Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Amino acid makeup, structural characteristics and substrate specificity of rabbit plasma kininogen]

Highly purified kininogen preparation with the activity of 16-18 int. units per mg was isolated from rabbit blood serum. Its molecular weight was estimated to be 54 000 by gel filtration through Sephadex G-200. Leucine was identified as N-terminal amino acid by the dansylation method. Rabbit kininogen consists of 394 amino acid residues (except tryptophane). Amino acid composition of kininogen is characterized by a high content of dicarbonic amino acids, proline and by a low content of methionine. Kininogen molecule does not contain SH-groups. 13.1-13.5 SH-groups were found in kininogen after the reduction of S-S bonds with beta-mercaptoethanol in the presence of 8 M urea, thus indicating the presence of 6-7 S-S bonds in kininogen molecule. Kininogen group does not occupy C-terminal position in the molecule, because the treatment of the protein with carboxypeptidase B does not change the content of bradykinine in it. Purified kininogen preparation is a substrate for kallikrein from rabbit blood plasma, human saliva and trypsin. Unlike trypsin, kallikreines from human blood plasma and saliva release kinines from kininogen with reduced S-S bonds. Under spontaneous reoxidation of reduced S-S bonds up to 90%, substate properties of kininogen for tripsin recover only by 50%. Rabbit kininogen is similar to beef kininogen II in its molecular weight, amino acid composition and the number of S-S bonds.     

Solubilization of angiotensin I-coverting enzyme from rabbit lung using trypsin treatment.

The solubilization of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was carried out using trypsin treatment. A good recovery of 76% was obtained. The enzyme from solubilized fraction was purified using colums of Sephadex G-200, hydroxyapatite and DEAE-cellulose. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 24.3 units/mg protein for hippurylhistidylleucyl hydroxide and 0.182 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment for 5 h could be divided into two components: (i) an enzyme of molecular weight 300 000 (peak II) and (ii) an enzyme of molecular weight 145 000 (peak III), by Sephadex G-200 gel filtration. The molecular weight of the denatured enzyme was found to be 155 000 by disc gel electrophoresis in the presence of sodium dodecyl sulfate. Km values of peak II and peak III fraction for Hippuryl-His Leu-OH were 2.6 mM.     

Purification, characterization and kinetic properties of the rabbit gastric lipase

Rabbit gastric lipase was purified from an acetonic powder of rabbit stomach fundus. 25 mg of pure rabbit gastric lipase (glycerol ester hydrolase, EC 3.1.1.3) was obtained from 30 rabbit stomachs after ammonium sulfate fractionation, Sephadex G-100 gel filtration and cation exchange (mono S column) using a fast protein liquid chromatography (FPLC) system. The pure enzyme obtained was resistant to acidic pH conditions, and had specific activities of 1200, 850 and 280 U/mg, using, respectively, short- (tributyroylglycerol (TC4)), medium- (trioctanoyl- to tridecanoylglycerol (TC8-TC10)) and long-chain (soybean oil) triacylglycerols. The amino-acid composition was determined, and the first 30 N-terminal amino-acid residues were sequenced. Interfacial denaturation and catalytic properties on triacylglycerol emulsions were studied. Rabbit gastric lipase turned out to be structurally and kinetically very similar to human gastric lipase.     

Characterization of two acid proteinases found in rabbit skin homografts.

Two types of acid proteinase activity found in rabbit skin homografts were characterized by studying the effect of temperature, pH and polyacrylamide-gel electrophoresis. Their chromatographic behaviour was characterized on DEAE-cellulose, Sephadex G-75, G-100 and G-200, and their molecular weights were estimated by gel filtration. One of the acid proteinases in the homograft resembled cathepsin D (EC 3.4.23.5) of normal skin. The other acid proteinase differed from cathepsin D with respect to heat inactivation, pH optimum and molecular weight; it was not inactivated on heating at 60 degrees C for 60 min, its pH optimum was 2.5 and its molecular weight measured by Sephadex G-100 chromatography was 100 000. In all these respects, the heat-stable proteinase resembles cathepsin E (EC 3.4.23.5) of rabbit polymorphonuclear leucocytes.     

Purification of N-hydroxy-2-acetylaminofluorene reductase from rabbit liver cytosol

N-Hydroxy-2-acetylaminofluorene reductase was purified from rabbit liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose, Sephadex G-200 and hydroxylapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 34,000 by the electrophoresis and by gel filtration on Sephadex G-200. The enzyme required cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, NADPH or NADH as an electron donor. The enzyme activity was inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide, cupric sulfate or disulfiram, but little by oxygen.     

Isolation and characterization of rabbit H of the alternative complement pathway

Rabbit factor H, a control protein of the alternative complement pathway, was isolated from rabbit serum by polyethylene glycol precipitation, DEAE-Sephacel chromatography, and gel chromatography on Sephadex G200. The protein migrated as a single-chain polypeptide with a molecular weight of 160,000 on sodium dodecyl sulfate-gel electrophoresis with Laemmli's buffer system, but hardly migrated into the gel with Fairbanks' buffer system. Physical and chemical properties of rabbit H were similar to those of human H, except that fragments produced by limited tryptic digestion from rabbit H had different molecular sizes from those produced from human H. Significant species-specificity was observed in the functional activity of factor H; activation of the alternative complement pathway was inhibited more efficiently with homologous H than with heterologous H. In contrast, factor H inhibited the hemolysis of homologous erythrocytes less than that of heterologous erythrocytes.     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

Characterization of Virus- and Endotoxin-induced Interferons Obtained from the Serum and Urine of Rabbits

Interferons induced in the rabbit by Newcastle disease virus or by endotoxin have been further characterized as to their physicochemical stability and molecular size by Sephadex G-100 gel filtration. Endotoxin-induced interferon obtained from serum was more labile than virus-induced interferon. Both endotoxin and virus induced interferons obtained from serum contained two peaks: a minor high molecular weight (>100,000) peak and a major lower molecular weight peak. The molecular weight of the major peak induced by endotoxin was 54,000, and that induced by Newcastle disease virus was 46,000. The gel filtration pattern of interferon recovered from the urine of animals inoculated with virus reflected faithfully the pattern found in serum except that there was proportionately less of the high molecular weight peak. However, the urine interferon from endotoxin-inoculated animals contained only one broad peak with a molecular weight of 35,000. This was not the peak fraction present in the serum of such animals. It is postulated that this may represent the basic unit of endotoxin-induced interferon, and that the serum components are either polymers or conjugates of this basic unit.     

Isolation and some properties of a new fiber-forming protein from Tetrahymena pyriformis

Isolation and characterization of a fibrous protein component which might be associated with contractile ring of a dividing Tetrahymena cell were attempted by making use of coprecipitation of the protein with rabbit skeletal muscle myosin. The protein was purified to homogeneity by ammonium sulfate fractionation between 40--70% saturation and column chromatography of Sephadex G-200, starting from KCl-extract of Tetrahymena acetone powder. Its molecular weight was calculated to be 38,000, based on the electrophoretic mobility in sodium dodecyl sulfate (SDS)-polyacrylamide gel, whereas molecular weight of its native state was determined to be 140,000 by gel filtration on Sephadex G-200, and its sedimentation coefficient was about 9S as estimated by sucrose density gradient centrifugation. The latter was a particle of 7.7 nm in diameter under an electron microscope and supposed to be a tetramer of the 38,000-dalton protein. The protein is considered to be a new, unique protein, since it is definitely different from the ubiquitous non-muscle actin in molecular weight, polymerizability in KCl solution and amino acid composition, and it also different from tropomyosin and tubulin in immunological characteristics and amino acid composition.     

Isolation and characterization of a sulfated glycoprotein from rabbit uterus.

Crude glycoproteins were extracted with 0.15 M NaCl from the pooled endometrial scrapings of rabbit uteri after treatment with estrogen. The crude glycoproteins were fractionated with ammonium sulfate, followed by DEAE-cellulose column chromatography, treatment with CM-Sephadex C-25 and gel filtration on Sephadex G-200. Subsequently, purification of an acidic glycoprotein was carried out by gel filtration on Sephadex G-200 and then Sepharose 4B. The results of electrophoresis and enzymatic digestion, together with analytical data and the infrared spectrum indicated that the acidic glycoprotein was a sulfated glycoprotein.     

Purification and Partial Characterization of Hypocalcemic Factor from Human Saliva

This study reports the isolation and partial purification of a polypeptide from human saliva which causes a significant serum calcium lowering when administered to mice. Purification was achieved by preparative electrophoresis, dialysis, two gel filtration steps on Sephadex G-150, and ion exchange chromatography on DEAE-cellulose. Homogeneity was determined by poly-acrylamide electrophoresis. Blood sampling was carried out by puncture of the orbital venous plexus and serum analyzed for calcium. The most active preparations lower serum calcium from 10–27% of initial value, producing tetany and convulsions in some cases. The molecular weight of this polypeptide was estimated to be 4, 260 by the use of a calibrated Sephadex G-75 column. This is a much smaller molecular weight than that expected from its initial exclusion from Sephadex G-150, and suggests that this hypocalcemic factor is associated with larger molecules through most of the purification procedure up to and including DEAE-cellulose chromatography. A second gel filtration on Sephadex G-150 separates two minor salivary protein contaminants (IgA and IgG immunoglobulin) in the excluded fraction from the smaller, hypocalcemically active polypeptide.

No hypocalcemia activity could be detected or isolated in a preliminary investigation on the saliva of a dysgammaglobuli-nemic (IgA deficient) patient.

The hypocalcemia induced does not differ significantly from that observed after administration of calcitonin to mice in that: 2) minimum values are reached in 1.5–2 hours and return to normal in 5–6 hours, b) magnitude of hypocalcemia response is dose dependent. The salivary hypocalcemia factor isolated in this study has the properties of a protein, in that its activity is destroyed by the proteolytic enzyme trypsin, it yields amino acids upon acid hydrolysis and it behaves on electrophoresis, gel filtration and ion exchange chromatography as a typical protein.     

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.     

Purification of multiple forms of adenosine deaminase from rabbit intestine.

Two forms of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), differing in molecular size, have been purified and obtained in homogeneous form from rabbit intestine. The purification procedures involved extraction with acetate buffer, pH 5.5, precipitation and fractional reextraction with (NH4)2SO4, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-75 and Sephadex G-200. Gel filtrations analysis gave molecular weight estimates of 265 000 and 32 000 for the large and small deaminases respectively. The two enzymes forms had similar pH optima and pH stability ranges.     

Highly acidic proteins from human brain: purification and properties of Glu-50 protein

Three extremely acidic proteins were isolated from human brain and purified to apparent homogeneity. One of them, Glu-50 protein, contained much glutamic acid (about 50% of the total amino acids). Its purification involved ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and gel filtration on Sephadex G-100 and G-75. Its molecular weight was determined to be 11,000 by SDS polyacrylamide gel electrophoresis and 34,000-36,000 by gel filtration on Sephadex G-75, suggesting that it consists of three identical polypeptide chains. Its isoelectric point was pH 3.9. Its N-terminal amino acid sequence was NH2-Asp-Glu-Pro-Pro-Asp-Glu and its C-terminal amino acid was Lys. It contained no detectable carbohydrate.     

Purification of hyaluronidase from human placenta.

Hyaluronidase [EC 3.2.1.35] was isolated from human placenta and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150. Its isoelectric point was at pH 5.2 and the molecular weight was 7 X 10(4) based on Sephadex G-200 gel filtration data. This enzyme was very stable at temperatures below 30 degree, but was almost completely inactivated at 60degree within 30 min. Its optimum pH was 3.9, a characteristic property of a lysosomal hyaluronidase. The Michaelis constant was 1.18 x 10(-1) mg per ml with purified hyaluronate. This enzyme depolymerized hyaluronate, chondroitin, chondroitin 4-sulfate and 6-sulfate, and the end product formed from hyaluronate was tetrasaccharide. Its biological diffusing activity was statistically significant on intracutaneous injection of 1.86 mU of the hyaluronidase into the back skine of a rabbit.     

Isolation,purification and partial characterisation of prealbumin from cerebrospinal fluid

Prealbumin from human cerebrospinal fluid was purified using a combination of ammonium sulphate precipitation, phenol precipitation, Polyacrylamide disc gel electrophoresis and gel filtration on Sephadex G-100. The homogeneity of the purified protein was established by Polyacrylamide gel electrophoresis and Immunoelectrophoresis. On the basis of its molecular weight (55,000), amino acid composition, electrophoretic mobility and immunological cross-reactivity, the prealbumin from cerebrospinal fluid showed complete identity with serum prealbumin. The cerebrospinal fluid prealbumin levels in various neurological disorders may have a diagnostic significance. Part of the Ph. D. thesis submitted by the first author.     

Purification and properties of a heat-stable protein inhibitor of phosphoprotein phosphatase from rabbit liver.

A heat-stable protein inhibitor of phosphoprotein phosphatase has been purified to homogeneity from rabbit liver extract by heating to 95 degrees followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The purified inhibitor showed a single band when examined by gel electrophoresis S20, w and Stokes radius values were 1.45 and 25.5, respectively. Using these two values, the molecular weight and frictional ratio was calculated to be 15,500 and 3.40, respectively. The molecular weight determined by sodium dodecyl sulfate-gel electrophoresis was found to be 14,200. The inhibition of phosphoprotein phosphatase was linear up to 40% inhibition with respect to inhibitor was constant with time of incubation for at least 30 min. The optimum pH for the inhibition was between 6.8 and 7.6. A kinetic analysis of the effect of the inhibitor on the dephosphorylation of [32P]phosphorylase a by rabbit liver phosphoprotein phosphatase indicated a noncompetitive inhibition with respect to phosphorylase a. Purified liver inhibitor inhibited the phosphoprotein phosphatase activity in all rat tissues examined. Utilizing purified rabbit liver phosphoprotein phosphatase, the presence of inhibitor activity was also demonstrated in all rat tissues tested.     

Characterization of bovine plasma retinol-binding protein and evidence for lack of binding between it and other bovine plasma proteins.

Bovine retinol-retinol-binding protein (RBP) was isolated from serum as a free, uncomplexed protein under experimental conditions in which human, rabbit, and chicken retinol-RBP are present as tight complexes with prealbumin (thyroxine-binding protein). Purified bovine retinol-RBP formed tight complexes with purified human and chicken prealbumin in physiological ionic strength buffers as judged by gel filtration chromatography, hyperchromic effect on the absorption spectrum of retinol-RBP, and changes in the circular dichroism spectrum. Addition of purified human prealbumin to whole bovine serum shifted the elution position of the specific retinol-RBP fluorescence from a gel filtration column, indicating complex formation in the whole bovine serum. It was concluded from this series of experiments that bovine serum lacks a protein with the binding properties of prealbumin and that bovine retinol-RBP has the normal potential binding to human, chicken, and presumably other prealbumins. Bovine retinol-RBP has a molecular weight, amino acid composition, absorption, and fluorescence spectra which are indistinguishable from that of human retinol-RBP, although the magnitude of the optical rotatory strength of the induced circular dichroism signal at 330 nm was 50% larger in the bovine than in the human material (1.65 and 1.1 Debye-Bohr magnetons, respectively). About 12 liters of bovine and human urine were concentrated by pressure dialysis and a search was made for retinol-RBP using gel filtration and ion exchange chromatography. No retinol-RBP was found in either of these species. This suggested that if, indeed, bovine retinol-RBP is filtered through the kidney's glomeruli due to small molecular size (molecular weight 21,000), there are efficient mechanisms of tubular reabsorption.