Isolation and properties of neutral SH-dependent proteinase from bovine spleen]

A neutral SH-dependent proteinase was isolated from bovine spleen by a slight modification of the previous method (1) and its action on some natural and synthetic substrates was studied. The activity of the enzyme was increased 2000--2500-fold as compared to that of the original extract. The enzyme hydrolyzed various histones (H1, H2a, H2b, H3), casein and protamine but did not split hemoglobin, serum albumin and 14C-tryptophane-labelled total protein from chicken embryos. The enzyme possessed neither collagenolytic nor elastase activity; it did not inactivate aldolase, hexokinase, glyceraldehyde phosphate dehydrogenase and glucose-6-phosphate dehydrogenase, which makes the enzyme different from cathepsin B1 and some other previously described proteinases. The enzyme did not split BAPA, BAEE, ATEE, Boc-Ala-ONP, Leu-beta-NA and some other peptides. The molecular weight of the enzyme was found to be about 15 000.     

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 characterization of thrombomodulin from bovine lung

Bovine thrombomodulin was isolated from the lung by Triton X extraction, affinity chromatography on diisopropyl phosphate-thrombin-agarose, and gel filtration on Ultrogel AcA-44. The final preparation was purified 6000-fold from the membrane extract with a yield of 21%. It showed apparent Mr of 78,000 and 105,000, before and after reduction, respectively, on polyacrylamide gel electrophoresis in SDS. The activity of the thrombomodulin was stable under the conditions of 1% SDS, 8 M urea, pH 2 and 10, and heat treatment at 60 degrees C for 30 min, but was unstable against treatment with 2-mercaptoethanol. Activation of protein C by thrombin in the presence of the thrombomodulin depended on Ca2+, and an equimolar complex formation between thrombin and thrombomodulin was required for the maximum rate activation. The rate of protein C activation by thrombin was increased 900-fold by thrombomodulin. Thrombomodulin inhibited the thrombin-induced fibrinogen clotting and platelet activation. However, it did not affect the inhibition of thrombin by antithrombin III with or without heparin, a protein C inhibitor or several synthetic inhibitors. These properties of bovine thrombomodulin were similar to those of rabbit thrombomodulin reported earlier.     

Isolation and properties of beta-endorphin-(1-27)-like peptide from bovine brains

A beta-endorphin-(1-27)-like peptide was isolated from bovine cerebral hemisphere extracts by gel filtration, ion-exchange chromatography, high performance liquid chromatography and paper electrophoresis. The peptide had tyrosine as the amino-terminal residue and its amino acid composition was nearly identical to that of equine pituitary beta-EP-(1-27). It had also the same mobility as equine pituitary beta-EP-(1-27) in paper electrophoresis. In radioimmunoassay and opiate receptor-binding assay, the brain peptide had 50% activity when compared with human beta-endorphin-(1-27). Evidence for the occurrence of NH2-acetylated form of beta-endorphin-(1-27) is also presented.     

Purification and properties of active atrial-natriuretic-peptide receptor (type C) from bovine lung.

Atrial-natriuretic-peptide (ANP) receptor, previously identified as a 140 kDa protein with a disulphide-linked homodimeric structure, was purified from bovine lung by (NH4)2SO4 fractionation and affinity chromatography on ANP-Affi-Gel 10. The purified receptor had a binding capacity of 4.2 nmol of ANP/mg of protein and an affinity constant of 6.5 pM. The isoelectric point of the receptor was 5.8, consistent with the acidic nature of the protein (amino acid analysis revealed a predominance of glutamic acid and aspartic acid residues). Treatment with endoglycosidase H and glycopeptidase F revealed that the receptor has three complex types of oligosaccharide chains per 70 kDa subunit. Deglycosylation of the receptor did not affect its binding activity. Reduction with dithiothreitol and reoxidation by dialysis revealed a strong tendency of the receptor subunits to dimerize via disulphide cross-linking; however, carboxymethylation of the reduced receptor indicated that the intersubunit disulphide bond is not necessary for the ligand-binding activity.     

Isolation of a novel chromium(III) binding protein from bovine liver tissue after chromium(VI) exposure

In the ongoing investigation into the biological importance and toxicity issues surrounding the bioinorganic chemistry of chromium, the accepted literature procedure for the isolation of the biological form of chromium, low molecular weight chromium binding protein (LMWCr) or chromodulin, was investigated for its specificity. When chromium(VI) is added to bovine liver homogenate, results presented here indicate at least four chromium(III) binding peptides and proteins are produced and that the process is non-specific for the isolation of LMWCr. A novel trivalent chromium containing protein (1) has been isolated to purity and initial characterization is reported here. Chromium(III) identification was determined by optical spectroscopy and diphenylcarbazide testing. This chromium binding protein has a molecular weight of 15.6kDa, which was determined from both gel-electrophoresis and mass spectrometry. The protein is comprised primarily of Asx, Glx, His, Gly/Thr, Ala, and Lys in a 1.00:2.51:0.37:2.09:0.39:1.17 ratio and is anionic at pH 7.4. In addition, the protein binds approximately 2.5 chromium(III) ions per molecule.     

Catalytic and molecular properties of a highly purified G type casein kinase from bovine lung tissue

We have determined, using polyacrylamide gel electrophoresis, that endothelial cell cultures derived from rabbit aorta synthesize a wide spectrum of heparan sulfate proteoglycans. The electrophoretically slower-moving heparan sulfate proteoglycans have been isolated from the endothelial cell culture medium. Antibodies to these proteoglycan species have been raised in the goat. The goat antiserum binds selectively the heparan sulfate proteoglycans that were used for the immunization and does not cross-react with the other (faster moving) species. Only a moderate level of cross-reactivity was observed with the heparan sulfate proteoglycans synthesized by another cell line (presumably smooth muscle cells) of vascular derivation. These results support the suggestion that structural differences in the heparan sulfate proteoglycans are responsible for certain differences in function between the various cell types of the vessel wall.     

Isolation and properties of oxaloacetate keto-enol-tautomerases from bovine heart mitochondria

Two highly purified proteins with quite different properties capable of oxaloacetate keto-enol-tautomerase activity (oxaloacetate keto-enol-isomerase, EC 5.3.2.2) were isolated from the bovine heart mitochondrial matrix. The first protein has an apparent molecular mass of 37 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-200 gel filtration. It is quite stable upon storage at 40 degrees C and reaches the maximal catalytic activity at pH 8.5 with a half-maximal activity at pH 7.0. The enzyme is specifically inhibited by oxalate and diethyloxaloacetate. When assayed in the enol----ketone direction at 25 degrees C (pH 9.0), the enzyme obeys a simple substrate saturation kinetics with Km and Vmax values of 45 microM and 74 units per mg of protein, respectively; the latter value corresponds to the turnover number of 2700 min-1. The second protein has an apparent molecular mass of 80 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-300 gel filtration. The enzyme is rapidly inactivated at 40 degrees C and shows a sharp pH optimum of activity at pH 9.0. The enzyme can be completely protected from thermal inactivation by oxaloacetate and dithiothreitol. The kinetic parameters of the enzyme as assayed in the enol----ketone direction at 25 degrees C (pH 9.0) are: Km = 220 microM and Vmax = 20 units per mg of protein; the latter corresponds to the turnover number of 1600 min-1. The enzyme activity is specifically inhibited by maleate and pyrophosphate. About 30% of the total oxaloacetate tautomerase activity in crude mitochondrial matrix is represented by the 37 kDa enzyme and about 70% by the 80 kDa protein.     

Isolation and properties of hyaluronic acid from bovine heart valves

1. A soluble extract of bovine heart valves was obtained after the tissue had been pulverized at liquid-nitrogen temperatures in a mill. 2. Hyaluronic acid was isolated from the crude extract by sedimentation equilibrium in a caesium chloride density gradient (Franek & Dunstone, 1966). 3. Analysis of the product indicated that it contained 15% of protein and the molar ratio of glucuronic acid to glucosamine was 1.27. 4. Its physicochemical properties, as determined by lightscattering, viscosity and sedimentation studies, suggested that its molecular size and configuration were similar to those of hyaluronic acid isolated from ox synovial fluid (Preston, Davies & Ogston, 1965).     

Isolation and properties of a T-kininogenase from bovine erythrocyte membranes

A kininogenase from bovine erythrocyte membranes has been purified 140-fold by affinity chromatography on pepstatin A-Agarose followed by ion exchange chromatography on CM Cellulose. The purified enzyme showed an apparent molecular weight of 31,000 daltons as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ItspH optimum is 7.5, and it was totally inhibited by soybean trypsin inhibitor, phenylmethylsulfonylfluoride, aprotinin, pepstatin, and dithiotreitol, suggesting the presence of a disulfide bond(s) whose integrity is(are) essential for maintaining the native three-dimensional structure. The referred enzyme was able to release kinin from a substrate partially purified from rat plasma. The kininogenase was activated by Zn²⁺, Ca²⁺, and cysteine-HCl.     

Isolation and properties of chondroitin sulphates from bovine heart valves

1. Several protein-polysaccharides were isolated from the soluble extracts of bovine heart valves by sedimentation equilibrium in a caesium chloride density gradient (Meyer, Preston & Lowther, 1969). 2. Compositional and structural studies indicated that the polysaccharide moiety was chondroitin sulphate. Differences in the protein content of the products were observed. There was no evidence suggesting the presence of keratan sulphate. 3. Sedimentation studies indicated that the molecular weights of the samples were between 4.2x10(4) and 6.5x10(4). The results are discussed in terms of a basic model for the protein-polysaccharides of two polysaccharide chains linked by a protein of variable size.     

Isolation and identification of alpha-(beta-alanyl)hypusine from bovine brain

A unique dipeptide was isolated from bovine brain using five steps of ion-exchange chromatography. Its acid hydrolysate contained equimolar amounts of beta-alanine and hypusine. The structure of the peptide was elucidated as alpha-(beta-alanyl)hypusine using dansylation technique. About 1 mumol of the compound was isolated from 1090 g of bovine brain.     

Isolation and characterization of two novel calcium-dependent phospholipid-binding proteins from bovine lung

Two calcium-dependent proteins of apparent Mr 32,000 and 34,000 were isolated from bovine lung. Approx. 70 mg/kg of each was obtained. Two-dimensional gel electrophoresis in the presence of 8 M urea showed their apparent p/values to be 5.1 and 5.0, respectively. Both proteins are related immunologically to calelectrin from Torpedo marmorata. They also have very similar amino acid compositions to calelectrin. Partial sequence information shows that both proteins contain the highly conserved sequence described for the annexins, a new family of calcium-dependent membrane-binding proteins. In common with other members of this family, the new proteins bind to acidic phospholipids in a calcium-dependent manner.     

Isolation of a neutral histone-hydrolyzing protease from bovine spleen]

Neutral histone-hydrolyzing protease has been isolated by fractionation of bovine spleen extract. The low level of the protease activity in the extract may be due to the presence of an inhibitor. The enzyme activity was increased 100--1200-fold during ammonium sulfate fractionations, gel filtration on Sephadex G-100 and G-75, chromatography on CM- and DEAE-celluloses. The protease was detected in the fraction with a molecular weight lower than 25000. The enzyme was markedly activated by dithiothreitol and EDTA and inhibited by p-chloromercuribenzoate and iodoacetic acid. It was also inhibited by N-tosyl-L-lysyl chloromethyl ketone, N-tosyl-L-phenylalanyl chloromethylketone, bovine blood serum and partially by soybean trypsin inhibitor DFP, trasylol and epsilon-amino caproic acid had no effect. Beside histone, the neutral protease hydrolyzed casein and gamma-globulin and fibrinogen in a low extent. The enzyme had no activity toward N-benzoyl-D,L-arginine p-nitroanilide, N-benzoyl-L-arginine ethyl ester and N-acetyl-L-tyrosine ethyl ester, collagen, elastin and fibrin. Some properties of the enzyme were similar to those of neutral SH-dependent proteases described by Hayashi and Lo Spalluto et al.     

Isolation of tyrosine-melanocyte-stimulating hormone release-inhibiting factor 1 from bovine brain tissue

Although Tyr-melanocyte-stimulating hormone release-inhibiting factor 1 (MIF-1) (Tyr-Pro-Leu-Gly-NH2) can exert a number of biological actions in the brain and elsewhere, it has never been isolated from any tissue. Accordingly, we attempted to purify it from acetic acid extracts of bovine brain tissue by gel filtration chromatography and several different high performance liquid chromatographic systems. Peptide content was followed by a specific and sensitive radioimmunoassay with an antibody that was generated against synthetic Tyr-MIF-1. In each of the five applied high performance liquid chromatographic systems, the immunoreactive fractions coincided exactly with the elution time of synthetic Tyr-MIF-1 in the control runnings. The structure of the isolated peptide was identified by microsequence analysis as the tetrapeptide Tyr-Pro-Leu-Gly-NH2 and shown to be biologically active.