Rat alpha 1-microglobulin. Purification from urine and synthesis by hepatocyte monolayers

Rat alpha 1-microglobulin was isolated from the urine of rats treated with sodium chromate, and was purified by the use of gel chromatography, affinity chromatography on concanavalin-A-Sepharose and ion-exchange chromatography. The protein was heterogeneous in charge, had a tendency to form dimers, and was associated with a brown-coloured chromophore. The size of the protein (25 kDa) was similar to guinea pig alpha 1-microglobulin but smaller than the human protein, when measured with sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunological cross-reaction with human and guinea pig alpha 1-microglobulin was demonstrated. The concentration of alpha 1-microglobulin in rat serum was 16.4 mg/l (SD = 8.5 mg/l, n = 13) and rat serum alpha 1-microglobulin was eluted from a gel chromatography column at two different positions corresponding to monomeric alpha 1-microglobulin and IgA. The latter alpha 1-microglobulin activity could be absorbed by anti-IgA serum. Rat alpha 1-microglobulin and albumin were continuously released into the medium of rat hepatocyte monolayers, and alpha 1-microglobulin was isolated from the medium by the use of immunoprecipitation with anti-(alpha 1-microglobulin). Tritiated leucine, added to the medium, was incorporated into the protein, suggesting a de novo synthesis of alpha 1-microglobulin by the hepatocytes. The size of hepatic alpha 1-microglobulin was similar to that of purified urinary rat alpha 1-microglobulin, when determined with sodium dodecyl sulfate/polyacrylamide gel electrophoresis.     

Structural and immunochemical characterization of human urine arylsulfatase A purified by affinity chromatography

Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was isolated from an ammonium sulfate precipitate of urinary proteins using two different affinity chromatography methods. One method involved the use of concanavalin A-Sepharose affinity chromatography at an early stage of purification, followed by preparative polyacrylamide gel electrophoresis. The other procedure employed arylsulfatase subunit affinity chromatography as the main step and resulted in a remarkably efficient purification. The enzyme had a specific activity of 63 U/mg. The final preparation of arylsulfatase A was homogeneous on the basis of polyacrylamide gel electrophoresis at pH 7.5, and by immunochemical analysis. However, when an enzyme sample obtained by either method of purification was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing or non-reducing conditions, peptide subunits, of 63.5 and 54.5 kDa, were observed. Immunological tests with 125I-labeled enzyme established the presence of a common protein component in both of the electrophoretically separable peptide subunits of human urine arylsulfatase. The amino acid analysis of homogeneous human urine arylsulfatase A showed only a few differences between it and the human liver enzyme. However, immunological cross-reactivity studies using rabbit anti-human urine arylsulfatase revealed immunological difference between the human urine and liver arylsulfatase A enzymes.     

Purification and NH2-terminal amino acid sequence of human thymidylate synthase in an overproducing transformant of mouse FM3A cells

Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows: Pro-Val-Ala-Gly-Ser-Glu-Leu-Pro-Arg-Arg-Pro-Leu-Pro-Pro-Ala-Ala-Gln-Glu- Arg-Asp -Ala-Glu-Pro-Arg-.     

Purification and properties of oxytocinase (cystine amino-peptidase) from monkey placenta.

Oxytocinase (cystyl-aminopeptidase) [EC 3.4.11.3] was isolated from monkey placenta in a purified form by a six-step prodedure comprising extraction from monkey placenta homogenate, ammonium sulfate fractionation, repeated chromatography on hydroxylapatite, chromatography on a column of DEAE-cellulose and gel filtration on a column of Sephadex G-200. The purified enzyme showed a single band on polyacrylamide disc electrophoresis. Oxytocin was inactivated by this enzyme preparation. The enzyme hydrolyzed several aminoacyl-beta-naphthylamides. A terminal amino group was required for enzyme activity. The molecular weight of the purified enzyme was estimated to be 87,000 by gel filtration and 83,000 by sodium dodecyl sulfate gel electrophoresis. Other properties of the enzyme, the effects of metal ions and various chemical reagents on the enzyme activity, the pH optimum, and Km values for a number of aminoacyl-beta-naphthylamides were also examined.     

Heterogeneity of hypoxanthine guanine phosphoribosyl transferase from human erythrocytes.

Hypoxanthine guanine phosphoribosyl transferase (E.C.: 2.4.2.8) has been purified 4000- to 4500-fold from normal human erythrocytes by three different schemes of protein fractionation. In one scheme, the enzyme was separated by preparative polyacrylamide gel electrophoresis in an LKB Uniphor system and purified by affinity column chromatography employing Sepharose/phosphoribosyl/pyrophosphate. In the second, the enzyme was isolated by isotachophoresis in the presence of Amphiline carrier ampholytes employing a Tris/phosphate/β-alanine ion system. The enzyme was then purified by isotachophoresis in the presence of carrier ampholytes using a Tris/acetate/glycine ion system. The hypoxanthine guanine phosphoribosyl transferase purified by affinity chromatography and isotachophoresis consisted, on immunoelectrophoresis, mainly of one component and had less than 5% impurities. When subjected to analytical polyacrylamide gel electrophoresis, such preparations were resolved into four isoenzymes. In the third scheme, the enzyme was isolated by isoelectric focusing. In this system, the enzyme was also resolved into four isoenzymes. Their isoelectric points were: 5.47, 5.63, 5.74, and 5.84. When subjected to analytical polyacrylamide gel electrophoresis each isoenzyme migrated at a different rate. In sodium dodecyl sulfate gel electrophoresis each isoenzyme yielded one major and one minor band. Protein appearing in the major and minor bands migrated at rates consistent with a molecular size of 33,500 and 26,500, respectively.     

Porcine A blood group-specific N-acetylgalactosaminyltransferase. I. Purification from porcine submaxillary glands.

The membrane-bound N-acetylgalactosaminyltransferase from porcine submaxillary glands which provides A blood group specificity to mucin has been purified 38,000-fold by affinity chromatography on UDP-hesanolamine-agarose in aqueous Triton X-100. Design of a suitable purification procedure was developed by assessing the strength of interaction between enzyme and affinity adsorbent using batch desorption. The pure transferase has an apparent molecular weight of 100,000 as judged by zonal centrifugation and by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of a reducing agent. The reduced and carboxymethylated protein has an apparent molecular weight of 46,000 and 57,000 as judged by sedimentation equilibrium and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, suggesting that the native enzyme contains two subunits. It is a glycoprotein with a specific activity of 30 micronmol/min/mg of enzyme, which is 55,000 times that reported for the same enzyme isolated from human serum.     

Biosynthesis of monoterpenes. Partial purfication and characterization of a bicyclic monoterpenol dehydrogenase from sage (Salvia officinalis)

Galactose 1-phosphate uridylyltransferase (uridine diphosphoglucose: α-d-galactose 1-phosphate uridylyltransferase, EC 2.7.7.12) was isolated from human red cells by DEAE-cellulose and hydroxylapatite chromatography. The enzyme consists. of two similar subunits of molecular weight 44,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the enzyme was found to be 67,000 by Sephadex G-200 chromatography and 88,000 by ultracentrifugation studies in sucrose density gradients. The specific activity of the purified enzyme was about 40 μmoles per min per mg of protein.     

Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.     

Purification and characterization of human plasma lecithin:cholesterol acyltransferase.

A highly purified (approximately 12 000-fold) homogeneous preparation of human plasma lecithin:cholesterol acyltransferase (LCAT) with 16% yield was obtained by a combination of density ultracentrifugation, high density lipoprotein affinity column chromatography, hydroxylapatite chromatography, and finally chromatography on anti-apolipoprotein D immunoglobulin-Sepharose columns to remove apolipoprotein D. This enzyme preparation was homogeneous by the following criteria: a single band by polyacrylamide gel electrophoresis in 8 M urea; a single band on sodium dodecyl sulfate gel electrophoresis with an apparent molecular weight of 68 000 +/- 1600; a single protein peak with a molecular weight of 70 000 on a calibrated Sephadex G-100 column. Its amino acid composition was different from human serum albumin and all other apoproteins isolated from lipoprotein fractions.     

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.     

Purification and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV

Dipeptidyl aminopeptidase IV (EC 3.4.14.-) was solubilized from a particulate membrane fraction of rat intestinal mucosa with Triton X-100. The solubilized enzyme was purified to homogeneity following ammonium sulfate fractionation, chromatography on DEAE-Sepharose and hydroxyapatite, gel filtration and preparative polyacrylamide gel electrophoresis. The final enzyme preparation had a specific activity of 55 units/mg protein representing a 1373 fold purification over the starting material. Purity was judged by polyacrylamide gel electrophoresis and double immunodiffusion. The molecular weight of the native undenatured enzyme was estimated to be 230000 by gel filtration and polyacrylamide gel electrophoresis. Electrophoresis under denaturing conditions (sodium dodecyl sulfate) indicated that the protein consists of two identical 98 kDa subunits. Dipeptidyl aminopeptidase IV is a glycoprotein containing approx. 8% carbohydrate by weight. A detailed analysis of the individual sugar components demonstrated that fucose, galactose, glucose, mannose, sialic acid and hexosamine sugars were present. The nature of the constituent asparagine linked oligosaccharide side chains was further examined following cleavage from the peptide backbone by hydrazinolysis. Following high voltage paper electrophoresis approx. 80% of the isolated oligosaccharide was found with the neutral fraction while the remaining 20% consisted of a single acidic component. Gel filtration of the neutral oligosaccharide fraction indicated that it contains approx. 19 sugar residues.     

Purification and immunological characterization of DNA polymerase-alpha from human acute lymphoblastic leukemia cells

DNA polymerase-alpha was purified from the cytosol of blast cells of a patient with acute lymphoblastic leukemia by ammonium sulfate fractionation and successive column chromatographies. The purified enzyme had a specific activity of 2943 units/mg protein with activated calf thymus DNA as a template. The enzyme sediments under high-salt conditions as a homogeneous band at 7.2 S and free from other DNA polymerases (beta, gamma) and terminal deoxynucleotidyl transferase activity. The native molecular weight of the enzyme from gel filtration and glycerol gradient centrifugation was found to be 175 000. The values of Stokes radius (53 A), diffusion coefficient (4.05 x 10(-7) cm2/s) and frictional ratio (1.42) determined by gel filtration suggest that the native enzyme is compact and globular. Antibodies to DNA polymerase-alpha were raised in rabbits. These antibodies, partially purified by 50% ammonium sulfate saturation and Sephadex G-200 chromatography, gave one precipitin band on immunodiffusion and inactivate DNA polymerase-alpha-. This antibody preparation also inhibited, in vitro, the activity of DNA polymerase-alpha from calf thymus, phytohemagglutinin-stimulated normal human lymphocytes, as well as that from other leukemic cells. Thus, DNA polymerase-alpha from calf thymus and human leukemic cells resemble each other in antibody specificity.     

Purification and chemical studies on rat urinary kallikrein.

A highly purified kallikrein was obtained from rat urine by chromatography on DE-32 cellulose, affinity chromatography on Bio-gel P-200-Aprotinin and gel filtration over Sephadex G-100 coarse and superfine. A molecular weight of 32,000 by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis was estimated. The aminoacid composition and the esterase activity of the purified material were determined. Biological characterization of the purified kallikrein was tested by liberation of a kinin from rat plasma kininogen, by direct action on the isolated rat uterus and by the lowering of rat arterial pressure after intravenous injection of the enzyme. The preparation of insoluble derivative of Aprotinin is described herein. The polymer used as insoluble support (Bio-gel P-200) was before changed to its corresponding azide, which reacts with Aprotinin; the product maintained the binding property of the Aprotinin with urinary kallikrein.     

Purification and properties of streptococcal nicotinamide adenine dinucleotide glycohydrolase.

Highly purified streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) was obtained by utilizing disodium tetrathionate to protect the enzyme by blocking the sulfhydryl groups of streptococcal proteinase. This was followed by two-step ion-exchange chromatography. The pure enzyme, demonstrated as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, had a specific activity of 11,200 NADase units per mg of protein and was devoid of hemolytic activity. NADase had a molecular weight of about 55,000 as determined by gel filtration, by summation of amino acid residues, and by sodium dodecyl sulfate/gel electrophoresis. The purified enzyme had optimal activity at pH 7.3 and at 40 C and a calculated Km of 5.1 times 10- minus 4 mM. It was inhibited by alpha-iodoacetamide.     

In vitro effects of some drugs on catalase purified from human skin

Catalase enzyme (H202: oxidoreductase; E.C. 1.11.1.6) was purified from human skin homogenate using ammonium sulfate precipitation and DEAE-Sephadex A50 ion exchange chromatography at 4 degrees C and some characteristics of the enzyme were investigated. The human skin enzyme, having a specific activity of 1354.5 EU/mg proteins was purified with a yield of 43.13% and 1110-fold. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band for the enzyme. Inhibition by piroxicam, ketoprofen, diclofenac sodium, sulfamethoxazole and nidazole occurred with I50 values of 0.414, 1.29, 1.8, 3.83, and 8.64 mM, respectively.     

Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.     

Characterization of lysosomal acid lipase purified from rabbit liver

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.     

Purification and properties of 3-ketovalidoxylamine A C-N lyase from Flavobacterium saccharophilum

3-Ketovalidoxylamine A C-N lyase was purified about 900-fold from the cell-free extract of Flavobacterium saccharophilum by ammonium sulfate fractionation, column chromatography on CM cellulose and gel filtration on Sephacryl S-200. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 36,000 by gel filtration on Sephacryl S-200 and by SDS polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The optimum pH was found at 9.0. The enzyme activity was inhibited by EDTA or ethyleneglycol bis(beta-aminoethylether)-N,N'-tetraacetic acid and the inhibition was reversed by Ca2+ ion. The enzyme was able to eliminate p-nitroaniline or p-nitrophenol from p-nitrophenyl-3-ketovalidamine (IV) or p-nitrophenyl-alpha-D-3-ketoglucoside (VI), but not from p-nitrophenyl-1-epi-3-ketovalidamine or p-nitrophenyl-beta-D-3-ketoglucoside. Apparent Km values for IV and VI were 0.24 mM and 0.5 mM, respectively.     

Purification and characterization of a tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp.

Tetrachloro-p-hydroquinone (TeCH) is the first intermediate in pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723. We previously purified a PCP hydroxylase that oxidized PCP to TeCH. Subsequently, we identified the reductive dehalogenation of TeCH to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone in a cell extract with the reduced form of glutathione as the reducing agent under anaerobic conditions. Here we report the purification of a TeCH reductive dehalogenase that reductively dehalogenated TeCH to trichlorohydroquinone and then to dichlorohydroquinone. The enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, and phenyl-agarose, anion-exchange, and gel filtration column chromatographies. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the protein has a molecular weight of about 30,000; nondenaturing polyacrylamide gel electrophoresis analysis suggests that the native enzyme exists as a dimer. The enzyme used glutathione but not NADPH, NADH, dithiothreitol, or ascorbic acid as the reducing agent. The optimal pH was close to neutral.     

Purification and characterization of a hyaluronidase associated with a temperate bacteriophage of group A, type 49 streptococci.

Urea treatment of a temperate bacteriophage from a type 49 strain of group A streptococcus (Streptococcus pyogenes) followed by ammonium sulfate fractionation, ion exchange, and affinity chromatography of solubilized proteins provided for the recovery (12%) and purification (44-fold) of the phage-associated hyaluronidase. The molecular weight of the homogeneous, purified enzyme was estimated to be 71,000 by polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulfate) and 75,000 by gel filtration with Sephacryl S-200. The enzyme has a pH optimum of 5.5, a Vmax of 0.1 absorbance unit/min per microgram of protein, and a Km of 4.8 X 10(-2) mg/ml with umbilical cord hyaluronic acid as substrate. Of the cations tested, calcium and magnesium were the only effectors of the enzyme. The enzyme is a glycoprotein (7.25% carbohydrate) containing glucose, galactose, and glucosamine. Analysis of the amino acid composition revealed a predominance of acidic amino acids and a relatively high content of cysteine. The partial specific volume, estimated from the amino acid and sugar analyses, was 0.725 cm3/g.