Acid sphingomyelinase of human placenta: purification, properties, and 125iodine labeling

Human placental acid sphingomyelinase was highly purified in the presence of Triton X-100. DEAE-Sephacel chromatography and chromatofocusing were the most effective steps in the purification procedure. Enzyme purification was 380,000 nmol/mg protein/h. Characterization and radioiodination were carried out with the chromatofocusing fraction containing highly purified enzyme. The purified enzyme contained no activity of eleven other lysosomal hydrolases but hydrolyzed bis-p-nitrophenyl phosphate slowly compared with [14C]sphingomyelin and chromogenic substrates. SDS-gel electrophoresis revealed two distinct protein bands with molecular weights of 70,500 and 39,800. This enzyme had a molecular weight of 200,000 as determined by analytical gel filtration. The pH optimum was 5.0 and Km was 52.6 x 10(-5) M for [14C]sphingomyelin. Highly purified sphingomyelinase was labeled with 125iodine by the use of Enzymobeads. Labeled sphingomyelinase preparation was rapidly cleared from blood with t1/2 of 1 min. It was absorbed mostly into the liver and presumably largely excreted from there. This labeled enzyme may be useful in metabolic studies in normal animals and animal models of genetic lysosomal storage disorders.     

Structural analogues of reactive intermediates as inhibitors of glucosamine-6-phosphate synthase and phosphoglucose isomerase

The active centers of phosphoglucose isomerase (PGI) and the hexose phosphate isomerase domain (HPI) of glucosamine-6-P (GlcN-6-P) synthase demonstrate apparent similarity in spatial arrangement of critical amino acid residues, except Arg272 of the former and Lys603 and Lys485 of the latter. Ten derivatives of d-hexitol-6-P, 5-phosphoarabinoate, or 6-phosphogluconate, structural analogues of putative cis-enolamine or cis-enolate intermediates, were tested as inhibitors of fungal GlcN-6-P synthase and PGI. None of the investigated compounds demonstrated equally high inhibitory potential against both enzymes. 2-Amino-2-deoxy-D-mannitol 6-P was found to be the strongest GlcN-6-P synthase inhibitor in the series, with an inhibition constant equal to 9.0 (+/-1.0) x 10(-6)M. On the contrary, 5-phosphoarabinoate (5PA) exhibited specificity for PGI, with K(i)=2.2 (+/-0.1) x 10(-6) M. N-acetylation substantially lowered the GlcN-6-P synthase inhibitory potential of 2-amino-2-deoxy-D-glucitol-6-P but strongly enhanced inhibitory potential of this compound towards PGI. Molecular modeling studies revealed that interactions of the C1-C2 part of transition state analogue inhibitors with the respective areas demonstrating different distribution of molecular electrostatic potential (MEP) inside HPI and PGI active centers determined enzyme:ligand affinity. In Escherichia coli HPI, a patch of the negative potential created by Glu488 aided by Val399, supposed to stabilize a putative positively charged intermediate, especially attracts ligands containing 2-amino function. The Arg272, Lys210, and Gly271 peptide bond nitrogen system, present in the corresponding space of rabbit PGI, creates an area of positive MEP, stabilizing cis-enolate intermediate and attracting its structural mimics, such as 5PA.     

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.     

Kinetic characterization of human glutamine-fructose-6-phosphate amidotransferase I: potent feedback inhibition by glucosamine 6-phosphate

Glutamine-fructose-6-phosphate amidotransferase (GFAT) catalyzes the first committed step in the pathway for biosynthesis of hexosamines in mammals. A member of the N-terminal nucleophile class of amidotransferases, GFAT transfers the amino group from the L-glutamine amide to D-fructose 6-phosphate, producing glutamic acid and glucosamine 6-phosphate. The kinetic constants reported previously for mammalian GFAT implicate a relatively low affinity for the acceptor substrate, fructose 6-phosphate (Fru-6-P, K(m) 0.2-1 mm). Utilizing a new sensitive assay that measures the production of glucosamine 6-phosphate (GlcN-6-P), purified recombinant human GFAT1 (hGFAT1) exhibited a K(m) for Fru-6-P of 7 microm, and was highly sensitive to product inhibition by GlcN-6-P. In a second assay method that measures the stimulation of glutaminase activity, a K(d) of 2 microm was measured for Fru-6-P binding to hGFAT1. Further, we report that the product, GlcN-6-P, is a potent competitive inhibitor for the Fru-6-P site, with a K(i) measured of 6 microm. Unlike other members of the amidotransferase family, where glutamate production is loosely coupled to amide transfer, we have demonstrated that hGFAT1 production of glutamate and GlcN-6-P are strictly coupled in the absence of inhibitors. Similar to other amidotransferases, competitive inhibitors that bind at the synthase site may inhibit the synthase activity without inhibiting the glutaminase activity at the hydrolase domain. GlcN-6-P, for example, inhibited the transfer reaction while fully activating the glutaminase activity at the hydrolase domain. Inhibition of hGFAT1 by the end product of the pathway, UDP-GlcNAc, was competitive with a K(i) of 4 microm. These data suggest that hGFAT1 is fully active at physiological levels of Fru-6-P and may be regulated by its product GlcN-6-P in addition to the pathway end product, UDP-GlcNAc.     

Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis.

D-Ribose isomerase, which catalyzes the conversion of D-ribose to D-ribulose, was purified from extracts of Mycobacterium smegmatis grown on D-ribose. The purified enzyme crystalized as hexagonal plates from a 44% solution of ammonium sulfate. The enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis. The molecular weight of the enzyme was between 145,000 and 174,000 by sedimentation equilibrium analysis. Its sedimentation constant of 8.7 S indicates it is globular. On the basis of sodium dodecyl sulfate gel electrophoresis in the presence of Mn2+, the enzyme is probably composed of 4 identical subunits of molecular weight about 42,000 to 44,000. The enzyme was specific for sugars having the same configuration as D-ribose at carbon atoms 1 to 3. Thus, the enzyme could also utilize L-lyxose, D-allose, and L-rhamnose as substrates. The Km for D-ribose was 4 mM and for L-lyxose it was 5.3 mM. The enzyme required a divalent cation for activity with optimum activity being shown with Mn2+. the Km for the various cations was as follows: Mn2+, 1 times 10(-7) M, Co2+, 4 times 10(-7) M, and Mg2+, 1.8 times 10(-5) M. The pH optimum for the enzyme was 7.5 to 8.5. Polyols did not inhibit the enzyme to any great extent. The product of the reaction was identified as D-ribulose by thin layer chromatography and by preparation of the O-nitrophenylhydrazone derivative.     

Purification of 5 beta-reductase from hepatic cytosol fraction of chicken

From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 4-en-3-oxosteroid 5 beta-reductase (EC 1.3.1.23) was purified by ammonium sulfate precipitation, followed by Butyl Toyopearl, DEAE-Sepharose, Sephadex G-75 and hydroxylapatite column chromatographies. The enzyme activity was quantitated from amount of the 5 beta-reduced metabolites derived from [4-14C]testosterone. During the purification procedures, 17 beta-hydroxysteroid dehydrogenase which was present in the cytosol fraction was separated from 5 beta-reductase fraction by the Butyl Toyopearl column chromatography. By the DEAE-Sepharose column chromatography, 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were able to be removed from 5 beta-reductase fraction. The final enzyme preparation was apparently homogeneous on SDS-polyacrylamide gel electrophoresis. Purification was about 13,600-fold from the hepatic cytosol. The molecular weight of this enzyme was estimated as 37,000 Da by SDS-polyacrylamide gel electrophoresis and also by Sephadex G-75 gel filtration. For 5 beta-reduction of 4-en-3-oxosteroids, such as testosterone, androstenedione and progesterone, NADPH was specifically required as cofactor. Km of 5 beta-reductase for NADPH was estimated as 4.22 x 10(-6) M and for testosterone, 4.60 x 10(-6) M. The optimum pH of this enzyme ranged from pH 5.0 to 6.5 and other enzymic properties of the 5 beta-reductase were examined.     

Purification and activation of brain sulfotransferase.

Galactosylceramide sulfotransferase (EC 2.8.2.11) catalyzes the biosynthesis of sulfatide from galactocerebroside and adenosine 3'-phosphate 5'-phosphosulfate (PAPS). This enzyme is developmentally controlled, reaching a maximum activity in the brains of mice corresponding to that of maximum myelination. The product, sulfatide, is an important component of myelin. This transferase from mouse brain has been purified 2600-fold using a combination of pyridoxal 5'-phosphate- and ATP-ligated columns. The purified enzyme yielded a single band following SDS-polyacrylamide gel electrophoresis with an apparent M(r) of 31,000. The entire purification procedure can be completed in 1 day. The pH optimum for the enzyme is 7.0. The Km for PAPS is 1.2 x 10(-6) M, and the Km for cerebroside is 2.6 x 10(-5) M. Cerebroside concentrations > 80 pmol/ml are inhibitory. Enzyme preparations were associated with several lipids. Vitamin K+P(i) activated purified preparations of the sulfotransferase and maintained enzyme activity during storage at -80 degrees C.     

Purification of lactoperoxidase from creek-water buffalo milk and investigation of kinetic and antibacterial properties

Water buffalo lactoperoxidase (WBLP) was purified with Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from skim milk. All purification steps of the WBLP were shown with SDS-PAGE and Rz (A412/A280) controlled the purification degree of the enzyme. Rz value for the purified WBLP was 0.8. To determine purification steps and kinetic properties, the activity of enzyme was measured by using 2,2-azino-bis-(3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a choromogenic substrate at pH=6. Km, Vmax, optimum pH, and optimum temperature for the WBLP were found by means of graphics for ABTS as substrates. Optimum pH and optimum temperature of the WBLP were 6 and 60 degrees C, respectively. Km value at optimum pH and optimum temperature for the WBLP was 0.82 mM. Vmax value at optimum pH and optimum temperature was 13.7 micromol/mL x min. Km value at optimum pH and 25 degrees C for the WBLP was 0.77 mM. Vmax value at optimum pH and 25 degrees C was 4.83 micromol/mL x min. The purified WBLP was found to have high antibacterial activity in a thiocynate-H2O2 medium for some pathogenic bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginose, Shigella sonnei, Staphylococcus saphrophyticus, Staphylococcus epidermidis, and Shigella dysenteriae and compared with well known antibacterial substances such as tetracycline, penicillin, and netilmicine.     

Glycosidases of molluscs. Purification and properties of alpha-L-fucosidase from Chamelea gallina L.

An alpha-L-fucosidase had been purified approximately 300-fold from the liver (hepatopancreas) of the marine mollusc Chamelea gallina L. (= Venus gallina L.). During the different steps of the purification procedure it was difficult to remove the contaminant N-acetylglucosaminidase activity; but, after electrofocusing, a final preparation free of this and other glycosidades present in the crude extract was obtained. The purified enzyme has a broad specificity; it hydrolyzes p-nitrophenyl alpha-L-fucoside and natural substrates such as oligosaccharides containing fucosidic residues with alpha 1--2, alpha 1--3 and alpha 1--4 linkages; also it hydrolyzes fucose-containing glycopeptides, such as thyroglobulin glycopeptide, and glycoproteins as procine submaxillary mucin (previously rendered free of sialic acid). The enzyme has a pH optimum of 5.2 +/- 0.2, with a Km of 7 X 10(-5) M using p-nitrophenyl L-fucoside as substrate. It is inhibited by Hg2+ and some sugars, and activated by CN-, Zn2+, Ca2+ and EDTA. It shows two peaks by isoelectric focusing (at 6.3 and 6.6). The molecular weight of the alpha-L-fucosidase by gel filtration was over 2000000.     

Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes: differential effects on glucosamine 6-phosphate, UDP-N-acetylglucosamine, and ATP levels

Glucose and glucosamine (GlcN) cause insulin resistance over several hours by increasing metabolite flux through the hexosamine biosynthesis pathway (HBP). To elucidate the early events underlying glucose-induced desensitization, we treated isolated adipocytes with either glucose or GlcN and then measured intracellular levels of glucose-6-P (G-6-P), GlcN-6-P, UDP-Glc-NAc, and ATP. Glucose treatment rapidly increased G-6-P levels (t((1/2)) < 1 min), which plateaued by 15 min and remained elevated for up to 4 h (glucose ED(50) = 4mm). In glucose-treated cells, GlcN-6-P was undetectable; however, GlcN treatment (2 mm) caused a rapid and massive accumulation of GlcN-6-P. Levels increased by 5 min ( approximately 400 nmol/g) and continued to rise over 2 h (t((1/2)) approximately 20 min) before reaching a plateau at >1,400 nmol/g (ED(50) = 900 microm). Thus, at high GlcN concentrations, unrestricted flux into the HBP greatly exceeds the biosynthetic capacity of the pathway leading to a rapid buildup of GlcN-6-P. The GlcN-induced rise in GlcN-6-P levels was correlated with ATP depletion, suggesting that ATP loss is caused by phosphate sequestration (with the formation of GlcN-6-P) or the energy demands of phosphorylation. As expected, GlcN and glucose increased UDP-GlcNAc levels (t((1/2)) approximately 14-18 min), but greater levels were obtained with GlcN (4-5-fold for GlcN, 2-fold for glucose). Importantly, we found that low doses of GlcN (<250 microm, ED(50) = 80 microm) could markedly elevate UDP-GlcNAc levels without increasing GlcN-6-P levels or depleting ATP levels. These studies on the dynamic actions of glucose and GlcN on hexosamine levels should be useful in exploring the functional role of the HBP and in avoiding the potential pitfalls in the pharmacological use of GlcN.     

Purification and characterization of a membrane-bound NADPH-cytochrome c reductase capable of catalyzing menadione-dependent O2- formation in guinea pig polymorphonuclear leukocytes

A membrane-bound NADPH-cytochrome c reductase, which is capable of forming the superoxide anion (O2-) in the presence of menadione, was highly purified from membrane fractions of disrupted guinea pig polymorphonuclear leukocytes by solubilization with 0.2% Triton X-100 and chromatographies on Sephacryl S-300 and 2',5'-ADP-agarose. The overall purification from the membrane fraction was over 110-fold, with a yield of about 6%. The purified preparation did not contain two other pyridine nucleotide-oxidizing enzymes: NADH- and NAD(P)H-oxidizing enzymes (J. Biochem. 94, 931-936, 1983). Besides cytochrome c, the purified enzyme was able to reduce menadione, Nitroblue tetrazolium (NBT) and 2,6-dichlorophenolindophenol. The reduction of menadione alone resulted in the formation of O2-. The purified enzyme preparation contained FAD. When assayed by measuring O2--generation in the presence of menadione, the enzyme showed an optimum pH at 7.0-7.4, and Km values for NADPH, NADH, and menadione were 25, 230, and 5.3 microM, respectively. The enzyme activity was not inhibited by NaN3 or dicumarol, but was by N-ethylmaleimide, EDTA, and quercetin; these inhibition profiles agree with those observed for the NADPH oxidase in the membrane fraction of phorbol-myristate acetate-stimulated leukocytes. Furthermore, when compared by means of the NBT-staining method combined with disc gel electrophoresis, the purified enzyme was electrophoretically indistinguishable from the NADPH-NBT reductase in the plasma membrane as well as phagosomes of the leukocytes. These results suggest that the purified NADPH-cytochrome c reductase is the putative flavoprotein of the NADPH oxidase system responsible for the respiratory burst.     

Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas

An ATP diphosphohydrolase (EC 3.6.1.5) from the pancreas of the pig has been characterized and purified. The enzyme which has an optimum pH between 8 and 9 is specific for diphospho- and triphosphonucleosides. The Km values for ADP and ATP are 7.4 and 7.3 x 10(-4) M, respectively, and the purified enzyme has specific activities of 13 and 15.2 mumol of Pi/min/m of protein, respectively. It requires calcium or magnesium ions and it is insensitive to ATPase inhibitors, namely oligomycin, ouabain, and ruthenium red, and to levamisole, an inhibitor of alkaline phosphatase. Denaturation experiments, by heat and trypsin treatments, indicated that only one enzyme is involved. This is confirmed by the solubilization and purification process and by polyacrylamide gel electrophoresis. A 270-fold purification was obtained by centrifugation and successive column chromatography on Sepharose 4B and Affi-Gel blue. It is a glycoprotein with a molecular weight of 65,000 as estimated by polyacrylamide gel electrophoresis.     

Dog liver glucose-6-phosphate dehydrogenase: purification and kinetic properties

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U x mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M(r) of 52.5 kDa suggested the faster moving band on native protein staining was the monomeric form of the enzyme.Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q10, for the enzymatic reaction were calculated to be 8.96, 8.34 kcal x mol(-1), and 1.62, respectively.The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 122 +/- 18 microM for glucose-6-phosphate (G6P) and 10 +/- 1 microM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (kcat/Km) of 1.86 x 10(6) and 5.55 x 10(6) M(-1) x s(-1), respectively. The intrinsic Km value for 2-deoxyglucose-6-phosphate was 24 +/- 4mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, Km d-ANADP was 23 +/- 3mM; Km for G6P remained the same as with NADP as coenzyme (122 +/- 18 microM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M(-1) x s(-1), respectively. Dog liver G6PD was inhibited competitively by NADPH (K(i)=12.0 +/- 7.0 microM). Low K(i) indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway.     

Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidine.

Pyridoxal kinase has been purified 2,000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Pyridoxal kinase, 60,000 molecular weight, catalyzes the phosphorylation of pyridoxal (Km = 2.5 x 10(-5) M) and pyridoxine (Km = 1.7 x 10(-5) M). Pyridoxamine is not a substrate of the purified kinase. Irradiation of the kinase in the presence of riboflavin leads to irreversible loss of catalytic activity. Riboflavin binds to the kinase with a KD = 5 microM as shown by fluorometric titrations. Singlet excited oxygen, generated by energy transfer from the lowest triplet of riboflavin to oxygen, acts as the oxidizing agent of approximately one histidine residue per mol of enzyme. The amino acid residues tyrosine, tryptophan, and cysteine are not photooxidized by the sensitizer bound to the enzyme. It is postulated that histidine is involved in the binding of the substrate ATP to the catalytic site of pyridoxal kinase.     

Purification of D-myo-inositol 1,4,5-trisphosphate 3-kinase from rat brain

The ATP-dependent, calmodulin-sensitive 3-kinase responsible for the conversion of D-myo-inositol 1,4,5-trisphosphate to D-myo-inositol 1,3,4,5-tetrakisphosphate has been purified 2,700-fold from rat brain to a specific activity of 2.3 mumol/min/mg protein. A method of purification is described involving chromatography on phosphocellulose, Orange A dye ligand, calmodulin agarose, and hydroxylapatite columns. Neither the highly purified enzyme nor enzyme eluting from the phosphocellulose column were activated by Ca2+. However, enzyme in the 100,000 x g supernatant from rat brain was activated by Ca2+ over the range from 10(-7) to 10(-6) M and Ca2+ sensitivity of the purified enzyme was restored by the addition of calmodulin. The enzyme has a catalytic subunit Mr of 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Size exclusion chromatography of the purified enzyme on a Superose 12 column gave a Mr value of 70,000, indicating that the purified enzyme was present as a monomer. In contrast, the 100,000 x g supernatant and the purified enzyme after addition of calmodulin and 10(-6) M Ca2+ chromatographed on size exclusion chromatography with a Mr of 150,000-160,000. These results imply that the native enzyme is a dimeric structure of two catalytic subunits plus calmodulin. The purified enzyme showed a Km of 0.21 +/- 0.08 microM for D-myo-inositol 1,4,5-trisphosphate and had a pH optimum of 8.5. Addition of calmodulin increased both the Km and the Vmax of the purified enzyme about 2-fold. The high affinity of the 3-kinase for D-myo-inositol 1,4,5-trisphosphate together with its activation by Ca2+/calmodulin suggests that this enzyme may exert an important regulatory role in inositol phosphate signaling by promoting the formation of additional inositol polyphosphate isomers.     

Solubilization of the Membrane-Bound Sialidase from Pig Brain by Treatment with Bacterial Phosphatidylinositol Phospholipase C

The total pellet from pig forebrain (from which the cytosolic sialidase was completely washed out) was treated with phosphatidylinositol phospholipase C (PIPLC) and centrifuged at high speed. The supernatant contained sialidase and 5'-nucleotidase activities. The greatest liberation of sialidase was obtained after incubation for 20 min with PIPLC at 37 degrees C using pH 6.0 and a ratio between PIPLC (as units) and protein of 1.6. Under these conditions, the release of sialidase, 5'-nucleotidase, and protein was 22, 50, and 18.5%, respectively. On treatment with PIPLC, a purified preparation of pig brain neuronal (synaptosomal) membranes released 28% of its sialidase whereas a purified preparation of pig brain lysosomes did not liberate any sialidase activity. The pH optimum of sialidase present in the supernatant obtained after PIPLC treatment of the total pellet was 4.2, the same as that of the enzyme embedded in the membrane. When this supernatant was subjected to ammonium sulfate fractionation, 88% of its sialidase, having a pH optimum of 4.2, was recovered in the fraction precipitated between 20 and 45% of salt saturation and subsequently dialyzed. Ammonium sulfate treatment caused the appearance of a second sialidase activity, having a pH optimum of 6.6 and behaving on fractionation similarly to the pH 4.2 sialidase. The Km and Vmax values of pH 4.2 and pH 6.6 sialidase were similar (1.48 x 10(-4) and 0.98 x 10(-4) M for Km and 1.6 and 1.4 mU/mg of protein for Vmax, respectively), whereas the stability on standing at 4 degrees C or exposure to freezing and thawing cycles was greater for pH 4.2 sialidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the purified microsomal FAD-containing monooxygenase from mouse and pig liver

The FAD-containing monooxygenase (FMO) has been purified from both mouse and pig liver microsomes by similar purification procedures. Characterization of the enzyme from these two sources has revealed significant differences in catalytic and immunological properties. The pH optimum of mouse FMO is slightly higher than that of pig FMO (9.2 vs. 8.7) and, while pig FMO is activated 2-fold by n-octylamine, mouse FMO is activated less than 20%. Compounds, including primary, secondary and tertiary amines, sulfides, sulfoxides, thiols, thioureas and mercaptoimidazoles were tested as substrates for both the mouse and pig liver FMO. Km- and Vmax-values were determined for substrates representative of each of these groups. In general, the mouse FMO had higher Km-values for all of the amines and disulfides tested. Mouse FMO had Km-values similar to those of pig FMO for sulfides, mercaptoimidazoles, thioureas, thiobenzamide and cysteamine. Vmax-values for mouse FMO with most substrates was approximately equal, indicating that as with pig FMO, breakdown of the hydroxyflavin is the rate limiting step in the reaction mechanism. Either NADPH or NADH will serve as an electron donor for FMO, however, NADPH is the preferred donor. Pig and mouse FMOs have similar affinity for NADPH (Km = 0.97 and 1.1 microM, respectively) and for NADH (Km = 48 and 73 microM, respectively). An antibody, prepared by immunizing rabbits with purified pig liver FMO, reacts with purified pig liver FMO but not with mouse liver FMO, indicating structural differences between these two enzymes. This antibody inhibited pig FMO activity up to 60%.     

Purification and some important characters of extracellular inulinase of Alternaria alternata (Fr.) Keissler

Protein precipitate of cell-free dialysate of extracellular inulinase (2,1-beta-fructan fructanohydrolase, EC 3.2.1.7) of A. alternata was maximally obtained by methanol. Such protein was fractionated by using 2-step column chromatography on Sephadex G150 and DEAE-cellulose. The partially purified enzyme had activity of 81 x 10(3) U/mg protein, with a yield of 69% of the original activity and the fold of purification was 62. Optimum temperature and pH for the activity of the purified enzyme were found to be 55 degrees C and 4.5, respectively. The enzyme was found to be stable up to 55 degrees C and in pH range of 4 to 5. Ba2+ and Ca2+ were found to stimulate the enzyme activity while Cu2+, Fe3+, Hg2+, and iodoacetate were recorded as strong inhibitors. T(1/2) of the enzyme was estimated to be two weeks and its apparent Km was calculated to be 0.066 M. The enzyme recorded hydrolyzing activity against sucrose and raffinose recording I/S ratio of 0.50. Molecular mass of the enzyme preparation was estimated by gel filtration and found to be 115 +/- 5 kDa.     

Thiamine Triphosphatase in the Membranes of the Main Electric Organ of Electrophorus electricus: Substrate-Enzyme Interactions

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate (TTP). Membrane fractions prepared from this tissue contain a thiamine triphosphatase that is strongly activated by anions and irreversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion transport inhibitor. Kinetic parameters of the enzyme are markedly affected by the conditions of enzyme preparation: In crude membranes, the apparent Km is 1.8 mM and the pH optimum is 6.8, but trypsin treatment of these membranes or their purification on a sucrose gradient decreases both the apparent Km (to 0.2 mM) and the pH optimum (to 5.0). Anions such as NO3- (250 mM) have the opposite effect, i.e., even in purified membranes, the pH optimum is now 7.8 and the Km is 1.1 mM; at pH 7.8, NO3- increases the Vmax 24-fold. TTP protects against inhibition by DIDS, and the KD for TTP could be estimated to be 0.25 mM, a value close to the apparent Km measured in the same purified membrane preparation. Thiamine pyrophosphate (0.1 mM) did not protect against DIDS inhibition. At lower (10(-5)-10(-6) M) substrate concentrations, Lineweaver-Burk plots of thiamine triphosphatase activity markedly deviate from linearity, with the curve being concave downward. This suggests either anticooperative binding or the existence of binding sites with different affinities for TTP. The latter possibility is supported by binding data obtained using [gamma-32P]TTP. Our data suggest the existence of a high-affinity binding site (KD of approximately 0.5 microM) for the Mg-TTP complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of inosine 5'-phosphate in activating glucose-bisphosphatase

Glucose-bisphosphate (G1c-1,6-P2) phosphatase has been purified greater than 200-fold from the cytosol of mouse brain. As reported earlier, the enzyme requires inosine monophosphate (IMP) and Mg2+ for activity [Guha, S.K., & Rose, Z. B. (1982) J. Biol. Chem. 257, 6634-6637]. Kinetic parameters and the role of IMP have been further investigated. When Glc-1,6-P2 and IMP are both varied, double-reciprocal plots of the data form a parallel line pattern. With 2 mM Mg2+, the Km obtained for G1c-1,6-P2 is 20 microM and the Ka for IMP is 9 microM. Co2+, Mn2+, and Ni2+ activate less effectively than Mg2+. The apparent Ka for Mg2+ decreases with increasing G1c-1,6-P2, and the observed Km of G1c-1,6-P2 decreases with increasing Mg2+. The extrapolated value of the Ka of Mg2+ at infinite substrate is 86 microM. Mg2+ does not affect the Ka of IMP. The phosphatase activity is optimal at pH 7. The phosphatase is not completely specific since mannose 1,6-bisphosphate is hydrolyzed and guanosine monophosphate activates. However, fructose 1,6-bisphosphate is no more than a poor inhibitor, and adenine nucleotides are neither activators nor inhibitors. The products of the reaction are glucose-1-P and glucose-6-P, in a ratio of 2:3, and Pi. Both glucose-P's are competitive inhibitors with respect to IMP [Ki(glucose-1-P) = 5 microM; Ki(glucose-6-P) = 18 microM]. Neither glucose-P competes with G1c-1,6-P2. The demonstration of an exchange reaction between G1c-1,6-P2 and glucose-6-P is evidence for the phosphorylation of the enzyme by the substrate. The exchange reaction requires Mg2+ and is inhibited by IMP. The observation of the exchange reaction and its elimination by IMP indicates that the low level of phosphoglucomutase activity that remains with the phosphatase throughout purification is an inherent property of the phosphatase. The requirement of glucose-bisphosphatase for the nucleotide IMP is consistent with possible roles for both G1c-1,6-P2 and IMP in the control of the ATP level in the brain.