Control of glycoprotein synthesis. Purification and characterization of rabbit liver UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I catalyzes an essential first step in the conversion of high mannose to hybrid and complex N-glycans (Schachter, H. (1986) Biochem. Cell Biol. 64, 163-181; Oppenheimer, C.L., and Hill, R.L. (1981) J. Biol. Chem. 256, 799-804), i.e. the addition of GlcNAc to (Man alpha 1-6(Man alpha 1-3)Man alpha 1-6)(Man alpha 1-3)Man beta 1-4GlcNAc-OR to form (Man alpha 1-6(Man alpha 1-3)Man alpha 1-6)(GlcNAc beta 1-2Man alpha 1- 3)Man beta 1-4GlcNAc-OR. The enzyme has been purified from Triton X-100 extracts of rabbit liver by chromatography on CM-Sephadex, Affi-Gel blue, UDP-hexanolamine-Sepharose, and a novel adsorbent in which UDP-GlcNAc is linked to thiopropyl-Sepharose at the 5-position of uracil. The enzyme exists in crude liver extracts in two molecular weight forms separable on Sephadex G-200. The low molecular weight form was purified 64,000-fold with a specific activity of 19.8 mumol/min/mg. The pure enzyme was free of N-acetylglucosaminyltransferase II-V activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single major band of Mr 45,000 and two minor bands of Mr 54,000 and 50,000. All three bands showed retarded elution from an affinity column in which the acceptor substrate for the transferase was covalently linked to Sepharose. Kinetic analysis indicated a largely ordered sequential mechanism with UDP-GlcNAc binding to the enzyme first and UDP leaving last. Studies with synthetic analogues of the substrate Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc showed that an unsubstituted equatorial hydroxyl on carbon 4 of the beta-linked Man residue was essential for enzyme activity.     

Solubilization and purification of a membrane-associated 3,3'',5-tri-iodo-L-thyronine-binding protein from rat erythrocytes.

3,3',5-Tri-iodo-L-thyronine (L-T3) binding sites from rat erythrocyte membranes were solubilized in an active form by using the zwitterionic detergent CHAPS or the anionic detergent lauroylsarcosine. The binding protein was successively purified by Sephadex G-200 and affinity chromatography. The purified material retained its binding activity and exhibited high affinity and specificity compared with those displayed in the original membrane. Yield was about 10% of the starting activity. The specific binding activity was enriched by approx. 100-fold, which represents a purity of only 0.1%. Analysis of the purified preparation on SDS/PAGE showed two major protein bands (Mr 64,000 and Mr 50,000), but these could not represent the binding protein since the purity obtained was low. However, affinity-labelling experiments with N-bromoacetyl-L-[125I]T3 in intact membranes showed that two proteins (also with Mr values of 64,000 and 50,000) bound the hormone specifically, suggesting a co-migration of hormone receptors and contaminants on gel electrophoresis.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

Complete purification and general characterization of FAD synthetase from rat liver

Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) was purified about 10,000-fold from the high-speed supernatant of rat liver by a sequence of ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex (A-50), chromatofocusing, FMN-agarose affinity, and Sephadex G-200. The specific activity of the purified enzyme was 133 units (nanomoles of FAD formed per min at 37 degrees C)/mg of protein. This preparation was free from contaminating FAD pyrophosphatase. The apparent molecular weight was estimated to be 97,000 by gel filtration on Sephadex G-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 53,000. Hence, the enzyme is a dimer of approximately 100,000. The enzyme was found most active at pH 7.1, requires Mg2+, and is essentially irreversible in the direction of FAD formation. Kinetic analysis gave Km values of 9.6 microM for FMN and 53 microM for ATP.     

Dimeric structure of rat skeletal muscle AMP-deaminase subunit

AMP-deaminase from rat skeletal muscle was purified by affinity chromatography on phosphocellulose and gel-filtration on Sephadex G-200. It was established that disulfide bridges and hydrogen bonds were not essential for stability of enzyme oligomeric structure. The dimeric structure of enzyme subunit with Mr 76 kDa (S1) was detected by means of PAGE in the presence of SDS: besides the S1 there were also exhibited two additional bands with Mr 42 (S2) and 33 (S3) kDa. Repeated SDS-PAGE of S1 has revealed the same three protein bands. These results indicate the possibility of dissociation of S1-subunit into two subunits with close Mr values.     

Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties

6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)(-1), was purified 344-fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and KM and Vmax values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G-200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Ki values and inhibition types were estimated by means of Lineweaver-Burk graphs obtained for NADPH and CO2 products.     

Isolation and purification of chloroplastic spinach (Spinacia oleracea) sedoheptulose-1,7-bisphosphatase.

Higher-plant sedoheptulose-1,7-bisphosphatase was isolated and purified over 200-fold from spinach (Spinacia oleracea) chloroplast stromal extracts to apparent electrophoretic homogeneity by DEAE-Fractogel, molecular sieving on Sephadex G-200 and Blue B dye-matrix affinity chromatography. It is a protein of Mr 66,000, made up of two apparently identical subunits (Mr 35,000). The enzyme is activated by reduced thioredoxin fb in the presence of dithiothreitol. Its specificity towards sedoheptulose 1,7-bisphosphate versus fructose 1,6-bisphosphate is high, but not absolute.     

Purification and properties of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes.

The peripheral high-affinity cyclic AMP phosphodiesterase from rat liver plasma membranes was purified to apparent homogeneity. The procedure used involved the initial purification of liver plasma membranes and the solubilization of the enzyme by using a high-ionic-strength medium. This was followed by chromatography of the enzyme on DEAE-cellulose, Affi-Gel Blue, a novel affinity column and Sephadex G-100. A 9500-fold purification of the enzyme with a 24% yield was achieved by this procedure. The purified enzyme was apparently monomeric (Mr 52000) as it exhibited identical molecular weights on analysis by gel filtration, sedimentation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It is suggested that the non-Michaelis kinetics exhibited by the enzyme are due to it obeying a mnemonical mechanism, where it displays Km 0.7 micrometer, Vmax. 9.1 units/mg of protein and Hill coefficient (h) 0.62. Cyclic GMP acts as a poor substrate for the enzyme, with Km 120 micrometer and Vmax. 0.4 unit/mg of protein, and also as an inhibitor of the enzyme, with I50 (concentration giving 50% inhibition) 150 micrometer when assayed at 0.4 micrometer-cyclic AMP. Inhibition by 5'-AMP is unlikely to be of physiological importance, as it is only a weak inhibitor of the enzyme (I50 47 mM assayed at 0.4 micrometer-cyclic AMP).     

The degradation of l-histidine in the rat. The formation of imidazolylpyruvate, imidazolyl-lactate and imidazolylpropionate

1. Soluble and mitochondrial forms of histidine-pyruvate aminotransferase were separated from rat liver preparations by chromatography on DEAE-cellulose. 2. These enzymes were characterized with respect to substrate specificity, substrate affinity, pH optimum, stability and molecular weight by chromatography on Sephadex G-200. 3. Each enzyme has a relatively broad specificity showing significant activity towards l-phenylalanine and l-tyrosine and catalysing transamination with a number of monocarboxylic 2-oxo acids. 2-Oxoglutarate is not a substrate for either enzyme. 4. The molecular weights of the two enzymes, by chromatography on Sephadex G-200, are in the range 130000-150000. 5. The formation in vitro of imidazolyl-lactate from imidazolylpyruvate and NADH was demonstrated by using liver preparations. 6. From a study of imidazolyl-lactate-NAD(+) oxidoreductase activity after electrophoresis of liver preparations on polyacrylamide gel, and from an examination of the activity of l-lactate-NAD(+) oxidoreductase (EC 1.1.1.27) towards imidazolylpyruvate, it is concluded that this latter enzyme is responsible for the formation of imidazolyl-lactate in the liver. 7. Preparations of bacteria obtained from rat faeces form imidazolylpropionate from l-histidine and urocanate without further subculture. The amount of imidazolylpropionate formed is increased under anaerobic conditions and more so in an atmosphere of H(2). It is suggested that the gut flora of the rat contribute largely, if not exclusively, to the formation of imidazolylpropionate normally found in the urine.     

Purification and properties of pig liver kynureninase.

Kynureninase [L-kynurenine hydrolase, EC 3.7.1.3] was purified from pig liver by a procedure including DEAE-cellulose chromatography, hydroxyapatite chromatography, ammonium sulfate fractionation, DEAE-Bio Gel chromatography, Sephacryl S-200 gel filtration, kynurenine-Sepharose affinity chromatography, and Sephadex G-200 gel filtration. The enzyme was found to be homogeneous by the criterion of disc-gel electrophoresis. The enzyme has a molecular weight of about 100,000 and exhibits absorption maxima at 280 and 420 nm. The optimum pH and the isoelectric point of the enzyme are 8.5 and 5.0, respectively. The Michaelis constants were determined to be as follows: L-kynurenine, 7.7 X 10(-4) M; L-3-hydroxykynurenine, 1.3 X 10(-5) M; and pyridoxal 5'-phosphate, 1.8 X 10(-6) M. L-3-Hydroxykynurenine is hydrolyzed more rapidly than L-kynurenine; the liver enzyme can be regarded as a 3-hydroxy-kynureninase.     

Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield. Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography. The method could be scaled up to produce gram quantities of purified enzyme. The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification. The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL. The enzyme had two major forms, of molecular weight 177,000 and 158,000. The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation.     

Crystallization and properties of human liver ornithine aminotransferase

Ornithine aminotransferase [EC 2.6.1.13] was purified and crystallized from human liver by a procedure involving heat treatment, chromatographies on DEAE-cellulose, Octyl-Sepharose CL-4B and Sephadex G-200, and crystallization. The purified enzyme appeared to be homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated as 44,000 by sodium dodecyl sulfate electrophoresis and as 177,000 by sucrose density gradient centrifugation, indicating that the enzyme is tetrameric. Various properties of the enzyme from human liver are similar to those of the enzyme from rat liver, including its molecular weight, pH optimum, Km values for ornithine, alpha-ketoglutarate and pyridoxal phosphate and specificity for amino acceptor from ornithine. The amino acid compositions of the two enzymes also have certain similarities, but the enzymes differ in electrophoretic mobility and antigenicity: the human enzyme moved more slowly to the anode, and on immunodiffusion analysis, the single precipitin lines formed between anti-human enzyme serum or anti-rat liver enzyme and the enzyme from human liver or lymphoblastoid cells and the rat liver enzyme fused with spur formation.     

Purification of a High Molecular Weight Kininogen from Rat Plasma

A high molecular weight kininogen has been isolated from rat plasma and purified. At each preparative step the kininogen concentration and purity were monitored by assay on the perfused isolated rat uterus in terms of bradykinin equivalents formed per mg protein following incubation of the plasma fractions with rodent acid protease for 24 hours at 37 and pH 4.0. Kinin formation by crystalline trypsin and human pancreatic kallikrein also was compared. Citrated rat plasma first was precipitated with 43% ammonium sulfate. The kininogen fractions then were subjected to a series of gel filtration ion exchange chromatographic columns that included G-200 Sephadex, G-200: G-100 Sephadex interconnected columns, DEAE-A50 Sephadex, and hydroxylapatite. The kininogen fractions finally were subjected to preparative polyacrylamide gel electrophoresis, resulting in a final purification of 92.9-fold compared to the initial rat plasma. A single major kininogen protein band and a minor band of protein impurity were obtained on disc gel electrophoresis. Only the pancreatic kallikrein did not form kinin from this purified kininogen. The apparent molecular weight was estimated by SDS polyacrylamide gel technique to be 110,000.     

Isolation and characterization of basic superoxide dismutase consisting of Mr-25,000 subunits in rat liver

1. A basic protein (pI = 9.0) exhibiting superoxide dismutase activity was purified to homogeneity from rat liver by DEAE-cellulose, CM-cellulose and S-hexylglutathione affinity gel chromatography, chromatofocusing and Sephadex G-150 gel filtration. 2. The purified enzyme had specific activity of 4700 units/mg protein. The activity was not affected by 2 mM KCN. Manganese was detected in the enzyme preparation; the content was 0.9 mol/mol subunit. The N-terminal sequence of the first 23 amino acids of the enzyme exhibited a strong homology (except at position 11) with the mature protein of human Mn-superoxide dimutase. It is, therefore, concluded that the purified enzyme is Mn-superoxide dismutase. 3. The N-terminal amino acid sequence showed that about 50% of tyrosine at position 11 was substituted by glutamine, suggesting the existence of microheterogeneity of the superoxide dismutase protein. 4. The superoxide dismutase purified here was found to consist of subunits with an apparent relative molecular mass of 25,000. This larger than the value hitherto reported for rat liver Mn-superoxide dismutase (Mr 2,400); the previous low value is attributed to differences in methods. 5. The enzyme was shown by immuno-blotting to be exclusively localized in the mitochondrial fraction in the liver. The tissue content of Mn-superoxide dismutase is organ-specific, and was the highest in heart. The precursor protein of the Mn-superoxide dismutase was not detectable in the liver cytosolic and mitochondrial fractions as well as in several extrahepatic organs (lung, heart, brain, muscle, kidney and testis), suggesting rapid transport across mitochondrial membranes and processing of the superoxide dismutase protein.     

Purification of rat chondrosarcoma xylosyltransferase

The chondroitin sulfate chain-initiating enzyme, UDP-d-xylose:core protein β-d-xylosyltransferase has been purified over 600-fold from the high-speed supernatant fraction of a rat chondrosarcoma. The purification procedure involved differential centrifugation, gel chromatography on Sephadex G-200, and affinity chromatography on a matrix consisting of core protein bound to Sepharose. The purified enzyme was homogenous by electrophoretic and immunological criteria, had a molecular weight between 95,000 and 100,000 and contained approximately 10% carbohydrate. The K_m value for UDP-xylose was 1 × 10^?5, m and for the core-protein acceptor was 330 mg/liter.     

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.     

Interactions of gold with cytosolic selenium-containing proteins in rat kidney and liver

Rats injected with aurothioglucose (ATG) for 5 days were subsequently injected with [75Se]selenious acid and killed after 3 days. Kidney and liver cytosols were chromatographed on Sephadex G-150. 75Se in kidney was associated with high molecular weight (HMW), 85,000 Mr, 26,000 Mr, and 10,000 Mr proteins and with a nonprotein fraction. The elution profile of liver cytosol was similar to that of kidney, but without a 26,000 Mr protein. ATG injection increased the association of 75Se with all fractions of kidney cytosol except the 85,000 Mr fractions, which contained Se-glutathione peroxidase (SeGSHPx) activity; 75Se in liver was increased only in HMW fractions. Unfractionated kidney cytosolic SeGSHPx activity was decreased 14% by ATG injection, but liver enzyme activity was not changed. However, Sephadex G-150 chromatography showed that total and specific activities, respectively, were decreased 28 and 23% in kidney and 25 and 16% in liver. Au coeluted with HMW and 10,000 Mr 73Se-containing kidney proteins; the latter contained 50% of the Au eluted from the column. DEAE Sephacel chromatography of the 10,000 Mr kidney protein showed that both Au and 75Se were tightly associated with metallothionein-like proteins. This study demonstrates the interaction of Au with rat liver and kidney 75Se-containing proteins.     

Purification of L-glutamate decarboxylase by affinity chromatography

L-Glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from rat brain synaptosomal extract was partially purified by affinity chromatography. On further purification by DEAE-Sephadex A 50 and Sephadex G-200, L-glutamate decarboxylase was purified to greater extent. It was found that a single affinity chromatography by appropriate elution gave a highly purified protein giving a single band of high specific activity on polyacrylamide gradient gel slab electrophoresis with minimal contamination. Substrate specificity of the purified enzyme was modified in the presence of 6-azauracil or phenylalanine resulting in decreased specificity to L-glutamate and increased specificity to L-aspartate.     

Purification and characterization of a rat liver cytosol neutral thiol peptidase that degrades glucagon, insulin, and isolated insulin A and B chains

A thiol peptidase that catalyzes at near neutral pH the hydrolysis of insulin, the isolated A and B chains of insulin, and glucagon was purified from rat liver cytosol by fractionation on Sephadex G-200, Affi-Gel Blue, and Spherogel TSK-G 3000 SW. The purified enzyme showed a single component by chromatography on a Spherogel TSK column and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 180,000 and consists of two subunits having pI's of 5.9 and 6.3. Studies on its substrate specificity showed that the purified enzyme degrades glucagon, insulin, insulin B chain, and insulin A chain, but it does not degrade proinsulin, ACTH, or denatured hemoglobin. Kinetic analyses were performed on three substrates. The Km values were: 34 nM for insulin, 276 nM for insulin B chain, and 3.5 microM for glucagon. The kcat and Vm/Km values were glucagon greater than B chain greater than insulin. Thus, the enzyme has the highest affinity/lowest efficiency for insulin, an intermediate affinity/intermediate efficiency for B chain of insulin and the lowest affinity/highest efficiency for glucagon. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. The enzyme activity was markedly inhibited by N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and was partially inhibited by dithiothreitol, by the chelating agents EDTA and EGTA, and by phenylmethylsulfonyl fluoride (PMSF). Bacitracin inhibited the activity of the enzyme, but the protease inhibitors aprotinin, leupeptin, pepstatin, and phosphoramidon had little or no effect. Reduced glutathione, iodoacetate, and N alpha,p-tosyl-L-lysine chloromethyl ketone (TLCK) also had little or no effect on the enzyme activity.     

Purification and properties of one component of acid phosphatase produced by Aspergillus niger.

One component, the i form, of acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) produced by Aspergillus niger was purified from the mycelial extract. The purified enzyme was homogenous on Sephadex G-200 gel filtration, disc electrophoresis and heat inactivation. The purified enzyme was studied and the following results were obtained: 1. The enzyme catalyzed the hydrolysis of a wide variety of phosphomonoesters, but not that of bis(p-nitrophenyl)phosphate, adenosine 3',5'-cyclic monophosphate, fructose 1,6-diphosphate, adenosine 5'-diphosphate or adenosine 5'-triphosphate. 2. Fluoride, orthophosphate, arsenate, borate, molybdate and (+)-tartrate acted as inhibitors. This enzyme was inactivated by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, and was not affected by p-chloromercuribenzoate, N-acetylimidazole, p-diazobenzenesulfonic acid and tetranitromethane. From these results, tryptophan was estimated to play an important role in the enzyme activity. 3. The apparent molecular weight was 310000 by Sephadex G-200 gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggested that the molecular weight of the subunit was approximately 89000. 4. The purified enzyme contained 29% carbohydrate consisting of glucosamine, mannose and galactose. The amino acid composition of this enzyme was not specific compared with other known acid phosphatases.