of this recombinant protease were also investigated. The whole complementary DNA （cDNA） of this novel protease was of 980 bp in length and consisted of an open reading frame of 861 bp encoding 286 aa. Sequence analysis of the deduced amino acid sequence revealed that the protease belongs to the serine protease family. The active enzyme of the pro posed A. cristata protease is composed of a signal peptide, a propeptide, and a mature polypeptide. The molecular weight of the recombinant mature protein was 26 kDa after overexpression in E. coli. The recombinant pro tein significantly inhibited cell growth and induced cell apoptosis of esophageal squamous cell carcinoma （ESCC） in vitro, and reduced tumorigenicity in vivo. Furthermore, administration of the recombinant protein led to the activa tion of caspase9 as well as downregulation of Mcl1 and Bcl2. Taken together, our findings indicated that the recom binant serine protease ofA. cristata could inhibit ESCC cell growth by mitochondrial apoptotic pathway and might act as a potential pharmacological agent for ESCC therapy.     

Purification and characterization of a low molecular weight zinc binding protein from human placenta

A low molecular weight, native zinc binding, cytosolic protein (LMZP) has been isolated, purified and characterized from human normal term placenta. Gel filtration of heat treated placental cytosol after sequential acetone precipitation (80% ppt) revealed a major zinc binding protein in the range of low molecular weight. This partially purified zinc binding fraction was further fractionated on DEAE-Sephadex A-25. The zinc was eluted in one of the three peak fractions. Further, the purity of zinc binding protein was confirmed on fast protein liquid chromatography (FPLC). The purified placental LMZP was homogenous on SDS-polyacrylamide gel electrophoresis with a single band. Ultraviolet (UV) spectrum of LMZP showed an absorption maximum at 257 nm which disappeared at pH 2. Molecular weight of LMZP as determined by gel chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis was 6 kDa. It was calculated that 1 g atom of zinc was bound to 1 mole of the LMZP. Unlike in classical metallothionein, the amino acid composition of placental LMZP revealed the presence of aromatic amino acids, lower content of cysteine and higher content of histidine, glutamic acid and aspartic acid (10, 9 and 5 residues/mole, respectively).     

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.     

Complete amino acid sequence of a beta-galactoside-binding lectin from human placenta

The complete amino acid sequence of a beta-galactoside-binding lectin from human placenta was determined at protein level. The lectin consists of 134 amino acids and its N-terminal alanine is blocked with acetate. The lectin shows about 50% similarity with chick 14K lectin, which was the first vertebrate beta-galactoside-binding lectin completely sequenced. Only 14 residues proved to be different from those of rat lung lectin, the sole mammalian lectin of which the complete sequence has been reported.     

9-LR-linoleyl hydroperoxide, a novel product from the oxygenation of linoleic acid by type-2 lipoxygenases from soybeans and peas.

Type-2 lipoxygenases from soybeans and peas, which have a pH optimum of 6--7 were examined for oxygenation activity at pH 9.0. The reaction velocity was found to be strongly dependent on substrate concentration. At higher substrate concentrations an inhibitory effect was observed, which is connected with the occurrence of a kinetic lag phase. On incubation of linoleic acid at pH 9.0 with either of these enzymes predominantly 9-LR-hydroperoxy-10-trans,12-cis-octadecadienoic acid is formed. The similarity of the product specificity with that of prostaglandin synthetase is discussed in view of the formation of prostaglandin-like substances by soybean lipoxygenase-2 (Bild, G.S., Bhat, S.G., Ramadoss, C.S. and Axelrod, B. (1978) J. Biol. Chem, 253, 21--23).     

Effects of prostaglandins on cyclic nucleotide phosphodiesterase activity in the human term placenta

Slices of human full-term placentas, obtained by elective cesarean section, were incubated in the absence or presence of prostaglandins (PGs) and the cyclic AMP phosphodiesterase (cAMP PDE) activity was measured. PGE1 and PGI2 were shown to stimulate cAMP PDE activity. The effect of PGE1 is related to an increase in the Vmax of the low Km activity without alteration of this apparent Km. Several findings suggest that the cAMP PDE is activated by its own substrate; PGE1 and PGI2, promote an increase of cAMP formation which is observed before the cAMP PDE activation. Dibutyryl cAMP or theophylline also activate cAMP PDE. In contrast, PGF2 alpha does not influence either adenylate cyclase or AMP PDE. In addition, we found that the ability of the placenta to degrade cAMP, increases after parturition. PG levels are higher in the foeto-placental unit during labor, and a causal relationship between these two phenomena is possible. Our data supporting the concept of hormonal control of cAMP PDE is consistent with the hypothesis that an accelerated cAMP metabolism in placenta contributes to the maintenance of a constant equilibrium of the cyclic nucleotide levels in the foeto-placental unit.