Purification, properties, and evidence for two subtypes of human placenta hexokinase type I

In human placenta 85% of total hexokinase activity (EC 2.7.1.1) was found in a soluble form. Of this, 70% is hexokinase type I while the remaining 30% is hexokinase type II. All the bound hexokinase is type I. Soluble hexokinase I was purified 11,000-fold by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The specific activity was 190 units/mg protein with a 75% yield. The enzyme shows only one band in nondenaturing polyacrylamide gel electrophoresis that stains for protein and enzymatic activity; however, two components (with Mr 112,000 and 103,000) were constantly seen in sodium dodecyl sulfate-gel electrophoresis. Many attempts were made to separate these two proteins under native conditions; however, only one peak of activity was obtained when the enzyme was submitted to gel filtration (Mr 118,000), preparative isoelectric focusing (pI 5.9), anion-exchange chromatography, hydroxylapatite chromatography, and affinity chromatography on immobilized dyes and immobilized glucosamine. The high and low molecular weight hexokinases show the same isoelectric point under denaturing conditions as determined by two-dimensional gel electrophoresis. Each hexokinase subtype was obtained by preparative sodium dodecyl sulfate electrophoresis followed by electroelution. Monospecific antibodies raised in rabbits against electroeluted high and low molecular weight hexokinases were not able to recognize the native enzymes but each of them detected both hexokinases on immunoblots. Amino acid compositions and peptide mapping by limited proteolysis of the high and low molecular weight hexokinases were also performed and suggested a strong homology between these two subtypes of human hexokinase I.     

PURIFICATION AND COMPARISON OF 2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASES FROM BOVINE BRAIN AND SPINAL CORD

The membrane-bound enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase has been purified from acetone powders of bovine white matter and spinal cord. Affinity chromatography on AMP-Sepharose has been used as the final step in the chromatographic purifications. The yield was about 3 mg of purified enzyme per 100 g of tissue in each instance. The enzymes from the two sources were indistinguishable by chromatography, gel electrophoresis, and amino acid analysis; the enzyme from spinal cord, however, has shown a specific activity of 225 units/mg compared to 342 units/mg for the enzyme from white matter. Both proteins had a molecular weight of 100,000 by gel filtration and 50,000 by sodium dodecyl sulfate-gel electrophoresis under reducing conditions. The properties of the enzyme, including amino acid composition determined on the purified soluble protein and on the protein purified by sodium dodecyl sulfate-gel electrophoresis, were those of a basic hydrophobic protein.     

Purification of a hexokinase-binding protein from the outer mitochondrial membrane.

Brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) binds selectively to the outer membrane of rat liver mitochondria but not to inner mitochondrial or microsomal membranes nor to the plasma membrane of human erythrocytes. A protein having subunit molecular weight of 31,000, determined by sodium dodecyl sulfate-gel electrophoresis, has been highly purified from the outer mitochondrial membrane by repetitive solubilization with octyl-beta-D-glucopyranoside followed by reconstitution into membranous vesicles when the detergent is removed by dialysis. When incorporated into lipid vesicles, the protein confers the ability to bind brain hexokinase in a Glc-6-P-sensitive manner as is seen with the intact outer mitochondrial membrane. Hexokinase binding ability and the 31,000 subunit molecular weight protein co-sediment during sucrose density gradient centrifugation. Both hexokinase binding ability and the 31,000 subunit molecular weight protein are resistant to protease treatment of the intact outer mitochondrial membrane while other membrane proteins are extensively degraded. It is concluded that this protein, designated the hexokinase-binding protein (HBP), is an integral membrane protein responsible for the selective binding of hexokinase by the outer mitochondrial membrane.     

Guanidoacetate methyltransferase. Purification and molecular properties.

Guanidoacetate methyltransferase has been purified about 140-fold from pig liver. Polyacrylamide gel electrophoresis of the purified enzyme showed four protein bands, each of which is associated with guanidoacetate methyltransferase activity. During gel electrophoresis at pH 3 in 8 M urea, guanidoacetate methyltransferase migrated as a single component. The molecular weight of the purified guanidoacetate methyltransferase was estimated to be 31,000 by sodium dodecyl sulfate-gel electrophoresis, which also showed only one protein component with guanidoacetate methyltransferase activity. This molecular weight is in agreement with that estimated by Sephadex G-75 chromatography. Guanidoacetate methyltransferase is inhibited by adenosylhomocysteine, 3-deazaadenosylhomocysteine, and sinefungin with Ki values of 16 microM, 39 microM, and 18 microM, respectively.     

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 of an autocatalytic protein-glycosylating enzyme from cell suspensions of Daucus carota L.

A glycosyltransferase was identified in the 174 000 · g membrane pellet and the supernatant from extracts of cell suspensions of Daucus carota L. The enzyme from the supernatant was enriched 475-fold, and sodium dodecyl sulfate-gel electrophoresis and fluorography of this purified sample showed that the only enriched protein band (40 000 Da) was simultaneously an enzyme and a glucose-acceptor. Gel filtration and electrophoresis under non-denaturing conditions proved that in vivo this protein provides the subunits for a very large molecule. Radio-gas-liquid chromatography demonstrated that only one glucosyl moiety was transferred from UDP-glucose to the protein.Abbreviations DEAE diethylaminoethyl - GT IsU glycosyltransferase I, soluble, substrate UDPglucose - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Human blood group glycosyltransferase. II. Purification of galactosyltransferase

A galactosyltransferase, which converts blood group O red bloodcells to B-cells, was purfied to homogeneity from plasma of blood group B subjects. The stepwise purification procedures include: (a) column chromatography with CM-Sephadex, followed by ammonium sulfate fractionation; (b) Sephadex G-200 gel filtration; (c) column chromatogr,phy with DEAE-Sephadex; and (d) column chromatography with hydroxylapatite. The procedures provided about a 400,000-fold increase of specific activity with a 40 to 50% yield. Further purification of the enzyme was performed by small scale preparative acrylamide gel electrophoresis at pH 4.3. The final enzyme preparation showed a single protein band which coincided with enzyme activity, in acrylamide gel electrophoresis, and revealed a single protein band in sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight, which was estimated by Sephadex gel filtration, and subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ for its activity and had a pH optimum at 7.0 to 7.5.     

Purification of messenger RNA coding for glycine methyltransferase from rat liver

Rat liver messenger RNA coding for glycine methyltransferase was associated preferentially with free polysomes. The mRNA was purified about 1000-fold over the total poly(A)-containing RNA by specific immunoadsorption of polysomes to protein A-Sepharose followed by oligo(dT)-cellulose column chromatography. Sodium dodecyl sulfate-gel electrophoresis of the in vitro translation products in a rabbit reticulocyte lysate system revealed only one major band which migrated to the position of the purified glycine methyltransferase subunit. The result shows that the mRNA isolated is nearly homogeneous and suggests that no precursor form of the enzyme existed. The mRNA sedimented at the position slightly smaller than 18 S rRNA in a sucrose density-gradient centrifugation and was shown to contain about 1,300 nucleotides by the Northern blot hybridization analysis with a cDNA probe.     

Ascaris suum hexokinase: purification and possible function in compartmentation of glucose 6-phosphate in muscle.

Hexokinase (EC 2.7.1.1) is present in a soluble and a bound form in homogenates of Ascaris suum muscle. Cellulose acetate electrophoresis, isoelectric focusing, and ion exchange chromatography confirmed the presence of only one molecular form of hexokinase in this muscle. A procedure for purifying hexokinase from Ascaris muscle has been developed utilizing ion-exchange chromatography, ammonium sulfate fractionation and gel filtration. The enzyme is a monomer with a molecular weight of 100 000 as determined by sodium dodecyl sulfate gel filtration. The Stokes' radius, diffusion coefficient, and frictional ratio have been determined. The apparent Michaelis constants for glucose and ATP are 4.7-10(-3) M and 2.2-10(-4) M, respectively. Ascaris hexokinase also exhibits end-product inhibition by glucose 6-phosphate and ADP. It is postulated that the kinetic parameters of the enzyme are the results of its function, that of generating glucose 6-phosphate primarily for glycogen synthesis.     

Human blood group glycosyltransferases. I. Purification of n-acetylgalactosaminyltransferase

An N-acetylgalactosaminyltransferase, which converts blood group O red blood cells to A cells, was purified to homogeneity from plasma of blood group A1 subjects. The enzyme was adsorbed on Sepharose 4B, and after washing out the impurities, the enzyme was eluted with UDP. This procedure resulted in a 70,000- to 100,000-fold increase in specific activity with recovery of about 80%. Further purification of the enzyme was achieved by Bio-Gel P treatment. The final enzyme preparation showed a single protein band, which coincided with enzyme activity, on acrylamide gel electrophoresis, and revealed a single protein band on sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight (90,000 to 100,000), which was estimated by Sephadex gel filtration, and the subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ and had optimum activity at pH 6.5 to 7.0.     

Purification and Characterization of Prosomatostatin from Rat Brain

A high molecular weight somatostatin-immunoreactive polypeptide, presumably prosomatostatin, was purified from rat brain and characterized. Purification steps included extraction with 2 M acetic acid, precipitation of contaminating proteins at pH 6.5, Sephadex G-50 chromatography, immunoaffinity chromatography, and HPLC steps (size exclusion and reversed-phase HPLC). The protein was purified more than 30,000-fold. It is heat stable. Sodium dodecyl sulfate-gel electrophoresis and immunoblotting revealed one major immunoreactive band of approximately 13,000 molecular weight which roughly corresponds to the size of prosomatostatin as derived from its DNA sequence. Isoelectric focusing and two-dimensional sodium dodecyl sulfate-gel electrophoresis gave a single immunoreactive spot at a pI of 5.4. The polypeptide did not bind to concanavalin A or to wheat germ lectin columns, suggesting lack of N-glycosylation in the molecule. Regional distribution of prosomatostatin varied between 6%, 10%, and 18% of total immunoreactivity in the brainstem, cortical areas, and striatum, respectively.     

Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the UDPglucose:glycogen 4-alpha-glucosyltransferase.

The Neurospora crassa glycogen synthase (UDPglucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) was purified to electrophoretic homogeneity by a procedure involving ultracentrifugation, DEAE-cellulose column chromatography, (NH4)2SO4 fractionation and 3-aminopropyl-Sepharose column chromatography. The final purified enzyme preparation was almost entirely dependent on glucose-6-P and had a specific activity of 6.9 units per mg of protein. The subunit molecular weight of the glycogen synthase was determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel to be 88 000--90 000. The native enzyme was shown to have a molecular weight of 270 000 as determined by sucrose density gradient centrifugation. Thus, the glucose-6-P-dependent form of the N. crassa glycogen synthase can exist as trimer of the subunit. Limited proteolysis with trypsin or chymotrypsin converted the glucose-6-P-dependent form of the enzyme into an apparent glucose-6-P-independent form. The enzyme was shown to catalyze transfer of glucose from UDPglucose to glycogen as well as to its phosphorylase limit dextrin, but not to its beta-amylase limit dextrin. Moreover, glucose, maltose and maltotriose were not active as acceptors.     

Purification and properties of the membrane-bound form of acetylcholinesterase from chicken brain. Evidence for two distinct polypeptide chains

The major molecular form of acetylcholinesterase (AChE) from chicken brain is a membrane-bound glycoprotein with an apparent sedimentation coefficient of 11.4 S. Analysis of the purified protein by gel filtration, velocity sedimentation, and sodium dodecyl sulfate-gel electrophoresis shows that the solubilized enzyme is a globular tetramer with an apparent Mr = 420,000. This membrane-bound form of AChE is hydrophobic and readily aggregates in the absence of detergent. These aggregates are concentration-dependent, relatively stable in the presence of high salt concentrations, yet readily dissociate upon addition of detergent to the 11.4 S form, indicating that the interactions are hydrophobic. Polyclonal and monoclonal antibodies raised against chicken brain AChE purified by ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis precipitate AChE enzyme activity. However, these antibodies do not cross-react with the enzyme from chicken muscle which preferentially hydrolyses butyrylcholine. Immunoprecipitation of isotopically labeled enzyme molecules from tissue cultured brain cells and analysis by sodium dodecyl sulfate-gel electrophoresis shows that AChE consists of two polypeptide chains with apparent Mr = 105,000 (alpha) and 100,000 (beta) in a 1:1 ratio. Immunoblotting of brain AChE with either the polyclonal or monoclonal antibodies indicates that the alpha and beta chains share antigenic determinants. Furthermore, both polypeptide chains can be labeled with [3H]diisopropyl fluorophosphate, indicating that they each contain a catalytic site. This is the first indication that globular forms of AChE may consist of multiple polypeptide chains.     

Phospholipase C from Clostridium perfringens: preparation and characterization of homogeneous enzyme

A new procedure for the purification of phospholipase C from Clostridium perfringens has been devised that results in essentially pure enzyme. The procedure consists of ammonium sulfate fractionation, ion-exchange chromatography on QAE-Sephadex, and affinity chromatography on phosphatidylcholine linked to Sepharose. The molecular weight of the enzyme, determined by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, and gel filtration, is 43,000; and the isoelectric point is pH 5.4. The enzyme was optimally active with phosphatidylcholine dispersed in sodium deoxycholate, although appreciable activity was observed with either phosphatidylcholine or sphingomyelin dispersed with ethanol. The requirement for metal ions in the assay could be met by a number of different ions. The pure enzyme was found to contain 2 mol zinc per mol enzyme, thus implicating it as a zinc metalloenzyme.     

Purification and properties of the membrane-bound acetylcholinesterase from adult rat brain

The membrane-bound acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from adult rat brain has been purified to homogeneity using sequential affinity chromatography on Con A-Sepharose and on dimethyl-aminoethylbenzoic acid-Sepharose 4B followed by DEAE-cellulose chromatography. The yield of the purified enzyme (specific activity: 3068 U/mg protein) is higher than 50%. Polyacrylamide gel electrophoresis in the presence of Triton X-100 gives only one band with acetylcholinesterase activity. With the exception of electrofocusing and pore gradient electrophoresis, where a multiple band pattern was detected (which seems to be artefactual), the enzyme appears to be homogenous. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X-100 give only one symmetrical peak, with a calculated molecular weight of 328 000. Since polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and mercaptoethanol gives only one band with a molecular weight of 74 500, a tetrametric structure can be postulated for the membrane-bound acetylcholinesterase from rat brain.     

Purification and properties of spinach leaf debranching enzyme

Starch debranching enzyme was purified from intact spinach (Spinacia oleracea L. cv Vital) chloroplasts and from a spinach leaf extract using affinity chromatography on Sepharose 6B-bound cycloheptaamylose (Schardinger β-dextrin). The enzyme from both sources was homogeneous upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Spinach leaf debranching enzyme appears to consist of a single polypeptide chain, since the molecular weight of the native protein (110,000 daltons) was not changed by treatment with sodium dodecyl sulfate. Only one spinach leaf debranching enzyme band could be detected after electrophoresis of a leaf extract on amylopectin-containing polyacrylamide gel, the retardation factor of which coincided with that of the single band seen with the chloroplast enzyme. The purified enzyme exhibited strong pullulanase activity, the specific activity being 69 units per milligram protein with pullulan and 22 units per milligram protein with amylopectin. Cycloheptaamylose is a potent competitive inhibitor of spinach leaf debranching enzyme. The pH optimum of the enzyme was found to be 5.5. The purified enzyme is rather unstable at both 20° and 0°C. Part of the activity lost under storage or at a suboptimal pH could immediately be restored by the addition of thiols. The reactivatable protein, being of the same molecular weight as the native enzyme, exhibited a somewhat altered electrophoretic mobility resulting in one or two minor bands on a zymogram.     

Isolation of lysyl hydroxylase, an enzyme of collagen synthesis, from chick embryos as a homogeneous protein.

Two procedures are reported for the purification of lysyl hydroxylase, both procedures involving (NH4)2SO4 fractionation, affinity chromatography on concanavalin A-agarose and elution of the column with ethylene glycol. The additional steps in procedure A consist of gel filtration and chromatography on a hydroxyapatite column, and in procedure B of affinity chromatography on collagen linked to agarose and gel filtration. The best preparations obtained with either of the two procedures were pure when examined by sodium dodecyl sulphate-polyacrylamide-disc-gel or slab-gel electrophoresis, but about half of the preparations obtained by procedure A had minor contaminants. The specific activity of a typical preparation purified by procedure B was 13 4000 times that of the 15 000 g supernatant of the chick-embryo homogenate, with a recovery of about 4%. The molecular weight of the pure enzyme was bout 200 000 by gel filtration, and that of the enzyme subunit about 85 000 by sodium dodecyl sulphate/polyacrylamide-disc-gel or slab-gel electrophoresis. It is suggested that the active enzyme is a dimer consisting of only one type of monomer, and that a previously described enzyme form with an apparent molecular weight of about 550 000 is a polymeric form of this dimer. The catalytic-centre activity of the pure enzyme, as determined with a saturating concentration of a synthetic peptide substrate and under conditions specified, was about 3-4 mol/s per mol.     

Purification and characterization of human factor II

Human plasma Factor II has been purified approximately 800-fold by a combination of barium citrate adsorption, ion-exchange chromatography and preparative polyacrylamide gel electrophoresis. The procedure is relatively simple and results in excellent yields of purified Factor II essentially free of Factor X activity. The purified factor behaved as a single component by analytical polyacrylamide gel disc electrophoresis at pH 8.9. No Factor V, VII or IX activity was detected in the purified Factor II. Its molecular weight was 7200±3000 as determined by analytical ultracentrifugation, electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on Bio-Gel P-200. An apparent molecular weight of 90 000–100 000 was observed on calibrated columns of Sephadex G-100, G-150, and G-200. The specific activity of human factor II was approximately 1300 N.I.H. units/mg as determined by the two-stage assay and 7 Ortho units/mg by the one stage assay. The purified protein contained by weight 2.8% neutral hexose, 2.3% sialic acids and 3.1% hexosamines.     

Pig kidney phosphofructokinase. Purification to homogeneity and qualitative basic characterization.

Phosphofructokinase has been purified from pig kidney by extraction with phosphate buffer at pH 8, followed by alcohol treatment, affinity chromatography on matrix-bound Cibacron blue F3G-A, and gel chromatography on Sepharose 6B. Using sodium dodecyl sulphate electrophoresis the enzyme was found to be homogeneous and to have a specific activity of about 80 units/mg protein. Like other phosphofructokinases, at pH 7.0 the enzyme exhibits a sigmoidal dependence in its activity on the fructose 6-phosphate concentration and is strongly inhibited by ATP. The degree of citrate inhibition is influenced by the concentration of the two substrates. ATP strengthens and fructose 6-phosphate relieves the inhibition by citrate. AMP and cAMP are able to overcome the ATP inhibition. The ADP activation curve is biphasic. The molecular weight of the subunit of pig kidney phosphofructokinase was determined to be 88 000 by means of sodium dodecyl sulphate electrophoresis.     

Isoelectric points and molecular weights of salt-extractable ribosomal proteins.

Rat liver ribosomes prepared in low salt buffer contain basic and acidic proteins not found on ribosomes washed in high salt buffer. Proteins extracted from liver ribosomes by 500 mM KCL were characterized by acid urea-polyacrylamide gel electrophoresis, sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel isoelectric focusing. The salt-solubilized proteins contain 12 polypeptides with a molecular weight over 67000, several polypeptides with molecular weights less than 67 000, and three polypeptides whose molecular weight exceeded 130 000. Ten to 12 of the proteins were basic, and about 24 acidic proteins were partially or wholly extracted from the ribosomes. Four of the acidic proteins have isoelectric points less than 4.5.