Purification and properties of dihydroxyacetone kinase from Gluconobacter suboxydans

Different carbon and nitrogen sources had little effect on the level of dihydroxyacetone kinase formed in the cells of Gluconobacter suboxydans. The enzyme was purified to homogeneity from cell-free extract of the organism by ammonium sulfate fractionation and chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200 (60-fold purification, 6% yield). Its molecular weight was 260,000; it was stabilized by addition of ATP, dithiothreitol, 2-mercaptoethanol or EDTA, and it reacted optimally at pH 6.5. d-Glyceraldehyde was equally as effective as DHA as a phosphate acceptor (K_m: 0.30 mM each). UTP showed 15% of the reactivity of ATP as a phosphate donor. K_m values for ATP were 0.33 mM in phosphorylation of dihydroxyacetone and 0.39 mM with d-glyceraldehyde. The enzyme activity was dependent on Mg²⁺ but not on Mn²⁺. The reaction with dihydroxyacetone as an acceptor was inhibited by d-glyceraldehyde. The inhibition was competitive with respect to dihydroxyacetone 3K_i=0.09 mM) and noncompetitive with respective to ATP (K_i=5.7 mM).     

Electron allocation to alternative substrates of Azotobacter nitrogenase is controlled by the electron flux through dinitrogenase

The electron flux through dinitrogenase (MoFe protein, protein containing Mo and Fe) from Azotobacter vinelandii controls the relative effectiveness of alternative substrates as electron acceptors in the nitrogenase system. The electron flux through dinitrogenase reductase (Fe protein) or the concentration of MgATP do not directly control electron allocation but rather control it via their influence on the electron flux through dinitrogenase. Kinetic properties of substrate reduction were studied as a function of the electron flux through dinitrogenase. N₂ was most effective at high electron fluxes, whereas H⁺ was the most effective acceptor at very low rates of electron flow through dinitrogenase. The K_m for acetylene was dependent on the electron flux through dinitrogenase, whereas the K_m for N₂ was much less sensitive to this electron flux. The lag period before the onset of acetylene reduction was proportional to the turnover time of dinitrogenase, and was approx. 12 times greater than the dinitrogenase turnover time. pH has effects on the electron allocation to substrates beyond that expected from the effect of pH on the electron flux; thus, pH may alter the relative ability of the nitrogenase enzyme system to reduce alternative substrates.     

Transport of Ammonium and Methylammonium Ions by Azotobacter vinelandii

Ammonium and methylammonium are rapidly taken up by cultures of Azotobacter vinelandii respiring in the presence of succinate. The rate of methylamine uptake increased with external pH from 5.5 to 7.5 but increasing the pH further to 8.5 had little effect on activity, indicating that methylammonium cation rather than uncharged methylamine is the permeant species. The kinetics of methylammonium entry followed the Michaelis-Menten relationship, yielding a K_m of 25 μM and a V_max of 3.8 nmol/min per mg of cell protein. At saturating concentrations ammonium was taken up at rates 30-fold higher than those for methylammonium. Ammonium was a competitive inhibitor of methylammonium uptake and gave an inhibition constant of 1 μM. Ammonium derivatives were inhibitors of methylammonium entry in order of effectiveness: hydrazine > methylhydrazine > formamidine > guanidine > dimethylamine > ethylamine; amides and amino acids did not block uptake. Likewise, metal cations inhibited in the order Tl⁺ > Cs⁺ > Rb⁺, whereas Na⁺, K⁺, and Li⁺ produced no significant effect. Methylammonium uptake was blocked in cells exposed to an uncoupler, p-trifluorome-thoxycarbonyl cyanide-phenyl hydrazone or gramicidin D, but not with dicyclo-hexylcarbodiimide or arsenate. Valinomycin stimulated methylammonium entry into cells in a K⁺-free medium but prevented entry in the presence of 10 mM K⁺. Monensin and nigericin had little effect on transport. These results indicate that methylammonium and ammonium ions enter A. vinelandii electrogenically via a specific transporter.     

Inhibition of glycollate oxidase from parsley leaves by HCO-3.

HCO₃^? is shown to be a noncompetitive inhibitor of glycollate oxidase from parsley leaves with respect ot glycolic acid. The K_i is found to be 20 mM at pH 7,7.     

Purification and partial characterization of the glutathione S-transferase of rat erythrocytes

The single glutathione S-transferase (EC 2.5.1.18) present in rat erythrocytes was purified to apparent homogeneity by affinity chromatography on glutathione-Sepharose and hydroxyapatite chromatography. Approx. 1.86 mg enzyme is found in 100 ml packed erythrocytes and accounts for about 0.01% of total soluble protein. The native enzyme (M_r 48 000) displays a pI of 5.9 and appears to possess a homodimeric structure with a subunit of M_r 23 500. Enzyme activities with ethacrynic acid and cumene hydroperoxide were 24 and 3%, respectively, of that with 1-chloro-2,4-dinitrobenzene. The K_m values for 1-chloro-2,4-dinitrobenzene and glutathione were 1.0 and 0.142 mM, respectively. The concentrations of certain compounds required to produce 50% inhibition (I₅₀) were as follows: 12 μM bromosulphophthalein, 34 μM S-hexylglutathione, 339 μM oxidized glutathione and 1.5 mM cholate. Bromosulphophthalein was a noncompetitive inhibitor with respect to 1-chloro-2,4-dinitrobenzene (K_i = 8 μM) and glutathione (K_is = 4 μM; K_ii = 11.5 μM) while S-hexylglutathione was competitive with glutathione (K_i = 5 μM).     

Properties of the solvent-stimulated ATPase activity of chloroplast coupling factor 1 from Chlamydomonas reinhardii

The effects of solvents on the ATPase activity of chloroplast coupling factor 1 (CF₁) isolated from wild-type Chlamydomonas reinhardii have been studied. Of the solvents examined, the following order summarizes their maximal ability to stimulate the ATPase activity of CF₁: ethanol > methanol>allyl alcohol >n-propanol > acetone≈dioxane > ethylene glycol. Glycerol inhibits the CF₁ activity at all concentrations. In the absence of organic solvents, 50% of the activity of the enzyme is irreversibly lost after a 10 min incubation at 65–70°C. Ethanol (23%) causes a 30°C drop in the temperature required for 50% inactivation. ATP partially stabilizes the CF₁ in the presence, but not in the absence, of ethanol. In the absence of organic solvents, both free Mg²⁺ and ADP inhibit the CF₁-ATPase. Mg²⁺ is a noncompetitive inhibitor with respect to MgATP, and the kinetic constants are: V, 6.3 μmol ATP hydrolyzed/mg protein per min; K_m(MgATP), 0.23 mM; K_ii(Mg²⁺), 27 μM; and K_is(Mg²⁺), 50 μM. In the presence of ethanol, double-reciprocal plots are no longer linear and have a Hill coefficient of about 1.8±0.1. V increases about 10–12-fold. The pattern of inhibition by Mg²⁺ appears to change from noncompetitive to competitive with respect to MgATP. In addition, ADP no longer inhibits the MgATPase activity of CF₁.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits

A procedure is described which allows the purification of fructokinase (EC 2.7.1.4) from young tomato fruit. The procedure yielded a 400-fold purification and two isoenzymes designated fructokinase I and II (FKI and FKII) were separated by anion-exchange chromatography. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass was estimated to be 35 kDa. Gel filtration on Sepharose-12 indicated that for both fructokinases the functional form is a dimer. Two dimensional isoelectric focusing/SDS-PAGE combined with immunoblotting showed that FKI has two components with isoelectric points (pIs) of 6.42 and 6.55, while four components with pIs from 6.07 to 6.55 were detected for FKII. A mixture of both fructokinases showed that the components of FKI match the more alkaline components of FKII. The activity of both fructokinases increased with increasing pH to around 8.0 and equal activity was observed from 8.0 to 9.5. Both fructokinases were specific for fructose with K _m values for fructose of 0.131 and 0.201 mM for FKI and FKII, respectively. At high concentrations (> 0.5 mM), fructose was also a strong inhibitor with inhibition constants (K _i) of 1.82 and 1.39 mM for FKI and FKII, respectively. The preferred phosphate donor for both isoforms was ATP, and K _m values of 0.11 and 0.15 mM were observed for FKI and FKII. At low concentrations (0.05–0.2 mM), fructose exhibited noncompetitive inhibition with respect to ATP for both fructokinases. This inhibition pattern changed to uncompetitive when higher fructose concentrations (0.5–10 mM) were used. These data indicated that substrate addition is ordered, with ATP adding first. Inhibition by ADP was also affected by the fructose concentrations. At 0.5 mM fructose, FKI showed non-competitive inhibition by ADP with respect to ATP and this inhibition changed to uncompetitive when 3 mM fructose was used. The isoform FKII showed a competitive inhibition pattern for ADP at 0.5 mM fructose which also changed to uncompetitive when 3 mM fructose was used. The features of the regulation of both fructokinases suggest that this enzyme might have a relevant role in carbon metabolism during tomato fruit development.     

Preparation and properties of 5′-nucleotidases from bovine milk fat globule membranes

The 5′-nucleotidase (5′-ribonucleoside phosphohydrolase, EC 3.1.3.5) from bovine milk fat globule membranes was partially purified. Two separate peaks of activity were obtained from a Sepharose column and the two fractions, designated V and VI in order of elution, were collected and characterized separately. Both V and VI exhibited pH optima between 7.0 and 7.5 for AMP, GMP and CMP in the absence of metal ions. In the presence of Mg²⁺, a second pH optimum at 10.0 was observed with both fractions. Low concentrations of MnCl₂ activated Fraction V but not Fraction VI. HgCl₂ was a potent inhibitor of both fractions. The relative rates of hydrolysis of various 5′-mononucleotides differed comparing the two fractions. Optimum temperature for P_i release was 69 °C for both fractions. Activation energies were 10 400 cal/mole and 9600 cal/mole for Fractions V and VI, respectively. For V, calculated K_m values for AMP, GMP and CMP were 0.94, 2.5 and 1.16 mM, respectively. Calculated K_m values for Fraction VI for AMP, GMP and CMP were 5.0, 3.95 and 1.73 mM, respectively. ATP was a competitive inhibitor of AMP hydrolysis by Fraction V and a noncompetitive inhibitor of AMP hydrolysis by Fraction VI. Both fractions contained chloroform-methanol-extractable phospholipid. The phospholipid distribution pattern of Fraction VI was similar to that of milk fat globule membranes. Fraction V contained only sphingomyelin and phosphatidylcholine. It is proposed that milk fat globule membranes contain two separate 5′-nucleotidases.     

A kinetic study of inosine nucleosidase from Azotobacter vinelandii

• 1.1. Inhibition of inosine nucleosidase from Azotobacter vinelandii by ATP and bases can be qualitatively and quantitatively accounted for by the partial noncompetitive inhibition mechanism with ligand exclusion model.
• 2.2. The enzyme has two binding sites for the substrate with equal affinity in the absence of the inhibitor. and two species of the inhibitor sites: I₁- and I₂-sites. The I₁-site may overlap part of each substrate binding sites, and the I₂-site is separated from the substrate sites.
• 3.3. ATP binds to the I₁-site of the enzyme, and prevents the substrate from binding to either of two identical sites, producing the cooperativity with inosine, whereas binding of ATP to the I₂-site causes a noncompetitive inhibition.
• 4.4. Adenine and hypoxanthine bind to the I₂-site of the enzyme, and the EIS complex is partially active, resulting in a partial noncompetitive inhibition with Michaelis-Menten kinetics.

     

Heme inhibition of ferrisiderophore reductase in Bacillus subtilis

Heme was a noncompetitive inhibitor (apparent K_i and K′_i = 0.043 mM) of a ferrisiderophore reductase purified from Bacillus subtilis; protoporphyrin IX had no effect. The cellular level of heme may partly regulate the function of this reductase to yield a controlled flow of iron into metabolism.     

Glyconamides as inhibitors of human beta-glucosidases and beta-galactosidases.

d-Gluconamide, d-gluconyl hydrazide, and N-(6-aminohexyl)-d-gluconamide were prepared from d-glucono-1,5-lactone by treatment with ammonia, hydrazine, and 1,6-diaminohexane, respectively. These d-gluconamide derivatives were tested for their inhibitory action on human liver lysosomal glucocerebrosidase and human spleen neutral aryl β-glucosidase. Analogous d-galactonamide derivatives were evaluated for their inhibition of human spleen galactocerebrosidase and G_M1-ganglioside β-galactosidase. d-Gluconyl hydrazide and d-gluconamide were effective inhibitors of the lysosomal glucocerebrosidase, attaining 50% inhibition at 5 and 12 mm, respectively. In contrast, N-(6-aminohexyl)-d-gluconamide did not inhibit the glucocerebrosidase. d-Gluconyl hydrazide was also the most effective inhibitor of human liver and spleen aryl β-glucosidase, 50% inhibition being achieved at 4 mm concentration (competitive inhibition, K_i = 0.4–0.9 mM). d-Galactonamide was the most effective inhibitor of spleen galactocerebrosidase; 4 mm d-galactonamide caused 50% inhibition of the enzyme activity (noncompetitive inhibition). N-(6-Aminohexyl)-d-galactonamide is a potent inhibitor (90% inhibition, 5 mm) of G_M1-ganglioside β-galactosidase but is without effect on galactocerebrosidase. It has, therefore, the potential usefulness in distinguishing between two of the galactosphingolipid β-galactosidases.     

Characterization of a spontaneous mutant of Azotobacter vinelandii in which vanadium-dependent nitrogen fixation is not inhibited by molybdenum

A spontaneous mutant derivative of Azotobacter vinelandii CA12 (ΔnifHDK), in which vanadium-dependent nitrogen fixation is not inhibited by molybdenum (A. vinelandii CARR), grows profusely on BNF-agar containing 1 μM Na₂MoO₄, alone or supplemented with 1 μM V₂O₅. The expression of A. vinelandii vnfH::lacZ and vnfA::lacZ fusions in A. vinelandii CARR was not inhibited by 1 mM Na₂MoO₄, whereas molybdenum at much lower concentration inhibited the expression of vnfH::lacZ and vnfA::lacZ fusions in A. vinelandii CA12. The mutant also exhibited normal acetylene reduction activity in the presence of 1 μM Na₂MoO₄. The expression of A. vinelandii nifH::lacZ fusion in A. vinelandii CARR was low even though the cells were cultured under non-repressing conditions with urea as nitrogen source in the presence of Na₂MoO₄. The molybdenum content of A. vinelandii CARR cells was found to be about one-fourth that of A. vinelandii CA12. No nitrate reductase activity could be detected in A. vinelandii CARR when the cells were cultured in the presence of 10 μM Na₂MoO₄, whereas A. vinelandii CA12 exhibited some activity even with 100 pM Na₂MoO₄.     

Purification and characterization of an intracellular N-terminal exopeptidase from Streptococcus durans

An intracellular N-terminal exopeptidase isolated from cell extracts of Streptococcus durans has been purified 470-fold to homogeneity (specific activity of 12.0 μmol/min per mg). In the absence of thiol compounds, the purified aminopeptidase undergoes a slow oxidation with a 70% loss of activity, which can be restored by the addition of 2 mM β-mercaptoethanol. The purified aminopeptidase (M_r 300 000) preferred L-peptide and arylamide substrates with small nonpolar or basic side chains. SDS electrophoresis yielded a single protein band corresponding to a molecular weight of 49 400, suggesting that the native enzyme is a hexameric protein. The enzyme-catalyzed hydrolysis of $\text{L}\text{-}\text{alanyl}\text{-p-}\text{nitroanilide}$ exhibited a bell-shaped pH dependence for log V_max/K_m(pK₁ = 6.35; pK₂ = 8.50) while the log V_max versus pH profile showed only an acid limb (pK = 6.35). Methylene blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino acid analysis indicated that this photooxidative loss of activity corresponded to the modification of one histidine residue per enzyme monomer. N-Ethylmaleimide (100 mM) caused a 78% reduction in enzyme activity. Treatment of the enzyme with 1.0 mM hydrogen peroxide resulted in the oxidation of two cysteine residues per enzyme monomer and caused a 70% decrease in the catalytic activity.     

Oxidative phosphorylation in pea cotyledon submitochondrial particles

Mitochondria and submitochondrial particles (SMP) from pea cotyledons were shown to catalyze oxidative phosphorylation as measured by ³²Pi uptake into phosphate esters. ATP synthesis was sensitive to the electron transport inhibitor KCN, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and the coupling factor inhibitor oligomycin. Experiments with the adenine nucleotide translocator inhibitor atractyloside indicated the SMP were inside-out. Mersalyl completely inhibited ATP synthesis by SMP, and a separate experiment indicated that mersalyl has a direct effect on the ATPase complex. The kinetics of ATP synthesis indicated a high affinity for phosphate (K_m = 0.18 millimolar). ADP kinetics gave a biphasic curve with K_m values of about 4.8 and 160 micromolar. O₂ uptake and ATP synthesis had a pH maximum of 7.6 while the ratio of micromoles phosphate esterified to microatoms O₂ taken up was highest at pH 7.2. Sodium chloride inhibited both ATP synthesis and O₂ uptake but stimulated the ATPase reaction. The SMP also catalyzed a slow ATP-phosphate exchange reaction.     

Catalytic properties of enzymes from Erwinia carotovora involved in transamination of phenylpyruvate

K _m for L-phenylalanine, L-glutamic acid, L-aspartic acid, and the corresponding keto acids were calculated, as well as V _max was measured for the following pairs of substrates: L-phenylalanine-2-ketoglutarate, L-phenylalanine-oxaloacetate, L-glutamic acid-phenylpyruvate, and L-aspartic acid-phenylpyruvate for aminotransferases PAT1, PAT2, and PAT3 from Erwinia carotovora catalyzing transamination of phenylpyruvate. The ping-pong bi-bi mechanism was shown for the studied aminotransferases. The substrate inhibition (K _s) of PAT3 with 2-ketoglutarate and oxaloacetate was 10.23 ± 3.20 and 3.73 ± 1.99 mM, respectively. It was shown that L-β-(N-benzylamino)alanine was a competitive inhibitor with respect to L-phenylalanine for PAT1 (K _i = 0.32 ± 0.07 mM, K _m = 0.45 ± 0.1 mM, V _max = 11. 6 ± 0.4 U/mg) at 25 mM concentration of 2-ketoglutarate in the reaction medium. L-β-(N-methylamino)alanine is a noncompetitive inhibitor with respect to L-phenylalanine for PAT3 (K _I = 138.4 ± 95.4 mM, K _m = 13.7 ±3.9 mM, V _max = 18.6 ± 4.1 U/mg) at 2 mM concentration of 2-ketoglutarate in the reaction medium. L-stereo isomers of nonprotein analogues of aromatic amino acids were studied as substrates for PAT1, PAT2, and PAT3. L-β-(2-Br-phenyl)alanine, L-β-(4-Br-phenyl)alanine, L-β-(2-F-phenyl)alanine, and L-(2-F)tryptophan were good substrates for all three aminotransferases; L-α-methyl-β-(2-Br-phenyl)alanine and L-O-benzyltyrosine were substrates only for PAT3; L-β-(4-F-phenyl)alanine was a substrate for PAT1 and PAT3. Thus, these analogues of aromatic amino acids can be stereoselectively synthesized using the studied aminotransferases in the presence of the corresponding keto acids.     

Acyl coenzyme A inhibition of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase: a comparison of the TPN and DPN linked reactions

The inhibitory effects of ATP, coenzyme A, and acetyl, malonyl, and oleyl derivatives of coenzyme A on the TPN and DPN dependent activities of Leuconostoc glucose-6-phosphate dehydrogenase are compared. At pH 7.8, 24°, saturating levels of DPN or TPN, and inhibitor concentrations of 2–4 mM only ATP has an appreciable effect on the TPN dependent reaction, but all were potent inhibitors of the DPN dependent reaction. Oleyl coenzyme A was the most effective (K_i ～ 0.15 mM against glucose-6-phosphate) while acetyl coenzyme A was least effective (K_i ～ 1.0 mM). A possible regulatory role of this inhibition in fatty acid synthesis is suggested.     

Comprehensive kinetic and structural studies of different flavonoids inhibiting diphenolase activity of mushroom tyrosinase

The effect of 4 flavonoids on the diphenolase activity of mushroom tyrosinase was studied using spectroscopic approach. Analysis of kinetic data demonstrated that flavonoids cause a reversible inhibition of the enzyme activity. Further study showed that gallic acid acted as noncompetitive inhibitor, whereas chrysin, naringin and quercetin inhibited the diphenolase activity of mushroom tyrosinase in a competitive fashion. Comparison of the inhibition constants revealed that the strength with which the inhibitors acted on the enzyme activity was ranking as follows: chrysin (K_i 7.90 mM) < quercetin (K_i 7.44 mM) < naringin (K_i 3.04 mM) < gallic acid (K_i 1.5 mM). These data, therefore, suggest that gallic acid is the most potent inhibitor of the enzyme compared to the other flavonoids used.     

Physiological Studies of Oxygen Protection Mechanisms in the Heterocysts of Anabaena cylindrica

The mechanism of O₂ protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O₂ inhibition of nitrogenase activity correlated with the O₂ tension of the medium in which heterocyst formation was induced. O₂ resistance also correlated with the apparent K_m for acetylene, indicating that O₂ tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O₂ that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O₂ sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O₂ but not in its absence. After 3 h of aerobic incubation in the presence of 20 mM azide, 75% of cross-reactive component I (Fe-Mo protein) in nitrogenase was lost; less than 35% was lost under microaerophilic conditions. Sodium malonate and monofluoroacetate, inhibitors of Krebs cycle activity, had only small inhibitory effects on nitrogenase activity in the light and on cross-reactive material. The results suggest that oxygen protection is dependent on both an O₂ diffusion barrier and active respiration by the heterocyst.     

Characteristics of α-glucan phosphorylase from Chlorella vulgaris

α-Glucan phosphorylase from Chlorella vulgaris has been partially purified. In the direction of glucan phosphorolysis the apparent K_m for Pi was ca 2.4 mM at pH 7.1. In the direction of glucan synthesis the K_m for G1P was ca 0.12 mM at pH 6.2. The enzymic activity was inhibited by physiological concentrations of ADP, ATP, ADPG and UDPG. In the direction of starch degradation in the presence of 2.4 mM Pi the I_0.5 values for ADP and ATP were ca 1.6 and 2.9 mM, respectively, while in the direction of synthesis in the presence of 0.12 mM G1P the values were ca 0.23 and 1.4 mM, respectively. The Hill plots for starch degradation showed n values of 2.2 for ADP and 2.2 for ATP and values of 1.5 and 1.2, respectively, for starch synthesis. Both ADPG and UDPG were linear competitive inhibitors either with respect to Pi or with respect to GIP. The K_i values for ADPG and UDPG in the direction of phosphorolysis were shown to be ca 0.11 and 0.51 mM, respectively, and those in the direction of synthesis 0.033 and 0.15 mM, respectively.