Human deoxycytidine kinase. Purification and characterization of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia patients.

A procedure for purifying human cytoplasmic and mitochondrial deoxycytidine kinase (NTP:deoxycytidine 5'-phosphotransferase, EC 2.7.1.74) was developed. Both purified isozymes have a similar molecular weight, activation energy and catalyze the reaction by a sequential mechanism. These two isozymes differ with respect to their substrate specificities. With cytoplasmic deoxycytidine kinase, ATP, GTP and TTP have the highest reaction velocity. Pyrimidine nucleoside triphosphates have higher affinity but lower V than purine nucleoside triphosphates. Cytidine and arabinosylcytidine can serve as substrates. With mitochondrial isozyme only ATP gives the highest reaction velocity. ATP and dATP have the same Km but different V values. Besides deoxycytidine, also deoxythymidine but not cytidine or arabinosylcytidine can serve as substrates. There are also differences between these two isozymes with respect to their sensitivity to inhibition. For cytoplasmic enzyme, Br5dCyd and Iodo5dCyd are not inhibitory. Both dCTP and UTP are competitive inhibitors (Ki 0.25 and 0.5 micronM, respectively) with respect to ATP. For mitochondrial isozyme both Br5dCyd and Iodo5dCyd are inhibitory and dCTP and TTP are competitive inhibitors (Ki 2 and 10 micronM, respectively) with respect to ATP.     

Metabolism of threo-beta-fluoroaspartate by H4 cells. Inhibition of adenylosuccinate lyase by fluoro analogs of its substrates

DL-threo-beta-Fluoroaspartate is a substrate for the two enzymes in de novo purine biosynthesis that use aspartate, namely 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) synthetase and adenylosuccinate synthetase. With both enzymes, Vmax with threo-beta-fluoroaspartate is about 50% of that observed with aspartate. The products of the two enzyme reactions, threo-beta-fluoro-SAICAR and threo-beta-fluoroadenylosuccinate, are inhibitors of adenylosuccinate lyase purified from rat skeletal muscle. In 20 mM phosphate buffer, pH 7.4, the KI values for threo-beta-fluoro-SAICAR are 5 and 3 microM and for threo-beta-fluoroadenylosuccinate are 3 and 1 microM, in the SAICAR and adenylosuccinate cleavage reactions, respectively. In 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, the KI values for threo-beta-fluoro-SAICAR are approximately 0.14 and 0.03 microM and for threo-beta-fluoroadenylosuccinate are approximately 0.05 and 0.015 microM, in the same two reactions, respectively. These KI values are one-half to one-hundredth of the Km values for SAICAR and adenylosuccinate, the two substrates of adenylosuccinate lyase. After an 8-h incubation with 45 microM threo-beta-fluoroaspartate, H4 cells contain 200-300 microM threo-beta-fluoro-SAICAR and 60-90 microM threo-beta-fluoroadenylosuccinate. These concentrations of fluoro analogs are sufficient to substantially inhibit adenylosuccinate lyase and hence the de novo synthesis of purines in H4 cells.     

Effect of Mg2, ATP and some analogs on phosphorylase phosphatase from adrenal cortex.

The effect of Mg2, ATP and some of its analogs was studied on the spontaneously active and the ATP-Mg-dependent forms of phosphorylase phosphatase extracted from adrenal cortex. Inhibition of the spontaneously active form was observed with Mg2 (Ki - 9mM), ATP (Ki = 9micronM), 2'-doxy-ATP (Ki = 8 micronM), AtetraP (Ki = 9 micronM), AMP(CH2)PP (Ki = 11 micronM), ADP(CH2)P (Ki = 19 micronM), ADP(NH)P (Ki = 16micronM) and ADP (Ki = 25micronM). Activation of the ATP-Mg-dependent form was obtained with Mg2 (Ka = 0.55mM) (to a lower extent) and with ATP (Ka = 2micronM), 2'-deoxy-ATP (Ka = 6micronM) or AtetraP (Ka = 15micronM) in the presence of 0.5mM Mg2. Activation with AMP(CH2)PP was only observed in the presence of high concentrations (5mM) of Mg2 (Ka = 13micronM). No activation at all was observed with ADP(CH2)P or ADP(NH)P. Even though the activation of the ATP-Mg-dependent form does not seem to involve a kinase reaction, the stimulation by ATP or its analogs is rather specific, since it does not occur with analogs in which a methylene group or a nitrogen is substituted for the oxygen between the beta- and gamma-phosphates.     

The effect of inhibitors on the oxygen kinetics of cytochrome c oxidase.

1. The oxygen kinetics of purified beef heart cytochrome c oxidase were investigated. 2. The effect of addition of various fixed concentrations of the inhibitors CO, HN3, HCOOH, HCN and H2S on the double reciprocal plot of respiration rate against oxygen concentration was studied. 3. CO is strictly competitive, azide and formate are uncompetitive, and cyanide and sulfide are non-competitive inhibitors towards oxygen. 4. Binding constants for the various inhibitors from secondary plots of the oxygen kinetics at pH 7.4 are: CO: Ki = 0.32 micronM, azide: Ki = 33 micronM; formate: Ki = 15 mM; cyanide: Ki = 0.2 micronM and sulfide: Ki = 0.2 micronM. 5. The possible significance of these results in the elucidation of the reaction mechanism is discussed.     

Phospholipid synthesis in isolated fat cells. Studies of microsomal diacylglycerol cholinephosphotransferase and diacylglycerol ethanolaminephosphotransferase activities.

Diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) activities were investigated in microsomes from isolated rat fat cells. Assays based on the conversion of CDP-[14C]choline of CDP-[14C]ethanolamine to phosphatidylcholine or phosphatidylethanolamine utilized ethanol-dispersed diacylglycerols and 1 to 5 microng of protein. Cholinephosphotransferase and ethanolaminephosphotransferase activities had similar dependences on MgCl2 and pH, and were inhibited similarly by CaCl2, organic solvents, Triton X-100, Tween 20, and dithiothreitol. Ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid stimulated both activities similarly. With 1,2-dioleoyl-sn-glycerol, the cholinephosphotransferase activity had an apparent Km for CDP-choline of 23.9 micronM and a V max of 8.54 nmol/min/mg. CDP-ethanolamine and CDP were competitive inhibitors of the cholinephosphotransferase activity (apparent Kl values of 227 micronM and 360 micronM, respectively). With 1,2-dioleoyl-sn-glycerol, the ethanolaminephosphotransferase activity had an apparent Km of 18.3 micronM for CDP-ethanolamine and a V max of 1.14 nmol/min/mg. CDP-choline appeared to be a noncompetitive inhibitor of the ethanolaminephosphotransferase activity (apparent Kl of 1620 micronM). Inhibition of the ethanolaminephosphotransferase activity by CDP appeared to be of a mixed type. The dependences on diacylglycerols containing fatty acids 6 to 18 carbons in length were investigated...     

Studies with GMP synthetase from Ehrlich ascites cells. Purification, properties, and interactions with nucleotide analogs.

GMP synthetase has been purified 57-fold from Ehrlich ascites cells. The enzyme was found to be stable and to have an approximate molecular weight of 85,000 (determined by gel filtration). Its activity was stimulated by dithiothreitol and inhibited by 2-mercaptoethanol, p-chloromercuribenzoate, and hydroxylamine. Both ammonia and glutamine could serve as amino group donors. While none of the 10 triphosphate purine and pyrimidine nucleotides studied were able to substitute for ATP as the energy donor for the reaction, all of these compounds were able to bind to the ATP site. The Ki values for CTP, beta-D-arabinofuranosyl-ATP, and 1-N6-ethenoATP were slightly lower than the Km of ATP (0.28 mM). Six monophosphate nucleotides were aminated by this enzyme. Listed in order of their substrate efficiency (Vmax/Km), they are: xanthosine 5'-phosphate(XMP) (28,000); 2'-dXMP (1,200); 8-azaXMP (320); 6-thioXMP (200); beta-D-arabinofuranosyl-XMP (72); 1-ribosyloxipurinol 5'-phosphate (0.5). 6-ThioXMP was a strong alternative substrate inhibitor with a Ki of 5 muM. The aminated products of the reaction (four studied) were competitive inhibitors with respect to XMP.     

The pur3 gene from the pur cluster encodes a monophosphatase essential for puromycin biosynthesis in Streptomyces

The pur3 gene of the puromycin (pur) cluster from Streptomyces alboniger is essential for the biosynthesis of this antibiotic. Cell extracts from Streptomyces lividans containing pur3 had monophosphatase activity versus a variety of mononucleotides including 3'-amino-3'-dAMP (3'-N-3'-dAMP), (N6,N6)-dimethyl-3'-amino-3'-dAMP (PAN-5'-P) and AMP. This is in accordance with the high similarity of this protein to inositol monophosphatases from different sources. Pur3 was expressed in Escherichia coli as a recombinant protein and purified to apparent homogeneity. Similar to the intact protein in S. lividans, this recombinant enzyme dephosphorylated a wide variety of substrates for which the lowest Km values were obtained for the putative intermediates of the puromycin biosynthetic pathway 3'-N-3'-dAMP (Km = 1.37 mM) and PAN-5'-P (Km = 1.40 mM). The identification of this activity has allowed the revision of a previous proposal for the puromycin biosynthetic pathway.     

New site-directed reversible inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase.

1. CoA-thioether analogues of 3-hydroxy-3-methylglutaryl-CoA containing an additional methyl group at positions 2, 6(methyl at C3) or 4 of the acyl residue were prepared. To probe for hydrophobic interaction, their inhibitory properties were determined with 3-hydroxy-3-methylglutaryl-CoA reductase purified from baker's yeast. The CoA-thioethers were purely competitive inhibitors whose affinity to the reductase was near to that of the physiological substrate. 2. CoA-sulfoxides derived from the CoA-thioethers displayed affinities to the reductase superior to that of the physiological substrate (Km = 7 microM). Depending on the degree of recognition of diastereomers by the enzyme, the inhibitor constants of the two best inhibitors vary from Ki = 200 nM and Ki = 80 nM (diastereomeric mixtures) to 25 nM and 20 nM, respectively (if only one diastereomer would interact with the enzyme).     

Kinetic studies on the ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase. Evidence for the K.S.T. mechanism with two enzyme-ATP complexes and two phosphorylated intermediates of high-energy type.

The kinetic properties of the [3H]ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase [EC 3.6.1,3] were investigated, using NaI-treated microsomes from bovine brain, and the following results were obtained. 1. The rates of the Na+-dependent exchange reaction in the steady state were measured in a solution containing 45 micronM free Mg2+, 100 mMNaCl, 80 micronM ATP, and 160 micronM ADP at pH 6.5 and 4-5 degrees. The rate and amount of decrease in phosphorylated intermediate on adding ADP, i.e., the amount of ADP-sensitive EP, were measured while varying one of the reaction parameters and fixing the others mentioned above. Plots of the exchange rate and the amount of ADP-sensitive EP against the logarithm of free Mg2+ concentration gave bell-shaped curves with maximum values at 50-60 micronM free Mg2+. Plots of the exchange rate and the amount of ADP-sensitive EP against pH also gave bell-shaped curves with maximum values at pH 6.9-7. They both increased with increase in the concentration of NaCl to maximum values at 150-200 mM NaCl, and then decreased rapidly with increase in the NaCl concentration above 200 mM. The dependences of the exchange rate and the amount of ADP-sensitive EP on the concentration of ADP followed the Michaelis-Menten equation, and the Michaelis constants Km, for both were 43 micronM. The dependence of the exchange rate on the ATP concentration also followed the Michaelis-Menten equation, and the Km value was 30 micronM. The amount of ADP-sensitive EP increased with increase in the ATP concentration, and reached a maximum value at about 5 micronM ATP. 2. The N+-dependent [3H]ADP-ATP exchange reaction was started by adding [3H]ADP to EP at low Mg2+-concentration. The reaction consisted of a rapid initial phase and a slow steady phase. The amount of [3H]ATP formed during the rapid initial phase, i.e. the size of the ATP burst, was equal to that of ADP-sensitive EP, and was proportional to the rate in the steady state. At high Mg2+ concentration, the rate of Na+-dependent exchange in the steady state was almost zero, and EP did not show any ADP sensitivity. However, rapid formation of [3H]ATP was observed in the pre-steady state, and the size of the ATP burst increased with increase in the KCl concentration. From these findings, we concluded that an enzyme-ATP complex (E2ATP) formed at low Mg2+ concentration is in equilibrium with EP + ADP, that the rate-limiting step for the exchange reaction is the release of ATP from the enzyme-ATP complex, that the ADP-insensitive EP (formula: see text) produced at high Mg2+ concentration is in equilibrium with the enzyme-ATP complex, and that the equilibrium shifts towards the enzyme-ATP complex on adding KCl. Actually, the ratio of the size of the ATP burst to the amount of EP was equal to the reciprocal of the equilibrium constant of step (formula: see text), determined by a method previously reported by us.     

Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase

The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.     

Thymidylate synthetase from Diplococcus pneumoniae, properties and inhibition by folate analogs.

Thymidilate synthetase (methylenetetrahydrofolate:dUMP C-methyltransferase) in crude extract from Diplococcus pneumoniae exhibits a partial but variable requirement for Mg-2+ depending upon the buffer. Optimum Mg-2+ concentration is between 0.014 and 0.02 M. The optimum pH for activity in a variety of buffers occurred as a broad peak between 7.0 and 7.7. In Tris/acetate buffer, but not in potassium phosphate buffer, the pH optimum was different in the presence and absence of Mg-2+. Methylation of uridylate, cytidylate and deoxycytidylate could not be demonstrated over a pH range of 5.0-8.0. The enzyme exhibited an apparent Km for deoxyuridylate of 3.08 - 10-5 M and an apparent Km for L-(+)(minus)-5,10-methylene tetrahydrofolate of 2.66 - 10-4 M. During molecular-sieve chromatography and sucrose density-gradient centrifugation, the enzyme was detectable only as a single catalytically active form of Mr 34 000-38 000. 2,4-Diamino quinazoline antifolates were better competitive inhibitors (Ki = 3-8 -10-6 M) of thymidylate synthetase than 2,4-diamino pteridines (Ki = 3- 10-5 M). 2-Amino-4-hydroxy-quinazolines were the best inhibitors (Ki = 1.3-2.9 - 10-6 M). All of the 2,4-diamino quinazolines and pteridines inhibited dihydrofolate reductase from D. pneumoniae in a nearly stoichiometric fashion (Ki = less than 10-10 M). The 2-amino-4-hydroxy-quinazolines were poor inhibitors of this enzyme (Ki = 10=5 M).     

Phosphate transport in Micrococcus lysodeikticus.

Phosphate accumulates in Micrococcus lysodeikticus cells against a concentration gradient, by an energy-dependent process. The phosphate transport is derepressed during phosphate deprivation. The depression process is inhibited by chloramphenicol. The apparent Km of phosphate transport is 4.3 micronM. The activation energy of the transport is 21 kcal per mol in the temperature range of 0-29degrees C, and 4.9 kcal per mol between 29 and 40degrees C. The rate of the transport increases in presence of K+ and Mg2+. Arsenate is a competitive inhibitor of phosphate transport, having an apparent Ki of 6.0 micronM. Sulfhydryl reagents, respiratory inhibitors and uncouplers of oxidative phosphorylation inhibit phosphate transport.     

Adenylosuccinate synthetase from Dictyostelium discoideum: effects of hadacidin analogs and binding of [14C]hadacidin

The enzyme adenylosuccinate (sAMP) synthetase has been partially purified from Dictyostelium discoideum using hadacidin-Sepharose 4B affinity chromatography, anion-exchange high-performance liquid chromatography (HPLC), and gel-filtration HPLC, resulting in a 2600-fold purification. Using a newly developed HPLC procedure to assay activity, it has been found that D. discoideum adenylosuccinate synthetase activity has apparent Km values for the substrates IMP, GTP, and aspartate of 36, 23, and 714 microM, respectively. The analog guanosine-5'-(beta, gamma-imino)triphosphate was found to be an inhibitor of GTP with a Ki of 15 microM, and IMP was competitively inhibited by its analog formycin B monophosphate with a Ki of 80 microM. An analysis of these kinetic data showed a pattern consistent with a fully random terter mechanism. Hadacidin, an analog of aspartate, was an inhibitor of that substrate at 86 microM. Other analogs of hadacidin were synthesized and examined for their effect on the sAMP synthetase activity. Compared to hadacidin, which produced 100% inhibition at 5 mM, it was observed that N-acetyl-N-hydroxyglycine, N-formylglycine, N-acetylglycine, and N-hydroxyglycine all inhibited between 50 and 75%, with N-(thiocarboxy)-L-aspartic anhydride less effective at 27%, and N-benzoylglycine at only 6%. N-Formylsarcosine, N-acetylmethionine, O-methylpyruvate oxime, and hadacidin methylester had no effect at this concentration. The adenylosuccinate synthetase activity was dependent on metal ions with maximum activity being obtained with Mg2+. The ability of the aspartate analog hadacidin to bind to the purified adenylosuccinate synthetase was demonstrated using anion-exchange HPLC and [formyl-14C]hadacidin. The radioactivity coeluted with the adenylosuccinate synthetase and the bound, radiolabeled hadacidin was displaced by excess aspartate.     

Characterization of a sialidase (neuraminidase) isolated from Clostridium chauvoei (Jakari strain)

A sialidase from Clostridium chauvoei (Jakari strain), an indigenous bacterial strain that causes blackleg in Nigerian cattle and other ruminants was isolated and partially purified by chromatography on DEAE cellulose, hydroxyapatite and phenyl agarose columns. The enzyme migrated as a 65-kDa protein after electrophoresis on sodium dodecyl sulphate polyacrylamide gels. It was optimally active at pH 4.5 and 40 degrees C with an activation energy (Ea) of 13.40 kJ mol(-1). It had Km and Vmax values of 170 microM and 200 micromole h(-1) mg(-1) respectively with fetuin as substrate. When sialyllactose (Neu5Ac2,3 lactose) was used as substrate the Km and Vmax values were 8 microM and 5 micromoles min(-1) mg(-1) respectively. The Clostridium chauvoei sialidase cleaved sialic acids from RBC ghosts of sheep, horse, goat, cattle, pig and mice as well as mouse brain cells, albeit at different rates. The enzyme was activated by Ca2+ and Mg2+ and inhibited by the group-specific reagents diethylpyrocarbonate (DEP) and N-ethylmalemide (NEM). The sialidase inhibitors, 2,3 didehydroneuraminic acid (Neu5Ac2,3en) and paranitrophenyl oxamic acid (pNPO) inhibited the enzyme competitively with Ki values of 40 and 30 microM respectively.     

Carbamate kinase of Lactobacillus buchneri NCDO110. I. Purification and properties

Carbamate kinase has been prepared from Lactobacillus buchneri NCDO110. An approximately 91-fold increase in the specific activity of the enzyme was achieved. The purified extract exhibited a single band following polyacrylamide gel electrophoresis. The apparent molecular weight as determined by gel electrophoresis was about 97,000. The enzyme is stable for 2 weeks at -20 degrees C. Maximum enzymatic activity was observed at 30 degrees C and pH 5.4 in 0.1 M acetate buffer. L. buchneri carbamate kinase requires Mg2+ or Mn2+; its activity is higher with Mn2+. The activation energy of the reaction was 4078 cal mol-1 for the reaction with Mn2+ and 3059 cal mol-1 for the reaction with Mg2+. From a Dixon plot a pK value of 4.8 was calculated. The apparent Km values for ADP with Mg2+ or Mn2+ were 0.71 X 10(-3) and 1.17 X 10(-3) M, respectively, and the apparent Km values for carbamyl phosphate with Mg2+ or Mn2+ were 1.63 X 10(-3) and 1.53 X 10(-3) M, respectively. ATP and CTP acted as inhibitors of this reaction and the following values were obtained: Ki (ATP)Mg2+ = 9.4 mM, Ki (ATP)Mn2+ = 6.2 mM, and Ki (CTP)Mg2+ = 4.4 mM.     

A study of the relationships of interactions between Asp-201, Na+ or K+, and galactosyl C6 hydroxyl and their effects on binding and reactivity of beta-galactosidase.

The interactions between Na+ (and K+) and Asp-201 of beta-galactosidase were studied. Analysis of the changes in Km and Vmax showed that the Kd for Na+ of wild type beta-galactosidase (0.36 +/- 0.09 mM) was about 10x lower than for K+ (3.9 +/- 0.6 mM). The difference is probably because of the size and other physical properties of the ions and the binding pocket. Decreases of Km as functions of Na+ and K+ for oNPG and pNPG and decreases of the Ki of both shallow and deep mode inhibitors were similar, whereas the Km and Ki of substrates and inhibitors without C6 hydroxyls remained constant. Thus, Na+ and K+ are important for binding galactosyl moieties via the C6 hydroxyl throughout catalysis. Na+ and K+ had lesser effects on the Vmax. The Vmax of pNPF and pNPA (substrates that lack a C6 hydroxyl) did not change upon addition of Na+ or K+, showing that the catalytic effects are also mediated via the C6 hydroxyl. Arrhenius plots indicated that Na+, but not K+, caused k3 (degalactosylation) to increase. Na+ also caused the k2 (galactosylation) with oNPG, but not with pNPG, to increase. In contrast, K+ caused the k2 values with both oNPG and pNPG to increase. Na+ and K+ mainly altered the entropies of activation of k2 and k3 with only small effects on the enthalpies of activation. This strongly suggests that only the positioning of the substrate, transition states, and covalent intermediate are altered by Na+ and K+. Further evidence that positioning is important was that substitution of Asp-201 with a Glu caused the Km and Ki values to increase significantly. In addition, the Kd values for Na+ or K+ were 5 to 8 fold higher. The negative charge of Asp-201 was shown to be vital for Na+ and K+ binding. Large amounts of Na+ or K+ had no effect on the very large Km and Ki values of D201N-beta-galactosidase and the Vmax values changed minimally and in a linear rather than hyperbolic way. D201F-beta-galactosidase, with a very bulky hydrophobic side chain in place of Asp, essentially obliterated all binding and catalysis.     

Pyrimidine-degrading enzymes. Purification and properties of beta-ureidopropionase of Euglena gracilis.

In photoorganotrophically grown, mid-log phase cells of Euglena gracilis, enzymes of pyrimidine degradation including uracil reductase, dihydrouracil dehydrogenase, dihydropyrimidinase, and beta-ureidopropionase, were detected in a crude extract. beta-Ureidopropionase (N-carbamoyl-beta-alanine amidohydrolase, EC 3.5.1.6) was purified 100-fold by heat treatment, ammonium sulphate fractionation and chromatography using Sepharose 6B and DEAE-Sephadex A-25. The enzyme follows Michaelis-Menten kinetics (Km of beta-ureidopropionase for beta-ureidopropionate 3.8 . 10(-5) M, Hill coefficient n = 1). Other enzyme properties are: pH optimum 6.25, temperature optimum 60 degrees C, stimulation by Mg2+, inhibition by Cu2+, Mr approximately 1.5--2 . 10(6). beta-Ureidoisobutyrate, the intermediate of thymine degradation, and beta-ureidopropionate are competing substrates of beta-ureidopropionase (Ki = Km of beta-ureidopropionase for beta-ureidoisobutyrate 1.8 . 10(-5) M). Structural analogues of beta-ureidopropionate, isobutyrate and propionate are competitive inhibitors (Ki of beta-ureidopropionase 0.3 and 0.16 mM, respectively). There were no indications of regulatory function of beta-ureidopropionase in pyrimidine degradation.     

Differences in substrate specificities of monoamine oxidase A from human liver and placenta.

The substrate specificities of monoamine oxidase (MAO) A isolated from human placenta and of human liver expressed in yeast have been compared in homogeneous preparations with respect to Vmax and Km values for natural and synthetic substrates and Ki values for competitive inhibitors. MAO A from these two sources is known to differ in at least 5 amino acid residues. While the Km and Ki values were found to be nearly identical in the enzymes from these two sources, the Vmax differed significantly on bulky synthetic substrates.     

5'-Nucleotidase from smooth muscle of small intestine and from brain. Inhibition of nucleotides.

5'-Nucleotidase prepared from muscle of small intesting of pig is strongly inhibited by nucleoside di- and triphosphates and their phosphonate analogs. Substrate kinetics appromate the Michaelis-Menten for for AMP, which shows a Km of 3-6 muM at pH 5.3-7.2. Inhibition is characterized as partial competitive, except at pH 5.3, where inhibition by ATP is noncompetitive. The Ki values for several inhibitors have been determined, and their departure from completeness of competitive inhibition has been studied. Inhibitor cooperativity of the type reported for the enzyme from sheep brain (P. L. Ipata (1968), Biochemistry 7, 507) was not observed for the enzyme from gut. In addition we failed to confirm sigmoid inhibition kinetics with 5'-nucleotidase from sheep brain.     

Characterization of microsomal choloyl-coenzyme A synthetase.

Choloyl-CoA synthetase (EC 6.2.1.7) was characterized for the first time under appropriated assay conditions. The p/ optimum for the reaction is pH 7.2.-7.3. The reaction has an absolute requirement for bivalent cation. Several different metal ions fulfil this requirement, but Mn2+ and Mg2+ were the most effective. The KAppm (apparent Km) for CoA, extrapolated from kinetic data, is 50 micronM, but in fact the rate of reaction is increased little by concentrations of CoA above 25 micronM. The KAppm for ATP is 600 micronM. High concentrations of ATP appear to cause substrate inhibition. The KAppm for cholate was 6 micronM. The enzyme was inhibited by treating the microsomal fraction with N-ethylmaleimide. The inclusion of various conjugated and unconjugated bile salts in the assay also inhibited the enzyme. Unconjugated bile salts were more potent inhibitors than the conjugated bile salts. High concentrations of oleic acid inhibited the enzyme. The properties of choloyl-CoA synthetase were not modified by alterations of the properties of the lipid phase of the microsomal membrane. Treatment with phospholipase A did not alter activity directly. Triton N-101 and Triton X-100 also were without effect on activity, and the enzyme was insensitive to temperature-induced phase transitions within the lipid portion of the membrane. The enzyme can be solubilized from the microsomal membrane in an active form by treatment with Triton N-101.