Purification and characterization of thiamine triphosphatase from bovine brain.

Soluble thiamine triphosphatase (EC 3.6.1.28) of bovine brain has been purified 68,000-fold to an electrophoretically homogeneous state with an overall recovery of 5.5% by hydrophobic chromatography on Toyopearl HW-60, Sephadex G-75 gel filtration, DEAE-Toyopearl 650M chromatography and Blue Sepharose CL-4B chromatography. The enzyme has an absolute specificity among thiamine and nucleoside phosphate esters for thiamine triphosphate and shows no nonspecific phosphatase activities. Thiamine triphosphatase is composed of a single polypeptide chain with molecular mass of 33,900 kDa as estimated by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 8.7 and is dependent on divalent metal ions. Mg2+ has been found to be the most effective among cations tested. A study of the reaction kinetics over a wide range of thiamine triphosphate concentrations has revealed a biphasic saturation curve being described by higher-degree rational polynomials.     

Purification and characterization of alkaline phosphatase from rat kidney.

Alkaline phosphatase [EC 3.1.3.1.] was purified about 250-fold from rat kidney, and its enzymological properties were studied. Kidney homogenate was extracted with n-butanol, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The peak from the DEAE-cellulose column was subjected to isoelectric focusing, and the alkaline phosphatase activity was separated into two peaks. The molecular weights of alkaline phosphatase in these peaks were 4.8.X10(4) and 1.0X10(5), as determined by SDS-polyacrylamide gel electrophoresis. Anti-serum against alkaline phosphatase from rat kidney was prepared, and was shown to neutralize the activity from kidney, liver or bone, but not that from intestine.     

Isolation and various properties of thiamine-binding protein from synaptosomes in the rat brain

A thiamine-binding protein (ThBP) with a specific activity of 8.21 nmoles/mg protein was isolated from rat brain synaptosomes by affinity chromatography and gel filtration on Sephadex G-200. The protein was purified 746-fold with a 40.5% yield. ThBP was homogeneous during sodium dodecyl sulfate gel electrophoresis; its molecular mass was determined by gel filtration on Sephadex G-200 and by sodium dodecyl sulfate gel electrophoresis and was equal to 107 and 103 kD, respectively. The pH optimum for the binding is 8.35. When the ability of ThBP to bind thiamine phosphates was tested, the latter decreased in the following order: thiamine monophosphate greater than thiamine triphosphate greater than greater than thiamine diphosphate.     

Isolation and characterization of 5'-nucleotidase inhibitor from rat liver.

5'-Nucleotidase (EC 3.1.3.5) is widely distributed in nature. However, it could not be detected in rat liver, because of the presence of specific inhibitors. Such inhibitors were also found in other tissues of rat, but at lower concentrations than that in the liver. The inhibitor activity was enriched in the membrane fraction and was also present in the cytosol fraction. It was sensitive to treatment with 6M urea and trypsin, while heating in a boiling water bath for 10 min or dialysis reduced the activity only slightly. Gel filtration or Sephadex G-50 yielded two types of inhibitors. Inhibitor I inhibited brain 5'-nucleotidase while inhibitor II inhibited both the brain and liver enzymes. Inhibitor II on further purification on CM Sephadex C-25 yielded five fractions with inhibitor activity of which inhibitor IIC was electrophoretically homogeneous. It had a molecular weight of 8500 by SDS gel electrophoresis, was rich in basic amino acids and had a high proportion of beta structure. Interaction of the inhibitor with 5'-nucleotidase brought about modifications in the secondary structure of the inhibitor as seen from the circular dichroism spectrum.     

Purification and characterization of alkaline phosphatase in cultured rat liver cells.

Alkaline phosphatase has been purified from cultured rat liver cells by butanol extraction, column chromatography on DEAE-cellulose and on Sephadex G-200, and preparative polyacrylamide gel electrophoresis. By electrophoresis on polyacrylamide, the purified enzyme was resolved into two active forms. Both forms have similar molecular weights of around 200,000. The subunit size was found to be 50,000 by SDS-polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase purified from cultured rat liver cells has a tetrameric structure. The optimum pH was found to be approximately 10.4, using p-nitrophenylphosphate as a substrate in a carbonate buffer system. The apparent Km was estimated to be 2.4 mM, using p-nitrophenylphosphate in carbonate buffer, pH 10.4.     

Induction of rat hepatic alkaline phosphatase and its appearance in serum: electrophoretic characterization of liver-membranous and serum-soluble forms

Simultaneous bile duct ligation and colchicine injection (2 mg/kg body weight) in rats caused a remarkable induction of alkaline phosphatase in the liver. Concomitantly, a marked elevation of the enzyme activity occurred in the serum, and three activity peaks (peaks I, II, and III) were separated by Sephadex G-200 gel filtration. By several criteria for alkaline phosphatase isoenzymes it was determined that the liver-derived enzyme was distributed in peak I (30% of total serum activity) as a vesicle-bound form and in peak II (65%) as a soluble form, while the intestinal enzyme was contained in peak III (5%). The serum alkaline phosphatase in peaks I and II was compared with the liver enzyme extracted from plasma membrane with n-butanol. Under non-reducing conditions, the soluble form of peak II showed an electrophoretic mobility different from that of the liver enzyme; in the presence of sodium dodecyl sulfate the serum-soluble form migrated a little more slowly than the liver one, while in the presence of Triton X-100 the former migrated much faster than the latter. The sedimentable fraction of peak I was found to contain two forms corresponding to the serum-soluble and liver-membranous forms. Neuraminidase treatment of these two forms reduced their mobilities but did not abolish the relative difference in their mobilities on gel electrophoresis in the presence of either Triton X-100 or sodium dodecyl sulfate. Under reducing conditions, however, each form (which was dissociated into single subunits) migrated with an identical mobility on sodium dodecyl sulfate gel electrophoresis. These results suggest that the hepatic alkaline phosphatase exists as conformationally different forms in the serum and the liver membrane (even solubilized), but the difference is no longer preserved after their denaturation into subunits.     

Purification and characterization of alkaline phosphatase from cultured rat ascites hepatoma cells.

Alkaline phosphatase of cultured rat ascites hepatoma cells has been purified by butanol extraction, DEAE-cellulose column chromatography, gel filtration through Sephadex G-200, concanavalin A-Sepharose affinity chromatography, and polyacrylamide gel electrophoresis. Affinity chromatography confirmed the glycoprotein nature of alkaline phosphatase from cultured rat ascites hepatoma cells. Electrophoresis on polyacrylamide gels of various concentrations indicated a molecular weight of 290,000. The molecular weight of the subunit was estimated to be 72,000 by SDS-polyacrylamide gel electrophoresis. These findings suggest that alkaline phosphatase of cultured rat ascites hepatoma cells is a tetramer with a subunit molecular weight of 72,000.     

Purification and properties of acid phosphatase from cultured tobacco cells

One component of acid phosphatase was purified from cultured tobacco cells. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The enzyme possesses high activity toward nucleoside di- and triphosphate, much less activity toward nucleoside monophosphates and sugar esters. The MWs of the phosphatase determined by Sephadex G-100 gel filtration and dodecyl sulfate gel electrophoresis were 74000 and 76000, respectively. The phosphatase showed high affinity for concanavalin A-Sepharose and single superimposed bands of protein and carbohydrate on gel electrophoresis, suggesting that it is a glycoprotein.     

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.     

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.     

Complete purification of choline kinase from rat kidney and preparation of rabbit antibody against rat kidney choline kinase

Choline kinase was purified from rat kidney to apparent homogeneity with respect to both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme showed a minimum molecular weight of 42,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the other hand, the molecular size of 75,000-80,000 was estimated through Sephadex G-150 gel filtration, indicating that the enzyme in rat kidney exists most likely in a dimeric form. Specific antibody was raised in rabbit against the highly purified rat kidney choline kinase protein, then immunochemical cross-reactivity was investigated between rabbit antiserum and choline kinase preparations from various rat tissues. The antiserum inhibited choline kinase activity almost completely in the crude preparation not only from kidney but also from lung, intestine, and normal untreated liver cytosol, but it could inhibit only partially the activity from either 3-methylcholanthrene- or carbon tetrachloride-induced rat liver cytosol. The overall results demonstrated that, although choline kinase protein appears to exist in multiple forms in rat tissues, most of them are immunochemically identical, and that either 3-methylcholanthrene- or carbon tetrachloride-inducible form(s) of choline kinase in rat liver could be quite different from a form or forms existing in normal untreated rat liver cytosol.     

Evidence for a new form of enolase in rat brain.

Rat brain enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) is unaffected by an antiserum raised against rat muscle enolase (isoenzyme 3) and an antiserum raised against rat liver enolase (isoenzyme 1) affects only 70% of the total brain activity. By gradient elution of QAE-Sephadex at pH 8.5, brain enolase is separated into two major peaks. The first is chromatographically and immunochemically identical with isoenzyme 1 whilst the second more complex peak is apparently specific for brain. Gel filtration chromatography on G-150 Sephadex shows no significant difference in molecular weight between these two components.     

Alkaline phosphatase of Blastocladiella emersonii: partial purification and characterization.

Alkaline phosphomonoesterase (EC 3.1.3.1) activity from Blastocladiella emersonii, while displaying typically broad substrate specificity for phosphorylated organic compounds, exhibited nearly complete substrate preference for N-acetylglucosamine-6-phosphate over N-acetylglucosamine-1-phosphate. Enzyme in zoospore extracts was purified 43-fold by differential centrifugation followed by gel filtration (Sephadex G-200) and then by ion-exchange chromatography (diethylaminoethyl-cellulose). The partially purified enzyme displayed an apparent molecular weight (Sephadex G-200) of approximately 170,000. The activity of partially purified enzyme exhibited a pH optimum of pH 8.5, did not require a metal divalent cation, but was inhibitable by ethylenediaminetetraacetic acid. During the life cycle of the organism, the specific activity of the phosphatase decreased slightly during germination and early exponential growth but then increased about 4.5-fold during sporulation. B. emersonii alkaline phosphatase does not appear to be a repressible enzyme.     

Thiamine triphosphatase activity in mammalian mitochondria

Mitochondrial preparations isolated from bovine kidney and brain as well as the liver and the brain of rat show thiamine triphosphatase (ThTPase) activity. The activity was determined from the particles by freezing-thawing suggesting that a soluble enzyme is involved. The liberation patterns of ThTPase and marker enzyme activities from mitochondria under osmotic shock or treatment with increasing Triton X-100 concentrations indicate the presence of ThTPase both in the matrix and intermembrane space. It was found, basing on gel filtration behavior, that the mitochondrial ThTPase has the same molecular mass as specific cytosolic ThTPase (EC 3.6.1.28). The enzymes, however, were clearly distinguishable in Km values, the mitochondrial one showing a higher apparent affinity for substrate. These results imply the existence of ThTPase multiple forms in mammalian cells.     

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.     

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.     

Purification and characterization of a lysosomal form and a variant form of beta-glucuronidase from the rat basophil leukaemia tumour.

Two isoenzyme of beta-glucuronidase from a rat basophil leukaemia tumour were co-purified 4067-fold by (NH4)2SO4 precipitation and sequential chromatography on concanavalin A--Sepharose, Sephadex G-200, DEAE-cellulose, CM-cellulose and phosphocellulose. The purity of the mixture was established by the coincidence of the peaks of enzyme activity and protein at a molecular weight of 300 000 on Bio-Gel P-300, the presence of only two protein bands, both of them enzymically active, in polyacrylamide gels after electrophoresis under non-denaturing conditions, and the presence of a single subunit species, of mol.wt. 75 000, after electrophoresis in polyacrylamide gels under a denaturing conditioning. The major isoenzyme co-migrated with the L form from rat liver during electrophoresis in alkaline polyacrylamide gels, whereas the minor isoenzyme migrated more rapidly than either the lysosomal form or the rat liver microsomal form and was designated the tumour (T) isoenzyme. A mixture of the purified isoenzymes from two preparations had an average specific activity of 1389 units/mg for phenolphthalein beta-D-glycopyranosiduronic acid. The L and T isoenzymes, which had pI5.9 and 5.7 respectively, could be obtained free of cross-contamination by isoelectric focusing and had similar specific activities. Although the T isoenzyme could be a catabolic product of the M or the L form, it could also be a unique tumour product, because it was not detected in extracts of normal rat tissues.     

Purification and characterization of adenylate kinase isozymes from rat muscle and liver

Isozymes of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) were purified from skeletal muscle and liver of rats to essentially homogeneous states by acrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. The isozyme from muscle was purified by acidification to pH 5.0, and column chromatography on phosphocellulose, Sephadex G-75 and Blue Sepharose CL-6B, while that from liver was purified by column chromatography on Blue Sepharose CL-6B, Sephadex G-75 and carboxymethyl cellulose. By these procedures the muscle isozyme was purified about 530-fold in 29% yield, and the liver isozyme about 3600-fold in 27% yield from the respective tissue extracts. The molecular weights of the muscle and liver isozymes were estimated as about 23 500 and 30 500, respectively, by both sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography, and no subunit of either isozyme was detected. The isoelectric points of the muscle and liver isozymes were 7.0 and 8.1, respectively. The Km values of the respective enzymes for ATP and ADP were similar, but the Km(AMP) of the liver isozyme was about one-fifth of that of the muscle isozyme. Immunological studies with rabbit antiserum against the rat muscle isozyme showed that the muscle isozyme was abundant in muscle, heart and brain, while the liver isozyme was abundant in liver and kidney.     

Thiamine Triphosphate and Membrane-Associated Thiamine Phosphatases in the Electric Organ of Electrophorus electricus

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate, which represents 87% of the total thiamine content in this tissue. The thiamine pyrophosphate concentration, however, is very low in the eel electric organ and skeletal muscle as compared with other eel or rat tissues. Furthermore, electroplax membranes contain a whole set of enzymes responsible for the dephosphorylation of thiamine tri-, pyro- and monophosphate. Thiamine triphosphatase has a pH optimum of 6.8 and is dependent on Mg2+. The real substrate of the enzyme is probably a 1:1 complex of Mg2+ and thiamine triphosphate. Thiamine pyrophosphatase is activated by Ca2+. The apparent Km for thiamine triphosphate and Vmax are found to be, respectively, 1.76 mM and 5.95 nmol/mg of protein/min. Thiamine triphosphatase activity is inhibited at physiological K+ concentrations (up to 90 mM) and increasing Na+ concentrations (50% inhibition at 300 mM). ZnCl2 (10 mM) inhibits 90% of the enzyme activity. ATP and ITP are also strongly inhibitory. No significant effect of neurotoxins is seen. Membrane-associated thiamine triphosphatase is affected differently by proteolytic enzymes and is partially inactivated by pretreatment with phospholipase C and neuraminidase. The physiological significance of thiamine triphosphatase is discussed in relation to a specific role of thiamine in the nervous system.