Structure and properties of calphobindin II, an anticoagulant protein from human placenta

The structure of human placental calphobindin-II (CPB-II) was investigated by amino acid composition and amino acid sequence analyses of peptides generated by protease digestion of the protein. The 45 peptides obtained from the lysyl endopeptidase digest of CPB-II, and the amino-terminal peptide prepared from its tryptic digest, were analyzed, and they accounted for over 98% of total amino acids of CPB-II. The structure of CPB-II determined by protein sequencing was identical to that previously predicted from its cDNA sequence (Iwasaki, A. et al. (1989) J. Biochem. 106, 43-49), except for the amino terminus. Since the amino terminus of CPB-II was blocked to Edman degradation, fast-atom-bombardment mass spectrometric analysis was used to demonstrate that the amino-terminal residue was acetyl-alanine. The carboxyl-terminal residue of CPB-II was identified as aspartic acid by the hydrazinolytic procedure. Calcium-binding studies indicated that 1 mol of CPB II binds 1 mol of calcium in the absence of phospholipid and 8 mol of calcium in the presence of phospholipid.     

Purification and characterization of a cytostatic factor with anti-viral activity from the bitter melon

We have previously reported that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic activities, and is a competitive inhibitor of guanylate cyclase activity. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses. In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity. The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This preparation was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons. The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse). As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system. DNA synthesis was slightly stimulated. The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h. The cytostatic component had no effect on cellular cyclic GMP metabolism.     

A novel blood coagulation factor IX/factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake): isolation and characterization

Using affinity chromatography on a column of factor X-Cellulofine, we have isolated a novel blood coagulation factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake). This anticoagulant protein was also purified by chromatography on Sephadex G-75 and S-Sepharose Fast Flow. The yield of the purified protein was approximately 16 mg from 400 mg of crude venom. The purified protein gave a single band on both analytical alkaline disc-gel electrophoresis and SDS-PAGE. This protein had a relative molecular weight (Mr) after SDS-PAGE of 27,000 before reduction of disulfide bonds and 14,000 after reduction of disulfide bonds. The protein prolonged the clotting time induced by kaolin or factor Xa. In the presence of Ca2+, it formed a complex with factor X, the molar ratio being 1 to 1. Similar complex formation was observed with factor Xa and factor IX/factor IXa, but not with other vitamin K-dependent coagulation factors, i.e., prothrombin, factor VII, protein C, protein S, and protein Z. The interaction of this anticoagulant protein with factor IX/factor X was dependent on gamma-carboxyglutamic acid (Gla) domains, since Gla-domainless derivatives of factor X and factor IXa beta' did not interact with this anticoagulant protein.     

A heat-stable protein inhibitor of cyclic 3',5'-nucleotide phosphodiesterase.

A heat-stable, non-dialyzable inhibitory factor of cyclic nucleotide phosphodieterase was detected in and partially purified from bovine retina. The factor appears to be a protein, since the inhibitory activity was abolished by trypsin digestion but not by DNAase or RNAase treatment. The protein inhibitor from bovine retina effectively inhibits the Ca2+-independent phosphodiesterase from several sources, including bovine retina, bovine rod outer segment, and a human lymphoblastic leukemia cell line, indicating lack of tissue and species specificity.     

Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody.

The use of bacterial signal peptides to target recombinant mammalian proteins to the periplasmic space of Escherichia coli (to promote proper disulfide bond formation) has met with variable success. We report the design and use of a bacterial expression vector to direct recombinant fusion proteins to the periplasmic space of E. coli: it contains the signal peptide from the pelB gene of Erwinia carotovora linked to a small peptide epitope for an unusual calcium-dependent antibody (HPC4). HPC4 binds to the epitope in a Ca(2+)-dependent manner, but the epitope itself does not bind Ca2+. We have used this system to express a biologically active, soluble form of tissue factor, the protein responsible for triggering the blood clotting cascade. Soluble tissue factor was secreted into the culture medium at 1-2 mg/liter, from which it could be readily purified using immobilized HPC4 antibody. The HPC4 epitope could be removed by digestion with thrombin or factor Xa, although a free amino terminus was not required for function since soluble tissue factor was equally active with the epitope still in place. This vector/epitope system permits large-scale expression and purification of recombinant soluble tissue factor and should be generally applicable to the isolation of other recombinant proteins. Furthermore, the epitope confers Ca(2+)-dependent binding of the fusion protein to HPC4 antibody while avoiding the creation of a new metal binding site on the fusion protein itself. Tb3+ can bind in this Ca2+ site near Trp, allowing this site to serve as a means of attaching a fluorescent probe to tissue factor.     

A heat-stable protein inhibitor of cyclic 3′,5′-nucleotide phosphodiesterase

A heat-stable, non-dialyzable inhibitory factor of cyclic nucleotide phosphodiesterase was detected in and partially purified from bovine retina. The factor appears to be a protein, since the inhibitory activity was abolished by trypsin digestion but not by DNAase or RNAase treatment. The protein inhibitor from bovine retina effectively inhibits the Ca²⁺-independent phosphodiesterase from several sources, including bovine retina, bovine rod outer segment, and a human lymphoblastic leukemia cell line, indicating lack of tissue and species specificity.     

Platelet factor 4 selectively inhibits binding of TGF-beta 1 to the type I TGF-beta 1 receptor.

A low molecular weight inhibitor of TGF-beta 1 binding was detected in partially purified human platelet extracts by using Hep 3B hepatoma cells in the binding assays. The inhibitory protein was purified to homogeneity and was identified as platelet factor 4 on the basis of its amino acid sequence. TGF-beta 1 binding to Hep 3B cells was almost completely inhibited by 100 nM concentrations of platelet factor 4, but TGF-beta 1 binding to NRK 49F fibroblasts was inhibited only slightly. Affinity cross-linking experiments revealed that these differences in the inhibition of TGF-beta 1 binding by platelet factor 4 were due to differences in the complements of TGF-beta 1 binding proteins present on these two cell types. In Hep 3B cells the majority of bound TGF-beta 1 was cross-linked to a complex which had an apparent molecular weight of 70 kDa. TGF-beta 1 binding to this protein was the most sensitive to inhibition by platelet factor 4. Based on its size and TGF-beta 1 binding properties, we believe this protein is the type I TGF-beta 1 receptor. Hep 3B cells also had a high-affinity TGF-beta 1 binding protein which appeared as an 80 kDa complex, and which we believe to be the type II TGF-beta 1 receptor. TGF-beta 1 binding to this protein was not inhibited by platelet factor 4. TGF-beta 1 was also cross-linked to complexes of higher molecular weights in Hep 3B cells, but it was not clear whether any of them represented the type III TGF-beta 1 receptor. In NRK 49F cells, the majority of bound TGF-beta 1 was cross-linked to a high molecular weight complex which probably represented the type III TGF-beta 1 receptor. NRK 49F cells also had type I TGF-beta 1 receptors and platelet factor 4 inhibited binding to these receptors in the NRK cells. Since the type I receptor contributed only a small percentage of total TGF-beta 1 binding, however, the overall effects of platelet factor 4 on TGF-beta 1 binding to NRK 49F cells were negligible. We were unable to demonstrate specific or saturable binding of platelet factor 4 to Hep 3B cells using either direct binding or affinity cross-linking assays. Thus, it is not clear whether platelet factor 4 inhibits TGF-beta 1 binding by competition for binding to the type I receptor. Modest concentrations of TGF-beta 1 reduced the adherence of Hep 3B cells to tissue culture dishes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Retinoic acid-binding protein in rat tissue. Partial purification and comparison to rat tissue retinol-binding protein.

When the 100,000 X g supernatant fractions of several rat organs are incubated with all-trans-[3H]retinoic acid, a binding component for retinoic acid with a sedimentation coefficient of 2 S can be detected by sucrose gradient centrifugation. This tissue binding protein for retinoic acid is distinct from the tissue binding protein for retinol which has been previously described. The tissue retinoic acid-binding protein has been partially purified from rat testis and this partially purified protein would appear to have a molecular weight of 14,500 as determined by gel filtration and high binding specificity for all-trans-retinoic acid. Binding of [3H]retinoic acid is not diminished by a 200-fold molar excess of retinal, retinol, or oleic acid but is reduced by a 200-fold excess of unlabeled retinoic acid. Tissue retinoic acid-binding protein can be detected in extracts of brain, eye, ovary, testis, and uterus but is apparently absent in heart muscle, small intestine, kidney, liver, lung, gastrocnemious muscle, serum, and spleen. This distribution is different than that observed for the tissue retinol-binding protein. Tissue retinol-binding protein was also purified extensively from rat testis. The partially purified protein has an apparent molecular weight of 14,000 and high binding specificity for all-trans-[3H]retinol as only unlabeled all-trans-retinol but not retinal, retinoic acid, retinyl acetate, retinyl palmitate, or oleic acid could diminish binding of the 3H ligand under the conditions employed. The partially purified protein has a fluorescence excitation spectrum with lambda max at 350 nm. In contrast, the retinol-binding protein isolated from rat serum and described by others has a fluorescence excitation spectrum with lambda max at 334 nm and an apparent molecular weight of 19,000. When partially purified tissue retinol-binding protein is extracted with heptane, the heptane extract has a fluorescence excitation spectrum similar to that of all-trans-retinol.     

Purification and Characterization of A Cytostatic Factor With Anti-Viral Activity From The Bitter Melon

We have previously reported -that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic active ties ^1,2, and is a competitive inhibitor of guanylate cyclase activity³. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses¹.

In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity.

The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This preparation was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons.

The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse).

As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system. DNA synthesis was slightly stimulated.

The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h.

The cytostatic component had no effect on cellular cyclic CMP metabolism.     

Purification and Characterization of a Cytostatic Factor with Anti-Viral Activity from the Bitter Melon

Abstract

We have previously reported that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic activi-ties ^1,2, and is a competitive inhibitor of guanylate cyclase activity³. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses¹.

In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity.

The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This pre-paration was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons.

The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse).

As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system⁴. DNA synthesis was slightly stimulated.

The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h.

The cytostatic component had no effect on cellular cyclic GMP metabolism.     

In vitro insulin-like actions of the growth factor from the tapeworm, Spirometra mansonoides

In vitro actions of purified plerocercoid growth factor (PGF) were compared with those of insulin and human growth hormone (hGH) in adipose tissue from normal male rats. Insulin-like effects were measured by the ability of PGF, insulin, or hGH to stimulate oxidation of [U-14C]glucose to 14CO2, to stimulate lipogenesis, and to inhibit epinephrine-induced lipolysis. PGF and insulin stimulated significant increases in glucose oxidation and lipogenesis in adipose tissue that had not been preincubated as well as in tissue that had been preincubated. hGH stimulated insulin-like effects only in tissue that had been preincubated for 3 hr. Insulin, hGH, and PGF inhibited epinephrine-induced lipolysis of preincubated (3 hr) adipose tissue. hGH produced a dramatic lipolytic response in tissue freshly removed from normal rats but no dose of PGF was lipolytic. PGF did not displace 125I-insulin from its receptors on adipocytes but did competitively inhibit 125I-hGH binding to adipocytes. These results suggest that PGF has direct insulin-like actions which are initiated by binding a GH receptor, but PGF had no anti-insulin action and the insulin-like activity of PGF was unaffected by refractoriness of adipose tissue to GH.     

Characterization of proteins from human synovium and mononuclear leucocytes that induce resorption of cartilage proteoglycan in vitro

Both human synovial tissue in culture and lectin-stimulated mononuclear leucocytes produced a protein that induced proteoglycan resorption in explants of bovine nasal cartilage and human articular cartilage. On gel filtration the protein had Mr 16000-20000 and on isoelectric focusing its pI was 5.2-5.3. The protein corresponded to catabolin, which has previously been identified as a product of cultured porcine synovial tissue and mononuclear leucocytes. The action of partially purified human catabolin was not inhibited by cortisol, although the activity of the leucocyte supernatants from which it had been isolated was inhibited. For this reason it is not possible to be sure that the active factor detected in the bioassay of the crude leucocyte culture supernatants is in fact catabolin.     

Characterization of polyclonal antibodies that distinguish acidic and basic fibroblast growth factors by using western immunoblotting and enzyme-linked immunosorbent assays

Rabbit polyclonal antibodies were raised against ovalbumin conjugates of purified bovine brain acidic fibroblast growth factor (aFGF) and a synthetic peptide containing the N alpha-terminal 1-24 amino acid sequence of bovine basic fibroblast growth factor (bFGF). These antibodies were used to specifically detect 1-ng quantities of aFGF and bFGF by using enzyme-linked immunosorbent assay (ELISA) and Western immunoblot procedures. Antibodies raised against aFGF recognized bovine brain aFGF and bovine recombinant aFGF but very poorly recognized recombinant bFGF or purified porcine or bovine pituitary bFGF with ELISA and Western immunoblot procedures. Antibodies raised against bFGF (1-24) recognized purified bovine, porcine, and recombinant human bFGF but only very poorly recognized aFGF with ELISA and Western immunoblot procedures. In vitro addition of anti-bFGF antibodies was able to partially neutralize bFGF-stimulated 3H-thymidine incorporation by COMMA-D mouse mammary epithelial cells while having no effect on aFGF or epidermal growth factor (EGF) stimulation. In vitro addition of anti-aFGF antibodies had no effect on bFGF- or EGF-stimulated 3H-thymidine incorporation, but surprisingly, had a potentiating effect on aFGF stimulation. Antibodies against aFGF immobilized on protein A-Sepharose were able to specifically and completely remove mitogenic activity from solutions containing aFGF but had no effect on removal of mitogenic activity from control solutions containing bFGF or EGF. Similarly, immobilized anti-bFGF antibodies completely removed mitogenic activity from solutions of bFGF, but not aFGF or EGF controls. These antibodies have been useful for the identification and characterization of growth factors from tissue and recombinant sources.     

Phosphorylation of tyrosine hydroxylase by calmodulin-dependent multiprotein kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated stoichiometrically by either cyclic AMP-dependent protein kinase or calmodulin-dependent multiprotein kinase from skeletal muscle, but not by five other protein kinases tested. The activity of tyrosine hydroxylase was elevated 3-fold by cyclic AMP-dependent protein kinase, but no activation was observed after phosphorylation by calmodulin-dependent multiprotein kinase. Phosphorylation produced by cyclic AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase was additive, suggesting different sites of phosphorylation. This was confirmed by high-performance liquid chromatography analysis of tryptic phosphopeptides which demonstrated that the major sites phosphorylated by each protein kinase were distinct. A calmodulin-dependent multiprotein kinase that had identical properties and substrate specificity to the skeletal muscle enzyme was partially purified from rat pheochromocytoma. The possibility that this protein kinase is involved in the regulation of tyrosine hydroxylase activity in adrenergic tissue in vivo is discussed.     

Reversible protein kinase activation of hormone-sensitive lipase from chicken adipose tissue

Hormone-sensitive lipase partially purified from adipose tissue of laying hens was markedly activated by cyclic AMP-dependent protein kinase. Activation was approximately 4-fold (ranging up to as great as 10-fold) compared with the much lower degree of activation obtained with analogous preparations from rat and human adipose tissues (59 and 86%, respectively). The partially purified preparations contained adequate endogenous protein kinase activity to effect complete activation with addition of cyclic AMP, ATP, and Mg(2+). Activation was blocked by protein kinase inhibitor (from rabbit skeletal muscle) but could be restored fully by addition of excess exogenous protein kinase (from bovine skeletal muscle). The fully activated lipase was slowly deactivated by dialysis at 4 degrees C and then rapidly and almost fully reactivated by addition of cyclic AMP and ATP-Mg(2+). Reactivation was blocked by protein kinase inhibitor. This deactivation-reactivation cycle was rapid at 23 degrees C with dialysis against charcoal and could be demonstrated repeatedly using a single preparation. The reversible deactivation of protein kinase-activated enzyme is presumed to reflect the action of a lipase phosphatase. Lipase prepared from tissue previously exposed to glucagon yielded a much smaller degree of activation than lipase prepared from tissue not exposed to the lipolytic hormone, indicating that the physiological hormone-induced activation is probably similar to or identical with the protein kinase activation demonstrated in the cell-free preparations. Under the conditions of assay used, the partially purified lipase fraction contained diglyceride, monoglyceride, and lipoprotein lipase activities. However, treatment with cyclic AMP-dependent protein kinase had virtually no effect on these lipase activities.     

Identification of neutral active phospholipase C which hydrolyzes choline glycerophospholipids and plasmalogen selective phospholipase A2 in canine myocardium

Two novel phospholipase activities have been identified in the cytosolic fraction of canine myocardium. Neutral active phospholipase C activity was partially purified by anion exchange, hydroxylapatite, chromatofocusing, and gel filtration chromatographies. The partially purified enzyme had similar maximum velocities (237 versus 241 nmol/mg X h) and apparent Michaelis constants (20 versus 14 microM) utilizing either plasmenylcholine or phosphatidylcholine as substrate. Myocardial phospholipase C had a pH optimum between 7 and 8, required divalent cations for maximal activity, and did not hydrolyze phosphatidylinositol or sphingomyelin. Myocardial cytosol contained a potent inhibitor of phospholipase C which masked enzymic activity until it was removed during the purification procedure. A plasmalogen selective phospholipase A2 activity was also identified in the cytosolic fraction of canine myocardium. The protein catalyzing this activity was partially purified by DEAE-Sephacel-hydroxylapatite tandem chromatography and exhibited a maximum velocity of 5 nmol/mg X h for plasmenylcholine but only 1 nmol/mg X h for phosphatidylcholine, had a pH optimum between 6 and 7 for both substrates, and did not require calcium ion for activity. These results constitute the first demonstration of a neutral active phospholipase C specific for choline and ethanolamine glycerophospholipids and a plasmalogen selective phospholipase A2 in mammalian tissue.     

Activator protein supporting the botulinum ADP-ribosyltransferase reaction

The ADP-ribosyl moiety of NAD was transferred to proteins with Mr values of 22,000 and 25,000 when bovine brain cytosol was incubated with a botulinum ADP-ribosyltransferase C3 (BT-C3) which was purified from the culture medium of a type C strain of Clostridium botulinum. Any protein fraction eluted from a chromatographic column to which the cytosol had been applied, however, was not significantly ADP-ribosylated by BT-C3, unless the reaction mixture was further supplemented with a small amount of the cytosol. Thus, substrate protein(s) could be partially purified based on their ability to be ADP-ribosylated by BT-C3 in the presence of the cytoplasmic activator(s). The rate of ADP-ribosylation of the substrates was extremely low by itself but was increased enormously and progressively when increasing amounts of cytosol were added, affording a reliable means for assay of the activator contained therein. The activator was separated from the substrate proteins and partially purified from the cytosol by sequential chromatography steps with an anion exchanger and a gel filtration column. The activity of the partially purified activator was heat-labile and protease-sensitive, suggesting that the activator was a protein or had a protein component necessary for activity. The action of the activator protein(s) was specific for BT-C3-catalyzed ADP-ribosylation; cholera toxin-catalyzed ADP-ribosylation of GTP-binding protein (Gs) was not supported by this activator. Thus, this is the first report to show that botulinum ADP-ribosyltransferase-catalyzed reaction can proceed significantly only in the presence of other protein factor(s), just as has been observed with an ADP-ribosylation factor required for cholera toxin-induced similar reaction.     

Probable occurrence of brain basic protein factor I, an activator of phosphoprotein phosphatases

Basic protein factor I (BPFI was purified to homogeneity from bovine brain by boiling and trichloroacetic acid-precipitation of tissue homogenate, followed by DEAE-cellulose, Sephadex G-150, Affi-Gel-phenothiazine, and Bio-Gel P-6DG chromatographic procedures. The preparation appeared as a single protein band in the SDS-polyacrylamide gel electrophoresis with a minimal Mr of 13,200. The factor was a basic protein as indicated by an estimated isoelectric point of pH 8.3 and a high content of amino acids including arginine, histidine, lysine and others. In the absence of Mn2+, the factor stimulated the phosphoprotein phosphatases (PPase) from rabbit brains. Unlike histones or protamine, the factor was a poor substrate for megamodulin-dependent protein kinase. In addition, the factor did not interact significantly with E. coli megamodulin.