Purification and properties of the Pichia guilliermondii acid nucleotide pyrophosphatase hydrolyzing flavin adeninine dinucleotide

Acid nucleotide pyrophosphatase was isolated from the cell-free extracts of Pichia guilliermondii Wickerham ATCC 9058. The enzyme was 25-fold purified by saturation with ammonium sulphate, gel-filtration on Sephadex G-150 column and ion-exchange chromatography on DEAE-Sephadex A-50 column. The pH optimum was 5.9, temperature optimum--45 degrees C. The enzyme catalyzed the hydrolysis of FAD, NAD+ and NADH, displaying the highest activity with NAD+. The Km, values for FAD, NAD+ and NADH were 1.3 x 10(-5) and 2.9 x 10(-4) M, respectively. The hydrolysis of FAD was inhibited by AMP, ATP, GTP, NAD+ and NADP+. The K1 for AMP was 6.6 x 10(-5) M, for ATP--2.0 X 10(-5) M, for GTP--2.3 X 10(-6) M, for NAD+--1.7 X 10(-4) M. The molecular weight of the enzyme was 136 000 as estimated by gel-filtration on Sephadex G-150 and 142 000 as estimated by thin-layer gel-filtration chromatography on Sephadex G-200 (superfine). Protein-bound FAD of glucose oxidase was not hydrolyzed by acid nucleotide pyrophosphatase. The enzyme was stable at 2 degrees C in 0.05 M tris-maleate buffer, pH 6.2. Alkaline nucleotide pyrophosphatase hydrolyzing FAD was also detected in the cells of P. guilliermondii.     

5'-Nucleotidase of human placental trophoblastic microvilli possesses cobalt-stimulated FAD pyrophosphatase activity

An enzyme with FAD pyrophosphatase activity was extracted from human placental syncytiotrophoblast microvilli and purified to near-homogeneity. The enzyme has been identified as 5'-nucleotidase by several criteria. Throughout purification, parallel increases in the specific activities of FAD pyrophosphatase and AMP phosphatase were observed. The enzyme was a glycoprotein with a subunit molecular weight of 74,000. EDTA treatment resulted in a marked decline in both activities, and restoration of FAD pyrophosphatase activity but not 5'-nucleotidase activity was accomplished by the addition of Co2+ or, to a lesser extent, Mn2+. The substrate specificity of the 5'-nucleotidase activity that we observed agreed closely with the results of others. The pyrophosphatase activity was relatively specific for FAD. ADP, ATP, NAD(H), and FMN were not hydrolyzed, and ADP strongly inhibited both activities. For FAD pyrophosphatase activity, a Km of 1.2 x 10(-5) M and a Vmax of 1.1 mumol/min/mg protein were determined in assays performed in the presence of Co2+. In the absence of added Co2+, the Vmax declined but the Km was unchanged. For 5'-nucleotidase (AMP as substrate) the Km was 4.1 x 10(-5) M and the Vmax 109 mumol/min/mg protein. Hydrolysis of FMN to riboflavin was observed in partially purified detergent extracts of microvilli that contained alkaline phosphatase activity and lacked FAD pyrophosphatase and 5'-nucleotidase activity. The presence of both FAD pyrophosphatase and FMN phosphatase activities in syncytiotrophoblast microvilli supports the view that the placental uptake of vitamin B2 involves the hydrolysis of FAD and FMN to riboflavin which is then absorbed, a sequence postulated for intestinal absorption and liver uptake.     

Purification and properties of the riboflavin kinase of the yeast Pichia guilliermondii]

Riboflavin kinase (E.C.2.7.1.26) was isolated from the cells of the yeast Pichia guilliermondii. The enzyme was 680-fold purified uzing ammonium sulphate fractionation, chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50 and gel-filtration through Sephadex G-75. Purified enzyme preparation was free from phosphatases and FAD-synthetase. The pH optimum was 8,7, the temperature optimum-45 degrees C. The enzyme was activated by Zn2+, Mg2+ and Co2+ ions. Km for riboflavin was 1,0x10(-5) M, for ATP -- 6,7X10(-6) M. Riboflavin kinase catalyzed the phosphorylation of riboflavin analogues with the substitution of methyl groups at positions 7 and 8. UTP, GTP, ADP and CTP, besides ATP, were phosphate donors. AMP inhibited the enzyme activity. Molecular weight of the enzyme was 28000, as estimated by gel-filtration through Sephadex G-150. Purified riboflavin kinase was stable under storage.     

A rat liver lysosomal membrane flavin-adenine dinucleotide phosphohydrolase: purification and characterization

An enzyme hydrolyzing flavin-adenine dinucleotide (FAD) to flavin mononucleotide and AMP was identified and purified from rat liver lysosomal (Tritosomal) membranes. The purified enzyme showed a single band on silver-stained denaturing gels with an apparent Mr 70,000. Periodate-Schiff staining after denaturing gel electrophoresis of whole membrane preparations revealed that this enzyme is one of the major glycoproteins in lysosomal membranes. FAD appeared to be the preferred substrate for the purified enzyme; equivalent concentrations of NAD or CoA were hydrolyzed at about one-half of the FAD rate. Negligible activity (less than or equal to 16%) was noted with ATP, TTP, ADP, AMP, FMN, pyrophosphate, or p-nitrophenylphosphate. The enzyme was inhibited by EDTA or dithiothreitol. It was stimulated by Zn, and was not affected by Ca or Mg ions, nor by p-chloromercuribenzoate. The pH optimum for FAD hydrolysis was 8.5-9 with an apparent Km of 0.125 mM. Antibodies prepared against the purified enzyme partially (50%) inhibited FAD phosphohydrolase activity in lysosomal membrane preparations but had no effect on the soluble lysosomal acid pyrophosphatase known to hydrolyze FAD. This enzyme could not be detected immunochemically in preparations of microsomes, Golgi, plasma membranes, mitochondrial membranes, or the soluble lysosomal fraction, suggesting that the enzyme is different from either soluble lysosomal acid pyrophosphatase or other FAD hydrolyzing activities in the liver cell.     

Purification and characterization of human seminal plasma aminopeptidase

A 50.4-fold purification of aminopeptidase is achieved by alcohol precipitation, DEAE-cellulose, CM-cellulose and finally Sephadex G-200 chromatography. On polyacrylamide gel electrophoresis of the purified enzyme after molecular sieving on Sephadex G-200, only one band was obtained, suggesting that the enzyme preparation was obtained almost homogeneous by three steps of column chromatography. Aminopeptidase showed highest activity at pH 7.0, using a buffer system, of 70 mM Na-phosphate. The enzyme was found to be active at 40 degrees C, even at 60 degrees C (80% activity), suggesting that the human seminal plasma enzyme is fairly thermostable. Amongst the various aminoacyl derivatives evaluated as substrates in the present study, L-alanine beta-naphthylamide hydrochloride was found to have the highest rate of hydrolysis. Ovalbumin showed effective cleavage in comparison to that of other natural substrates. The Km value for the purified seminal plasma aminopeptidase towards L-alanine beta-naphthylamide hydrochloride was 4 x 10(-4) M. Hg+2 showed highest inhibitory effect than other metal ions tested in the present study. Concentration causing 50% inhibition of the enzyme (I50) by Hg2+ was 4.7 x 10(-6) M. Inhibition by EDTA at 1 mM concentration in the incubation system was higher than by EGTA and sodium azide, suggesting that the enzyme contains a metallo group at the active site. A 50% inhibition of the enzyme by EDTA was obtained at 5.11 x 10(-3) M. The Ackerman and Potter plot for EDTA inhibition suggests that EDTA is a reversible inhibitor of seminal plasma aminopeptidase. A single molecular form of aminopeptidase was found to be present in human seminal plasma as shown by polyacrylamide activity gel electrophoresis.     

Diguanosinetetraphosphatase from rat liver: Acitivity on diadenosine tetraphosphate and inhibition by adenosine tetraphosphate.

The hydrolysis of diadenosine tetraphosphate, a compound previously described by others to occur in liver at concentrations of around 0.1 mu M, is carried out by a specific enzyme. This enzyme has been partially purified from rat liver extracts, and the following properties have been found. The Km value for diadenosine tetraphosphate is 2 mu M; the products of hydrolysis are ATP and AMP; the Km value for diguanosine tetraphosphate is 2 mu M; none of the following substances were substrates of the enzyme: diadenosine triphosphate, diguanosine di and triphosphates, adenosine tetraphosphate, ATP, ADP, NAD+, NADP+ and bis-p-nitrophenylphosphate. Cyclic AMP was not an inhibitor of the reaction. The enzyme requires Mg2+ ions, is maximally active at a pH value of approximately 8, and has a molecular weight of 22000 as estimated by filtration on Sephadex G-100. The activation energy of the reaction was of 10250 cal times mol-1 (42886 J times mol-1). Particularly striking is the inhibition by adenosine tetraphosphate (Ki equals 48 nM) and guanosine tetraphosphate (Ki equals 14 nM). Other nucleotides tested were also competitive inhibitors with Ki values in the 10--100 mu M range.     

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.     

Three diadenosine 5',5'-P1,P4-tetraphosphate hydrolytic enzymes from Physarum polycephalum with differential effects by calcium: a specific dinucleoside polyphosphate pyrophosphohydrolase, a nucleotide pyrophosphatase, and a phosphodiesterase

Two new enzymes that hydrolyze diadenosine tetraphosphate (Ap4A) have been isolated from the acellular slime mold Physarum polycephalum. Both enzymes are different from the Physarum Ap4A symmetrical pyrophosphohydrolase previously described on the basis of their substrate specificities, reaction products, molecular weights, and divalent cation requirements. One enzyme is a nucleotide pyrophosphatase that asymmetrically hydrolyzes Ap4A to AMP and ATP. This enzyme hydrolyzes several mono- and dinucleotides with the corresponding nucleotide monophosphate as one of the products. The percentage hydrolysis of NAD+, Ap4A, and Ap4G, each at 10 microM, was 100, 56, and 51, respectively. A divalent cation is required for activity, with Ca2+ yielding 20-30 times greater activity than Mg2+ or Mn2+. Values of Km for Ap4A and Vmax are similar to the corresponding values for Ap4A symmetrical pyrophosphohydrolase. The second enzyme is a phosphodiesterase I with broad substrate reactivity. This enzyme also asymmetrically hydrolyzes Ap4A, but it does not hydrolyze NAD+. Activity of the phosphodiesterase I is stimulated by divalent cations, with Ca2+ being 50-60 times more stimulatory than Mg2+ or Mn2+. The apparent molecular weights of the nucleotide pyrophosphatase and phosphodiesterase are 184,000 and 45,000, respectively. In contrast, the Ap4A pyrophosphohydrolase hydrolyzes Ap4A to ADP, is inhibited by Ca2+ and other divalent cations, and has an apparent molecular weight of 26,000 as previously reported.     

Purification and physico-chemical properties of collagenase synthesized by a bacterium of the type Acinetobacter sp.]

The Acinetobacter spec collagenase has been almost completely purified. This enzyme is a true collagenase the activity of which is high on collagen. The enzyme is active on insoluble collagen, gelatin and the synthetic Pz-peptide, but has no proteolytic activity on casein or bovine serum-albumin. The collagenase was obtained on a simple medium with gelatin and yeast extract. The enzyme was purified by (NH4)2SO4 precipitation. DEAE cellulose column chromatography, Sephadex G 200 gel-filtration. The molecular weight of the enzyme was found to be 102 000 daltons, and its isoelectric point was found to be 7,7 +/- 0,2. The optimum pH and temperature for insoluble collagen hydrolysis were 7.6 and 37 degrees C, respectively; so, this collagenase corresponds to true collagenase. Hydrolysis of Pz-peptide is activated by Ca2+ and inhibited by metal ions (Cu2+, Fe3+, Zn2+, Pb2+, Hg2+). EDTA and o-phenanthroline induced a very significant reduction in enzyme activity. Iodoacetate and p-CMB induced a slight reduction in enzyme activity only at high concentrations (10-2M). The collagenase is most stable for temperatures less than or equal to 50 degrees C.     

Purification and characterization of cathepsin L-like proteinase from goat brain

Cathepsin L-like proteinase was purified approximately 1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation, Sephadex G-100 column chromatography and ion-exchange chromatography on CM-Sephadex C-50 at pH 5.0 and 5.6. The molecular weight of proteinase was found to be approximately 65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mbetaNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-beta-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mbetaNA was hydrolyzed maximally by the enzyme (Km value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mbetaNA, Z-Phe-Val-Arg-4mbetaNA, Z-Arg-Arg-4mbetaNA and Z-Ala-Arg-Arg-4mbetaNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and beta-mercaptoethanol. It was strongly inhibited by p-hydroxymercuribenzenesulphonic acid, iodoacetic acid, iodoacetamide and microbial peptide inhibitors, leupeptin and antipain. Leupeptin inhibited the enzyme competitively with Ki value 44 x 10(-9) M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg(2+), Ca(2+), Cu(2+), Li(2+), K(+), Cd(2+), Ni(2+), Ba(2+), Mn(2+), Co(2+) and Sn(2+) also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40 degrees C. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mbetaNA was approximately 50-55 degrees C with an activation energy Ea of approximately 6.34 KCal mole(-1).     

Characterization of Haemophilus influenzae nucleotide pyrophosphatase. An enzyme of critical importance for growth of the organism

A nucleotide pyrophosphatase isolated from Haemophilus influenzae was purified to electrophoretic homogeneity and characterized with respect to molecular weight, substrate specificity, pH profile, thermal stability, functional group involvement, and effectiveness of selective inhibition. The enzyme catalyzes the hydrolysis of NAD to NMN and AMP and appears located appropriately to facilitate the internalization of NAD needed to satisfy the V-factor growth requirement of the organism. In the processing of NAD and structurally related substrates, the enzyme exhibited negative cooperativity. Structural alterations in the purine moiety of these dinucleotide substrates had pronounced effects on the negative cooperativity of the enzyme. AMP, ADP, and several related nucleotides were observed to be effective substrate-competitive inhibitors of the enzyme. Several of the dinucleotides serving as substrates for the nucleotide pyrophosphatase were evaluated with respect to substituting for NAD in supporting growth of the organism. AMP and ADP inhibited growth of the organism when NAD served as V-factor, and this inhibition correlated well with the inhibitory effects of these nucleotides on the purified nucleotide pyrophosphatase.     

Isolation and characterization of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum

A new enzyme that hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate has been purified by a factor of 250 from the acellular slime mold Physarum polycephalum. Activity was assayed radioisotopically with [3H]Ap4A. Isolation of the enzyme was facilitated by dye-ligand chromatography. The enzyme symmetrically hydrolyzes Ap4A to ADP and exhibits biphasic kinetics for the substrate with values for the apparent Km of 2.6 micro M and 37 micro M. The two values of Vmax differ by a factor of 10. Mg2+, Ca2+, and other divalent cations inhibit the activity with 40-80% inhibition occurring at 0.5 mM. Mg2+, at 0.5 mM, decreases both values of Vmax by 50%, decreases the low Km value by about 30%, and increases the high Km value by about 100%. (Ethylenedinitrilo)tetraacetic acid (EDTA) and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), at 10 mM, inhibit the activity by 50%. ADP, ATP, Ap4, and Gp4 are equipotent inhibitors with 50% inhibition occurring at 30 micro M. AMP is a relatively weak inhibitor. The molecular weight of the enzyme is 26000 on the basis of elution of activity from a calibrated Sephadex G-75 column.     

Purification and characterization of arylamidase from monkey brain.

Arylamidase [EC3.4.11.2] was isolated from monkey brain extract and purified about 2100-fold in approximately 11% yield by a six-step procedure comprising extraction from monkey brain homogenate, ammonium sulfate fractionation, first hydroxylapatite chromatography, DEAE-cellulose chromatography, Sephadex G-200 gell filtration and second hydroxylapatite chromatography. The enzyme showed a single band on polyacrylamide disc electrophoresis and consisted of a single polypeptide chain, as judged by disc electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was strongly inhibited by PCMB, TPCK, and puromycin. Puromycin competitively inhibited the enzyme and the Ii value was about 5 x 10(-7)M. Treatment with EDTA resulted in a loss of enzyme activity. The enzyme activity was restored by addition of Zn2+, Co2+, Mn2+. Among various amino acid beta-naphthylamides, L-alanine beta-naphthylamide was most rapidly hydrolyzed and N-carbobenzoxyl-L-leucine beta-naphthylamide was not hydrolyzed by this enzyme preparation. The molecular weight of the enzyme was 92,000 as determined by gel filtration on Sephadex G-200.     

Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays.

An activity that inhibited both glutamine synthetase (GS) and nitrate reductase (NR) was highly purified from cauliflower (Brassica oleracea var. botrytis) extracts. The final preparation contained an acyl-CoA oxidase and a second protein of the plant nucleotide pyrophosphatase family. This preparation hydrolysed NADH, ATP and FAD to generate AMP and was inhibited by fluoride, Cu2+, Zn2+ and Ni2+. The purified fraction had no effect on the activity of NR when reduced methylviologen was used as electron donor instead of NADH; and inhibited the oxidation of NADH by both spinach NR and an Escherichia coli extract in a time-dependent manner. The apparent inhibition of GS and NR and the ability of ATP and AMP to relieve the inhibition of NR can therefore be explained by hydrolysis of nucleotide substrates by the nucleotide pyrophosphatase. We have no evidence that the nucleotide pyrophosphatase is a specific physiological regulator of NR and GS, but suggest that nucleotide pyrophosphatase activity may underlie some confusion in the literature about the effects of nucleotides and protein factors on NR and GS in vitro.     

Studies on nucleotidases in plants. Dimerization of the crystalline mung bean nucleotide pyrophosphatase by 5'-adenosine monophosphate and the properties of the dimerized enzyme.

The addition of AMP to the crystalline and homogeneous mung bean nucleotide pyrophosphatase [EC 3.6.1.9]altered its electrophoretic mobility. AMP was tightly bound to the enzyme and was not removed on passage through a column of Sephadex G-25 or on electrophoresis. The molecular weight of the native and AMP-modified enzymes were 65,000 and 136,000, respectively. The properties of the native enzyme such as the pH (9.4) and temperature (49 °C) optima, inhibition by EDTA, reversal of EDTA-inhibition by Zn²⁺ and Co²⁺, were not altered on dimerization by AMP. The AMP-modified enzyme had a linear time-course of reaction, unlike the native enzyme which exhibited a biphasic time-course of reaction. The AMP-modified enzyme was irreversibly denatured by urea. AMP concentrations larger than 100 μM inhibited linearly the activity of the AMP-modified enzyme. ADP and ATP inhibited the activity in a sigmoidal manner. K_m and V of the native and AMP-modified enzymes were, 0.25 mm and 0.58 mm; and 3.3 and 2.5, respectively.     

Aminopeptidase PC from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica

Homogeneous aminopeptidase PC was isolated with yield 67% and purification degree 237 from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica by ion-exchange chromatography on DEAE-Sepharose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephadex G-150. The enzyme is a homodimer with a molecular mass 220 kD (110 x 2). Aminopeptidase PC has pI = 4.1. It hydrolyzes Leu-pNA optimally at pH 6.0 and at the optimum temperature 36-40 degrees C; in the presence of Ca2+ the enzyme is stable at pH 5.5-8.0. Aminopeptidase PC is activated by Ca2+, Mg2+, and Fe2+; it is completely inhibited by EDTA, o-phenanthroline, and bestatin. The enzyme contains four Zn atoms per molecule and is therefore a metalloaminopeptidase. The aminopeptidase PC can effectively cleave N-terminal Arg and Lys residues as well as Leu, Phe, and Met residues. Km and kcat values for hydrolysis of Leu-pNA were 0.075 mM and 0.19 sec-1 and for hydrolysis of Arg-pNA 0.078 mM and 0.48 sec-1, respectively. D-Amino acid residues cannot be cleaved. Thus, aminopeptidase PC of the Kamchatka crab has a mixed substrate specificity which is characteristic of some microbe aminopeptidases. Its N-terminal sequence ESVEIELPEGLSPLV is 46% coincident with that of yeast vacuolar aminopeptidase YSCA.     

Inhibition of the malic enzyme from Acinetobacter calcoaceticus by NADPH and NADH]

The malic enzyme enriched from Acinetobacter calcoaceticus is inhibited by NADPH and NADH. The inhibition afforded by the reduced coenzymes is not affected by NAD+, AMP and 3'.5'-AMP. Against L-malate, NADPH inhibits the enzyme in a noncompetitive linear fashion (Ki = 1.5 x 10(-4) M), against NADP+, competitively linearly (Ki = 5.0 x 10(-5) M). While NADPH acted as a product inhibitor, NADH seems to be an allosteric effector of the malic enzyme, because with L-malate as the variable substrate in the double reciprocal plot, a nonlinear curve is obtained.     

Acid proteinase from the roe of the trout Salmo gairdneri R

Acidic proteinase from the trout spawn is 640 fold purified (yield 22%). Purification includes autolysis, acid treatment, ammonium sulphate fractionation, G-100 Sephadex gel-filtration, ion-exchange chromatography on DEAE-cellulose. Molecular mass of the enzyme under study is 70 kDa according to the data of gel-filtration. Acidic proteinase displays its greatest activity towards hemoglobin (pH 4.0, 37 degrees C) and is inhibited completely by EDTA, by 50%--by Pb2+ and soya inhibitor of trypsin and 2.8 times activated by Zn2+. Enzyme activity is not affected by dithiotreitol, iodine acetate, phenylmethylsulphonylfluoride parachloromercurybenzoate, Hg2+, Na+, Co2+, Ca2+.     

Purification and Characterization of β-d-Galactosidase and α-d-Mannosidase from Papaya (Carica papaya) Seeds

β-D-Galactosidase was purified 115-fold from a saline extract of papaya seeds by fractionation with ammonium sulfate, DEAE-Sephadex chromatography and gel-filtration on Sephadex G-75, G-150, and G-100. The purified β-D-galactosidase (MW, 56,000 daltons) had an isoelectric point (pI) at pH 8.4 and the optimal pH for its activity was 3.5 to 4.5. The enzyme activity was inhibited by Cu²⁺,Ag⁺,Hg²⁺,Pb²⁺,NaAsO₂ and р-chloromercuribenzoate at concentrations of 1x10^-3 M. Among the various mono- and oligosaccharides tested, D-galactose, D-galacturonic acid, D-galactono-γ-lactone and melibiose significantly inhibited the enzyme activities at concentrations of 2xl0^-3 to 1X10^-2M. The purified enzyme hydrolyzed β-nitrophenyl β-D-galactoside (Km = 1.0X10^-3M), methyl β-D-galactoside (Km=1.6x10^-2M), aminoethyl β-D-galactoside (Km =3.3X10^-2M) and lactose (Km = 9.1X10^-2M). β-(l→3)-Linked galactotetraosyl-eryth itol and asialo-glycopeptide isolated from fetuin were also hydrolyzed to the extent of 78 and 75%, 4respectively, on the basis of their galactose contents.

∝-D-Mannosidase from papaya seeds was also purified 130-fold by ammonium sulfate fractionation, DEAE-Sephadex chromatography, gel-filtration on Sephadex G-150 and hydroxylapatite chromatography. The purified enzyme (MW, 156,000 daltons), consisting of two subunits (78,000x2), was inhibited by Hg²⁺,Ag⁺,Cu²⁺, р-chloromercuribenzoate, D-glucose, D-glucosamine and D-mannose at concentrations of lx10^-3 to 1x10^-2M. The ∝-D-mannosidase hydrolyzed р-nitrophenyl ∝-D-mannoside (Km=5.6x10^-3M), methyl ∝-D-mannoside (Km=2.8X10^-2M), ∝-D-mannosyl-D-mannitol (Km=2.2X10^-2M), ∝-(l→2)linked D-mannobiosyl-D-mannitol (Km=6.3x10^-3M) and D-mannotriosyl-D-mannitol (Km=5.3x10^-3 M).     

Pyridine nucleotide interaction with rat liver dihydropteridine reductase.

The interactions of a homogeneous preparation of rat liver dihydropteridine reductase with NADH, NADPH, NAD+, NADP+, and the 1-N6-ethenoadenine derivative of NAD+ have been investigated by fluorescence titration, circular dichroism, equilibrium dialysis, Sephadex G-25 chromatography, and polyacrylamide gel electrophoresis. The procedures indicate that the dimeric enzyme has a definite preference for NADH, but binds only 1 mol of this nucleotide per mol of enzyme. The binary complex of enzyme with NADH is only partially stable to exhaustive dialysis and gel electrophoresis, where it shows greater mobility (0.26) than the free enzyme (0.21); however, the complex can be isolated by Sephadex G-25 chromatography, and characterized with respect to its absorbance spectrum. No ternary complexes are observed when samples of reductase, preincubated with excess NADH, and either the reaction product, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, or the inhibitor, methotrexate, are subjected to polyacrylamide gel electrophoresis.