Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions

1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37°C for 1h released about 60% of the membrane-bound UDP-galactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with α-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn²⁺ that could not be replaced by Ca²⁺, Mg²⁺, Zn²⁺ or Co²⁺, and was active over a wide pH range (6–8) with a pH optimum of 6.5. The apparent K_m for UDP-galactose was 10.8μm. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000–70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2–5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-ε-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the ε-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction.     

The molecular and catalytic properties of galactosyltransferase from fetal calf serum

A soluble galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyl-transferase) was purified to apparent homogeneity from fetal calf serum with an overall increase in specific activity of 19,600-fold. The enzyme exhibited the following properties: specific activity, 8.5 units/mg of protein; acceptor specificity, N-acetylglucosamine/ ovalbumin = 3.3; diffusion coefficient, 5.56; sedimentation coefficient, 3.2; and molecular weight, 47,800. Comparison of the structural and catalytic properties of the fetal calf serum enzyme with purified galactosyltransferase from bovine milk indicated that the enzymes from the two bovine sources are very similar and possibly identical.     

Specific Selenium-Containing Macromolecules in the Marine Diatom Thalassiosira pseudonana

Thalassiosira pseudonana Husedt (Hasle and Heimdal) clone 3H was grown in axenic culture in artificial seawater medium containing 10⁻⁸ molar Na₂⁷⁵SeO₃. Biochemical distribution of radiolabeled Se was determined by solvent extraction techniques, gel filtration, and polyacrylamide gel electrophoresis. Of the total cellular Se, 51% was protein bound. Two soluble macromolecules of 21 and 29 kilodaltons contained ⁷⁵Se. These results are the first to provide evidence of specific Se-containing compounds in a photosynthetic organism. Glutathione peroxidase (GSH-Px) activity was measured in cell-free extracts and on nondenaturing polyacrylamide gels by a glutathione-reductase coupled assay. Two enzymes showing GSH-Px activity were present. One enzyme was active with H₂O₂ and tert-butyl hydroperoxide (tBOOH); consistent with known Sedependent GSH-Pxs, but the other enzyme was only active with tBOOH. Co-migration of the H₂O₂-active GSH-Px and ⁷⁵Se on nondenaturing polyacrylamide gels provides evidence that T. pseudonana contains a Sedependent GSH-Px. The molecular weight of one of the ⁷⁵Se-labeled macromolecules is identical with the weight of previously characterized GSH-PX subunits. We conclude that the obligate requirement for Se in Thalassiosira pseudonana is due in part to the presence of the selenoenzyme glutathione peroxidase.     

Galactosyltransferase activities in pancreas, liver and gut of the developing rat

Two galactosyltransferase activities (1 and 2) were measured in the pancreas, liver and gut of the developing rat embryo. 1. N-Acetylglucosamine:Galactosyltransferase. UDP [¹⁴C]galactose + N-acetylglucosamine → [¹⁴C]galactosyl-β-(1 → 4)-N-acetylglucosamine + UDP. 2. N-Acetylgalactosamine-protein:Galactosyltransferase. UDP [¹⁴C]galactose + N-acetylgalactosamine-protein → [¹⁴C]galactosyl-β-(1 → 3)-N-acetylgalactosamine-protein + UDP. Galactosyltransferases 1 and 2 increased in the pancreas, about 10- and 40-fold in specific activity, respectively, from 11 to 12 days in utero to birth. During this period the activities of both transferases in the liver were somewhat variable, but showed no definite trend. A drop in the level of galactosyltransferase 1 in the pancreas occurred at birth or shortly thereafter. The “Golgimarker” enzyme for liver, galactosyltransferase 1, may be absent or present at low levels in adult rat pancreas.Zymogen granule membrane preparations apparently are devoid of these galactosyltransferase activities. Bromodeoxyuridine, which inhibits the development of the synthetic capability of the specific exocrine proteins, had essentially no effect on the normal accretion of the galactosyltransferase activities in organ cultures of pancreatic rudiments from 13-day rat embryos.     

Purification and properties of the NAD+-dependent (type D) and O2-dependent (type O) forms of rat liver xanthine dehydrogenase.

The xanthine-oxidizing enzyme of rat liver has been purified as an NAD⁺-dependent dehydrogenase (type D) and as the O₂-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD⁺ with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The K_m for xanthine is not significantly different for the two forms.     

Gel-Electrophoretic Separation, Detection, and Characterization of Plant and Bacterial UDP-Glucose Glucosyltransferases

We have developed procedures for detection and characterization of UDP-glucose: glucosyltransferases following electrophoretic separation in nondenaturing polyacrylamide gels. Using digitonin-solubilized membrane protein preparations from a variety of plants and two cellulose-producing bacteria, activity can be demonstrated for several UDP-glucose:β-glucan synthases with an in situ assay following gel electrophoresis. These enzymes can be characterized within the gels with respect to effector requirements and products produced, and several advantages of this assay over solution assays are demonstrated. For example, the clear dependence of plant UDP-glucose:(1→3)-β-glucan synthase on both Ca²⁺ and a β-linked glucoside is shown; bacterial cellulose synthases show direct stimulation within the gel by guanyl oligonucleotide, and the Acetobacter xylinum enzyme appears more stable in the gel assay than in solution assay.     

Purification and properties of an ld-dipeptidase FROM Bacillus sphaericus

• 1.1. An ld-dipeptidase (EC 3.4.13.-) that hydrolyzes the unrelated dipeptides l-Ala-d-Glu (sp. act. 0.85 μmol·min⁻¹·mg⁻¹) and l-Lys-d-Ala (sp. act. 11 μmol · min⁻¹·mg⁻¹) has been purified 250-fold from the sporulation medium of Bacillus sphaericus with a 4% recovery of lytic activity.
• 2.2. Throughout the purification steps, followed with both substrates, the enzyme peaks of activities were congruent and the ratios of activities were constant. Both activities were activated 50-fold by cobalt. Polyacrylamide gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. The data are consistent with those activities being due to a single enzyme.
• 3.3. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band (M_r 38,000).
• 4.4. This dipeptidase hydrolyzes some other ld-dipeptides with a free amino and carboxyl group. Although dipeptides having a di-amino acid as the amino terminus are the best of the substrates tested, the hydrolysis occurs also when neutral amino acids are N-terminal. The activity is higher with neutral C-terminal residues such as Gly or d-Ala than with a di-acid residue such as d-Glu.
• 5.5. This enzyme may have a function in peptidoglycan metabolism.

     

Respiration and protein synthesis in Escherichia coli membrane-envelope fragments. VI. Solubilization and characterization of the electron transport chain

Membranes obtained from Escherichia coli have been solubilized with deoxycholate. The solubilized dehydrogenases and cytochromes are not sedimented at 105,000 g. These components readily penetrate the "included space" of Sepharose 4B (Pharmacia Fine Chemicals Inc., Uppsala, Sweden) and polyacrylamide gels and have been fractionated on the basis of molecular size. Solubilization destroys nicotinamide adenine dinucleotide, reduced form (NADH) oxidase and D-lactate oxidase activities, but leaves an appreciable part of the original succinoxidase activity intact. Evidence for a succinate dehydrogenase-cytochrome b ₁ complex is given. Menadione added to the solubilized preparation does not elicit NADH oxidase activity nor stimulate the existing succinoxidase activity, but does provoke an active D-lactate oxidase activity. This D-lactate oxidase activity, however, does not use cytochromes and is not sensitive to cyanide.     

Biosynthesis of keratan sulfate: purification and properties of a galactosyltransferase from bovine cornea.

A soluble galactosyltransferase was purified 22,000-fold from bovine cornea. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-acetyl-d-glucosamine, α- and β-glucosaminides, bovine cornea and nasal septum agalactokeratan, and to glycoproteins containing terminal nonreducing N-acetylglucosaminyl units. When N-acetyl-d-glucosamine served as acceptor, the product formed by the cornea transferase contained galactose glycosidically linked to carbon atom 4 of N-acetyl-d-glucosamine; the same glycosidic linkage was found in [¹⁴C]keratan preparations isolated from reaction mixtures where keratan containing terminal nonreducing N-acetylglucosaminyl units served as acceptor. The cornea enzyme exhibited a markedly lower K_m with keratan than with N-acetyl-d-glucosamine. The physical and kinetic properties of the cornea galactosyltransferase and of the milk A-protein (A-protein + α-lactalbumin = lactose synthase), including modulations of acceptor specificity by α-lactalbumin, were compared. The results of these studies strongly suggest that the two glycosyltransferases are similar, if not identical. Efforts to demonstrate the presence of other soluble galactosyltransferases in cornea were unsuccessful; no change in the ratios of products formed with several acceptors was observed at any stage of purification. It is suggested that in bovine tissues a single galactosyltransferase participates in the synthesis of both high and low molecular weight galactosides including the assembly of the repeating disaccharide [O-β-galactopyranosyl-(1 → 4)-N-acetylglucosamine] of cornea keratan sulfate.     

In vitro phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme a reductase: Analysis of 32P-labeled,inactivated enzyme

Rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase was inactivated with Mg²⁺ and [γ-³²P]ATP, then solubilized and purified to homogeneity. The ³²P radioactivity was precipitated by antibody to homogeneous rat liver reductase and comigrated with nonprecipitated, homogeneous reductase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nondenaturing conditions, ³²P radioactivity comigrated with reductase protein and activity on polyacrylamide gels. These results provide direct support for the concept that the enzyme is covalently phosphorylated during the in vitro incubation of microsomes with Mg²⁺ and ATP.     

Purification of uridine diphosphate-galactose:glucosyl ceramide, beta 1-4 galactosyltransferase from human kidney.

A galactosyltransferase that transfers galactose from UDP-galactose to glucosylceramide was purified 440-fold to apparent homogeneity from normal human kidney "buffy coat" preparation employing detergent extraction, ultrafiltration, and Sepharose Q column chromatography. On reducing and nonreducing gels, the enzyme resolved into two bands with apparent molecular weights on the order of 60,000 and 58,000, respectively. The activity of the enzyme was also associated with these two bands following separation on polyacrylamide gels. Analytical isoelectric focusing revealed that the pI of this enzyme is approximately 4.55. Product characterization and substrate specificity studies employing chromatography, enzymatic digestion with various glycosidases, and use of a variety of glycosphingolipid substrates revealed that the major product synthesized by this enzyme was Cer1-1 beta Glc4-1Gal, and Cer1-1 beta Glc was the preferred substrate. Digestion of the 60- and 58-kDa proteins with Staphylococcus aureus (V-8) protease revealed at least six peptides having identical electrophoretic migration. This finding suggests that the two proteins may be related to each other. Western immunoblot assays revealed that the antibody against UDP-galactose:GlcCer, beta 1-4 galactosyltransferase (GalT-2) but not galactosyltransferase UDP-Gal:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyltransferase (EC 2.4.1.38) (B-GT) immunoprecipitated (recognized) the kidney GalT-2. In contrast, antibody against B-GT did not immunoprecipitate GalT-2. Thus our data indicate that GalT-2 and B-GT are two distinct enzymes. The availability of the enzyme GalT-2 and corresponding antibody will allow functional studies in the near future.     

Cancer-associated serum galactosyltransferase activity. Demonstration in an animal model system.

Two different lines of solid tumors were produced in outbred hamsters by subcutaneous injection of polyoma transformed BHK cells. Growth of the tumors correlated with the appearance in serum of an electrophoretically distinct peak of galactosyltransferase: NeuAc-, Gal-free fetuin acceptor activity on polyacrylamide gels. This slow moving peak of enzyme activity (GT-HH) was detected before solid tumors could be grossly observed and the amount of activity in this peak was also found to be linearly related with growth of the tumor. GT-IIH was not detectable in control animals and separated from a faster migrating major area of serum galactosyltransferase activity (GT-IH) found in sera of both control and tumor-bearing hamsters. These two activities were shown to maintain their respective mobilities on re-electrophoresis. Solubilized enzyme derived from excised tumors demonstrated an electrophoretic mobility on polyacrylamide gels identical to that for GT-IIH present in serum from tumor-bearing animals. In contrast, enzyme activity solubilized from livers of both control or tumor-bearing hamsters showed a mobility similar to that of the faster moving serum galactosyltransferase enzyme activity, i.e. GT-IH. In addition, medium derived from nonconfluent BHKpy cells in tissue culture contained galactosyltransferase activity which co-electrophoresed with the slower migrating characteristics of galactosyltransferase activities derived from serum (control and tumor-bearing), solid tumors, liver and BHKpy cells in tissue culture were compared. All kinetic properties were similar with the exception that the Km UDP-galactose of GT-IIH (1.0 X 10(-5) M) was half that of GT-IH (2.0 X 10(-5) M).     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

A silver staining procedure for nucleic acids in polyacrylamide gels without fixation and pretreatment

Silver staining of nucleic acid has been widely used in molecular marker analysis such as simple sequence repeat (SSR), single-strand conformation polymorphism (SSCP), and amplified fragment-length polymorphism (AFLP). Many alternatives to silver staining methods have been described, but these methods are not efficient or cost-effective. Here we report a silver staining method that requires less than 10 min for one gel and can save chemicals as well. It has a detection limit of approximately 5.6 pg of DNA/mm² in nondenaturing polyacrylamide gels and 12.8 pg/mm² in denaturing polyacrylamide gels.     

Studies on the purification and properties of UDP-galactose glycoprotein galactosyltransferase from rat liver and serum.

1. Rat liver microsomal preparations incubated with 200mM-NaCl at either 0 or 30 degrees C released about 20-30% of the membrane-bound UDP-galactose-glycoprotein galactosyl-transferase (EC 2.4.1.22) into a 'high-speed' supernatant. The 'high-speed' supernatant was designated the 'saline wash' and the galactosyltransferase released into this fraction required Triton X-100 for activation. It was purified sixfold by chromatography on Sephadex G-200, and appeared to have a higher molecular weight than the soluble serum enzyme. 2. Rat serum galactosyltransferase was purified 6000-7000-fold by an affinity-chromatographic technique using a column of activated Sepharose 4B coupled with alpha-lactalbumin. The purified enzyme ran as a single broad band on polacrylamide gels and contained no sialytransferase, N-acetylglucosaminyltransferase and UDP-galactose pyrophosphatase activities. 3. The highly purified enzyme had properties similar to those of both soluble and membrane-bound galactosyltransferase. It required 0.1% Triton X-100 for stabilization, but lost activity on freezing. The enzyme had an absolute requirement for Mn2+, not replaceable by Ca2+, Mg2+, Zn2+ or Co2+. It was active over a wide pH range (6-8) and had a pH optimum of 6.8. The apparent Km for UDP-galactose was 12.5 x 10(-6) M. Alpha-Lactalbumin had no appreciable effect on UDP-galactose-glycoprotein galactosyltransferase, but it increased the specificity for glucose rather than for N-acetylglucosamine, thus modifying the enzyme to a lactose synthetase. 4. The possibility of a conversion of higher-molecular-weight liver enzyme into soluble serum enzyme is discussed, especially in relation to the elevated activities of this and other glycosyltransferases in patients with liver diseases.     

Purification and properties of methylmalonyl coenzyme A mutase from human liver

Methylmalonyl coenzyme A (CoA) mutase has been purified to apparent homogeneity from human liver by a procedure involving column chromatography on DEAE-cellulose, Matrex-Gel Blue A, hydroxylapatite, and Sephadex G-150. The overall purification achieved is 500- to 600-fold, yield 3–5%. Electrophoresis of the native purified protein on nondenaturing polyacrylamide gels shows a single diffuse band coincident with the enzyme activity; dodecyl sulfate/polyacrylamide gels show a single protein band with an apparent molecular weight of 77,500. The native protein has a molecular weight of approximately 150,000 by Sephadex G-150 chromatography, suggesting that it is composed of two identical subunits. The activity of the purified enzyme is stimulated only slightly (10–20%) by the addition of its cofactor, adenosylcobalamin, indicating that the purified enzyme is largely saturated with coenzyme. The spectrum of the enzyme is consistent with the presence of about 1 mole of adenosylcobalamin per mole of subunit. The enzyme displays complex kinetics with respect to dl-methylmalonyl CoA; substrate inhibition by l-methylmalonyl CoA appears to occur. The enzyme activity is stimulated by polyvalent anions (PO₄^3? > SO₄^2? > Cl^?); monovalent cations are without effect, but high concentrations of divalent cations are inhibitory. The enzyme activity is insensitive to N-ethylmaleimide, is rapidly destroyed at temperatures > 50 °C, and shows a broad pH optimum around pH 7.5.     

Phosphatidylcholine biosynthesis in castor bean endosperm : purification and properties of cytidine 5'-triphosphate:choline-phosphate cytidylyltransferase

Cytidine 5′-triphosphate:choline-phosphate cytidylyltransferase (EC 2.7.7.15) has been purified to near homogeneity (3350-fold) from castor bean (Ricinus communis L. var Hale) endosperm. The steps of purification included a differential solubilization of this enzyme with n-octyl β-d-glucopyranoside (OGP) and column chromatography on sequential DEAE-sepharose, sepharose-6B, and second DEAE-sepharose columns. The uses of appropriate concentrations of the detergent, OGP, in each step were crucial to obtain the highly purified enzyme. The purified enzyme gave a single protein band on nondenaturing polyacrylamide gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed one major protein band of 40 kilodaltons. Gel filtration chromatography indicated that native cytidylyltransferase was approximately 155 kilodaltons, suggesting that it exists naturally as a tetramer. The purified enzyme used methylethanolamine-phosphate as a substrate but not ethanolamine-phosphate and dimethylethanolamine-phosphate. ATP and other nucleotides tested showed little effect on the purified enzyme. The purified enzyme activity was stimulated by both phospholipids extracted from castor bean endosperm and phosphatidylcholineoleate vesicles.     

Phosphorylation ofEscherichia coli NADP+-specific glutamate dehydrogenase

Glutamate dehydrogenase fromEscherichia coli is phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein, when exposed to acid conditions, releases the phosphate; this implies that the phosphorylation site is not on a serine, tyrosine, or threonine residue(s). Treatment of glutamate dehydrogenase with diethyl pyrocarbonate, a highly specific histidine-modifying reagent, blocks incorporation of³²P-phosphate from [-³²P]ATP into the enzyme, suggestive that the phosphorylation site is a histidine residue(s). The phosphorylated glutamate dehydrogenase was identified on the basis of its comigration with highly purified glutamate dehydrogenase, isolated fromE. coli, on denaturing, nondenaturing, and isoelectric focusing polyacrylamide gels and by sequence analysis.     

Investigation of the pH dependence of proton uptake by porcine heart mitochondrial malate dehydrogenase upon binding of NADH

The pH dependence of proton uptake upon binding of NADH to porcine heart mitochondrial malate dehydrogenase (l-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) has been investigated. The enzyme has been shown to exhibit a pH-dependent uptake of protons upon binding NADH at pH values from 6.0 to 8.5. Enzyme in which one histidine residue has been modified per subunit by the reagent iodoacetamide (E. M. Gregory, M. S. Rohrbach, and J. H. Harrison, 1971, Biochim. Biophys. Acta253, 489–497) was used to establish that this specific histidine residue was responsible for the uptake of a proton upon binding of NADH to the native enzyme. It has also been established that while there is no enhancement of the nucleotide fluorescence upon addition of NADH to the iodoacetamide-modified enzyme, NADH is nevertheless binding to the modified enzyme with the same stoichiometry as with native enzyme. The data are discussed in relation to the involvement of the essential histidine residue in the catalytic mechanism of “histidine dehydrogenases” recently proposed by Lodola et al. (A. Lodola, D. M. Parker, R. Jeck, and J. J. Holbrook, 1978, Biochem. J.173, 597–605) and the catalytic mechanism of “malate dehydrogenases” recently proposed by L. H. Bernstein and J. Everse (1978, J. Biol. Chem.253, 8702–8707).     

Determination of human serum galactosyltransferase using a kinetic spectrophotometric technic

A kinetic spectrophotometric method in which galactose transfer is coupled to the production of NADH, has been adapted to the assay of galactosyltransferase activity in human serum. Under the described conditions, the rate of NADH production is linear with regard to enzyme concentration, and directly depends upon the various biochemical factors which control galactosyltransferase activity.