A method for the determination of the concentration of catalytic centres in low activity and unstable preparations of acetylcholinesterase.

A simple and rapid method has been developed for the titration of catalytic centres of acetylcholinesterase of low activity and stability in homogenates of larvae of the cattle tick Boophilus microplus. It is based on the difference in uptake of the labeled organophosphate inhibitor [¹⁴C]coroxon between substrateprotected and unprotected enzyme. The excess coroxon is removed rapidly by solvent extraction of the acidified enzyme medium with acetone and toluene. The method was validated by the use of bovine erythrocyte acetylcholinesterase, with only 6 × 10^?12 catalytic centre mole equivalents of this enzyme being required for a single accurate assay. The turnover number at pH 7.6 and 37°C was 1.22 × 10⁶ molecules of acetylcholine hydrolysed per min per active centre. The catalytic efficiency of enzyme of larvae of the cattle tick was markedly different, being onetenth of that of bovine erythrocyte enzyme. Advantages of the method are discussed.     

Large-scale preparation of Na, K-ATPase from ox brain.

A method for the isolation of membrane-bound Na,K-ATPase in quantity from brain gray matter is described. The method permits a large amount of enzyme to be obtained rather quickly with about 60% of the original activity of Na,K-ATPase of the tissue being recovered. The enzyme is stable, it has a specific activity of about 200 μmoles ATP split per mg protein per hour. Mg-ATPase comprises about 1% of the total ATPase activity. The enzymatic properties of this Na,K-ATPase do not differ from those in the literature; the turnover number is about 9300 min^?1.     

Bound nucleotides and phosphorylation in Rhodospirillum rubrum.

Chromatophores from $\text{Rhodospirillum rubrum}$ contain 12 × 10^?3 mol ATP and 8.3 × 10^?3 mol ADP per mol chlorophyll, tightly bound to the coupling ATPase. Under energised conditions, these exchange slowly with added nucleotide. Using single turnover light flashes, it is demonstrated that the release of bound ATP is too slow to be on the direct pathway of photophosphorylation.     

Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN^?-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN^?) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min^?1 · 10^?11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min^?1 · 10^?11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.     

Two forms of phosphoinositol kinase from germinating mung bean seeds

Phosphoinositol kinase, the key enzyme responsible for the biosynthesis of higher inositol phosphates has been isolated from the cotyledons of mung beans germinated for 24 hr and has been resolved into two different forms, phosphoinositol kinase A and phosphoinositol kinase B. Both forms were purified to homogeneity and characterized. The K_m values for ATP with phosphoinositol kinase A (1.78 × 10^?4 M) and phosphoinositol kinase B (3.12 × 10 ^?5 M) showed that phosphoinositol kinase B had a greater affinity for ATP. ATP could be partially replaced as phosphate donor by UTP and phosphoenolpyruvate in the case of phosphoinositol kinase A but not in the case of phosphoinositol kinase B.     

The induction kinetics of bacterial photophosphorylation. Threshold effects by the phosphate potential and correlation with the amplitude of the carotenoid absorption band shift

1. ATP synthesis (monitored by luciferin-luciferase) can be elicited by a single turnover flash of saturating intensity in chromatophores from Rhodopseudomonas capsulata, Kb1. The ATP yield from the first to the fourth turnover is strongly influenced by the phosphate potential: at high phosphate potential (?11.5 kcal/mol) no ATP is formed in the first three turnovers while at lower phosphate potential (?8.2 kcal/mol) the yield in the first flash is already one half of the maximum, which is reached after 2–3 turnovers.2. The response to ionophores indicates that the driving force for ATP synthesis in the first 20 turnovers is mainly given by a membrane potential. The amplitude of the carotenoid band shift shows that during a train of flashes an increasing ΔΨ is built up, which reaches a stationary level after a few turnovers; at high phosphate potential, therefore, more turnovers of the same photosynthetic unit are required to overcome an energetic threshold.3. After several (six to seven) flashes the ATP yield becomes constant, independently from the phosphate potential; the yield varies, however, as a function of dark time (t_d) between flashes, with an optimum for t_d = 160–320 ms.4. The decay kinetics of the high energy state generated by a long (125 ms) flash have been studied directly measuring the ATP yield produced in post-illumination by one single turnover flash, under conditions of phosphate potential (?10 kcal/mol), which will not allow ATP formation by one single turnover. The high energy state decays within 20 s after the illumination. The decay rate is strongly accelerated by 10^?8 M valinomycin.5. Under all the experimental conditions described, the amplitude of the carotenoid signal correlates univocally with the ATP yield per flash, demonstrating that this signal monitores accurately an energetic state of the membrane directly involved in ATP synthesis.6. Although values of the carotenoid signal much larger than the minimal threshold are present, relax slowly, and contribute to the energy input for phosphorylation, no ATP is formed unless electron flow is induced by a single turnover flash.7. The conclusions drawn are independent from the assumption that a ΔΨ between bulk phases is evaluable from the carotenoid signal.     

Analysis of the binding of phenylalanine to phenylalanyl-tRNA synthetase

Using the complete rate equation for the PP_i-ATP exchange reaction at equilibrium, the dissociation constants of phenylalanine (10^?5m), phenylalanine butyl ester (8 × 10^?5m), benzyl alcohol (6 × 10^?4m), phenylalaninol (2 × 10^?4m), hydrocinnamic acid (3 × 10^?3m) and glycine (>1 m) with the phenylalanyl-tRNA synthetase (Escherichia coli K12) were determined. Taking the model of Koshland (1962) for the estimation of the configurational free energy change due to proximity and orientation, and decomposing the process of binding into several thermodynamic steps, the contribution to binding of the benzyl group, glycine unit, protonated amino group, carboxylate group and joint interactions were estimated. The results are: (1) the standard free energy contributions for binding phenylalanine are benzyl group (?8.2 kcal/mol), glycine unit (?2.5 kcal/mol), protonated amino group (?0.8 kcal/mol) and carboxylate group (1 kcal/mol). (2) The standard free energy change due to the change in the interaction between the protonated amino group and carboxylate group when they are transferred from the aqueous environment to the enzyme environment is ?2.7 kcal/mol. (3) A dissociation constant for glycine of 7.5 m is calculated without the hypothesis that a conformational change occurs in the enzyme when the benzyl unit of phenylalanine binds, permitting an interaction of the enzyme with the protonated amino and/or carboxylate groups.The detection of E·AA² and E·ATP shows that a sequential addition of substrates is not necessary for binding. A comparison of the dissociation constants of E·AA (10^?5m), E·ATP (1.5 × 10^?3m), E·PP (5.5 × 10^?4m), E·I (8 × 10^?5m) and the mixed complexes E·I·ATP (6 × 10^?8m²), E·I·PP (5 × 10^?8m²) and E·AA·PP (7 × 10^?9m²), with phenylalanine butyl ester as the inhibitor, indicates no strong interaction between the binding of ATP or PP_i with the binding of phenylalanine.     

Factors which may affect removal of protamine from sperm DNA during fertilization in the rabbit

In order to derive information about possible mechanisms by which the sperm head is converted into the male pronucleus during fertilization in the rabbit, unfertilized egg homogenate was assayed for two enzyme activities. Protamine was extracted from rabbit sperm, purified, and labelled with [¹⁴C] in an in vitro reaction and used as a probe to assay for a protein kinase which could transfer [³²P]PO₄ from [γ-³²P]ATP onto the substrate. A kinase with a pH optimum of approximately 8.0 to 8.5 is described. Assays for the enzyme glutathione reductase were performed using homogenates from eggs or embryos at three early stages of development. Results suggest that oocytes can oxidize 2.58 × 10^?6 μmol NADPH per minute per oocyte, unfertilized eggs 5.16 × 10^?7 μmol NADPH per minute per ovum, and 20- to 24-hour postcoitus fertilized eggs 2.30 × 10^?6 μmol NADPH per minute per ovum. The relevance of these observations to male pronuclear formation is discussed.     

RELATIONSHIP BETWEEN NUCLEOSIDE DIPHOSPHATE KINASE ACTIVITY AND LIGHT-LIMITED GROWTH RATE IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Light-limited cultures of the marine diatom Thalassiosira pseudonana (Hustedt) Hasle and Heimdal (3H clone) were grown over a range of growth rates between 0.06 and 1.64 d^?1. Variations in cell volume, cell quotas of carbon, nitrogen, and protein, and maximal activity of the enzyme nucleoside diphosphate kinase (NDPK) were measured and examined as a function of growth rate. NDPK from T. pseudonana showed K_m values of 0.24 and 0.68 mM for thymidine 5′-diphosphate and adenosine 5′-triphosphate (ATP), respectively, which are similar to those found for NDPK from a variety of organisms, from bacteria to mammals. An apparent activation enthalpy of 3.52 kCal·mol^?1 was determined from Arrhenius plots. No thermodynamic transition points were noted over a temperature range from 10° to 25°C. NDPK activity was significantly correlated with growth rate but not with cell volume, carbon, nitrogen, or protein; for interspecific comparisons, normalization of enzyme activity to cell number may be most meaningful. NDPK activity per cell versus growth rate followed a U-shaped relationship, being relatively constant between 0.5 and 1.0 d^?1 and rising at higher and lower growth rates. Over this range, enzyme activity may be regulated by substrate concentration (ATP or other nucleoside triphosphates) or by adenylate energy charge. At higher growth rates where energy charge and substrate concentrations are probably high, changes in enzyme concentration appear to be required. The reasons for a rise in enzyme activity at low growth rate is unclear. Simultaneous measurement of nucleoside di- and triphosphate levels alongside NDPK measurements may help clarify the relationship, but these preliminary experiments indicate that NDPK is of limited usefulness as an index of in situ growth rate.     

Affinity labelling of tryptophanyl-transfer RNA synthetase

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme.Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, α₂ An ATP analogue, γ-(p-azidoanilide)-ATP does not serve as a substrate for enzymatic aminoacylation of tRNA^Trp but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K₁ = 1 × 10^?3m (K_mfor ATP = 2 × 10^?4m). The covalent photoaddition of azido-ATP³ results in complete loss of enzymatic activity in both the ATP-[³²P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme.Two binding sites for tRNA^Trp have been found from complex formation at pH 5.8 in the presence of Mg²⁺. The two tRNA molecules bind, with K_dis = 3.6 × 10^?8m and K_dis = 0.9 × 10^?6m, respectively, pointing to a strong negative co-operativity between the binding sites for tRNA.N-chlorambucilyl-tryptophanyl-tRNA^Trp and TRSase form a complex with K_dis = 5.5 × 10^?8m at pH 5.8 in the presence of 10 mm-Mg²⁺. This value is similar to the value of K_dis for tryptophanyl-tRNA of 4.8 × 10^?8m. Under the same conditions a 1:1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilyl-Trp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of tRNA^Trp whereas the tRNA devoid of tRNA^Trp does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the complex formation between TRSase and the tRNA analogue. Photoaddition of the azido-ATP completely prevents the reaction of the enzyme with the tRNA analogue, although the non-covalent complex formation is not affected.Exhaustive alkylation of TRSase partially inhibits the reaction of ATP [³²P]pyrophosphate exchange and completely blocks the aminoacylation of tRNA^Trp. Cleavage of the tRNA which is covalently bound to TRSase restores both the ATP-[³²P]pyrophosphate exchange and aminoacylation activity.The TRSase which is covalently-bound to R-Trp-tRNA is able to incorporate only one ATP molecule per dimeric enzyme into the active centre. This doubly modified enzyme is completely enzymatically inactive. Removal of the tRNA residue from the doubly modified enzyme results in the formation of the derivative with one blocked ATP site. Therefore, a “single-site” TRSase may be generated either by alkylation of the enzyme with Cl-R-Trp-tRNA or after the removal of covalently bound tRNA from the doubly labelled protein.Tryptophanyl-tRNA synthetase containing blocked ATP and/or tRNA binding site(s) seems to bo a useful tool for investigation of negative co-operativity and may help in the elucidation of the structure function relationships between the active centres.     

Calcium inhibition of adenylate cyclase: Studies in turkey erythrocyte and S49 CYC− cell membranes

We studied the mechanism of calcium inhibition of adenylate cyclase using partially purified components of the enzyme complex and computer analysis of free metal and substrate concentrations. A sigmoidal relationship was observed between percentage maximal adenylate cyclase activity with 1-isoproterenol/guanylyl-β,γ-imidodiphosphate and the calculated free calcium. Fifty percent inhibition occurred at 2.5 × 10^?4m free calcium. This inhibition appeared to be independent of calmodulin. Calcium inhibited the holocatalytic enzyme in a manner indentical to that of the native enzyme, but did not affect the ability of 1-isoproterenol and guanylyl-β,γ-imidodiphosphate to promote the formation of the holocatalytic state. There was no effect of calcium on the conformation of the activated G unit nor on the holocatalytic enzyme as determined by sedimentation velocity analysis. Calcium did not cause detectable dissociation of the activated G unit from the catalytic unit, nor convert activated G unit to an inactive form. Calcium inhibition of the catalytic unit of adenylate cyclase was studied in S49 CYC^? lymphoma cell membranes. High concentrations of calcium inhibited manganese-stimulated CYC^? enzyme, but this could be explained by competition between calcium and manganese for ATP. With addition of forskolin, CYC^? adenylate cyclase utilized MgATP^2? as substrate and was shown to have a separate binding site for free magnesium. Calcium inhibited forskolin-stimulated CYC^? enzyme by competing with free magnesium for its regulatory site. Calcium inhibition was noncompetitive with respect to MgATP^2?. We conclude that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit.     

Purification and Properties of Pantothenate Kinase from Brevibacterium ammoniagenes IFO 12071

Pantothenate kinase (ATP: pantothenate 4′-phosphotransferase, EC 2.7.1.33) was purified about 200-fold from the cell extract of Brevibacterium ammoniagenes IFO 12071 by ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-150 gel filtration. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was calculated approximately 45,000. The enzyme catalyzed the formation of pantothenic acid 4′-phosphate and ADP from pantothenate and ATP in the presence of Mg²⁺ ATP could be substituted for, partly, by ITP, GTP, and UTP. The enzyme phosphorylated not only pantothenate, but also pantothenoylcysteine, pantetheine, and pantothenyl alcohol. Apparent Km values were 6.7×10^?5 m for pantothenate, 3.5×10^?5 m for ATP, and 10^?3 m for Mg²⁺. The reaction was inhibited by the intermediates of CoA biosynthesis, of which CoA itself was a most effective inhibitor. Other properties of the enzyme were also investigated.     

A microsomal ATP-activated pyridine nucleotide transhydrogenase

An ATP-activated transhydrogenase which catalyzes the reduction of TPN⁺ by DPNH has been demonstrated in the microsomal fraction from the endosperm of immature Echinocystis macrocarpa seeds. The activity is specifically dependent on the presence of ATP (K_m of approximately 0.1 mm) of several nucleotides tested. The reaction is stimulated by MgCl₂ addition up to concentrations of 6 mm. When 10^?2m EDTA is added to the assay mixture in the absence of added MgCl₂, a transhydrogenation reaction is observed which no longer shows any dependence on added ATP. A TPN⁺-dependent ATPase activity can be demonstrated in these preparations, but no fixed stoichiometry between ATP cleavage and TPNH formation could be established. A lag in attaining the maximal rate of transhydrogenation is seen unless the enzyme is preincubated for 10 min with ATP before initiating the reaction. It can further be shown that preincubation of the enzyme with ATP followed by removal of the ATP on a Dowex 1 column produces an enzyme capable of catalyzing the transhydrogenation without the further addition of ATP. 2,4-Dinitrophenol and thyroxin are effective inhibitors of the transhydrogenase and 2,4-dinitrophenol was shown to inhibit the activating effect of ATP during the preincubation period. It is concluded that the role of ATP is in the modification of the enzyme rather than direct participation in the transhydrogenation. The transhydrogenase is inhibited by ADP and AMP. This results in a response of the enzyme to adenylate energy charge in a manner characteristic for regulatory enzymes which participate in ATP-utilizing metabolic sequences.     

Characterization of a 5′-AMP binding site in purified membranes from Dictyostelium discoideum

Although D.discoideum amoebae do not bind AMP at their surface if they are not disrupted, total cell lysates display high levels of AMP binding activity specifically associated with the plasma membrane. The binding of AMP is not competed by adenosine and only poorly by ADP and ATP. The AMP binding sites have a single affinity of 0.6 μM for AMP; the association and dissociation rate constants are respectively 8×10³ sec^?1M^?1 and 4.8 ×10^?3sec^?1. The AMP binding occurs at a site distinct from the cAMP binding site and from the catalytic site of a membrane bound enzyme.     

Kinetic Properties of Fungal Polyamine Oxidases and Their Application to Differential Determination of Spermine and Spermidine

Polyamine oxidase from Penicillium chrysogenum oxidized spermine rapidly and spermidine slightly at pH 7.5. The apparent Km values for spermine and spermidine were calculated to be 2.25 × 10^?5 m and 9.54 × 10^?6 m, respectively. The relative maximum velocities for spermine and spermidine were 3.37 × 10^?3 m (H₂O₂) per min per mg of protein and 2.08 × 10^?4 m (H₂O₂) per min per mg of protein, respectively. Spermine oxidation of the enzyme was competitively inhibited by spermidine and putrescine. The apparent Ki values by spermidine and putrescine were calculated to be 3.00 × 10^?5 m and 1.80 × 10^?8 m, respectively. On the other hand, polyamine oxidase from Aspergillus terreus rapidly oxidized both spermidine and spermine at pH 6.5. The apparent Km values for spermidine and spermine were 1.20 × 10^?8 m and 5.37 × 10^?7 m, respectively. The relative maximum velocities for spermidine and spermine were 1.55 × 10^?2 m (H₂O₂) per min per mg of protein and 6.20 × 10^?3 m (H₂O₂) per min per mg of protein, respectively.

Differential determination of spermine and spermidine was carried out using the two enzymes. The initial rate was assayed with Penicillium enzyme and the end point was measured afte addition of Aspergillus enzyme. Small amounts of polyamines (25 to 200 nmol of spermine and 25 to 250 nmol of spermidine) were assayed by solving two simultaneous equations obtained from the rate assay method and the end point assay method. The calculated values were in close agreement with those obtained by an amino-acid analyzer.     

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% β-guanidinopropionic acid (GPA) for 6–10 weeks to deplete their skeletal muscle of creatine. ³¹P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 μmol·g^?1) and the [ATP] to 50% (3.3 μmol·g^?1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 μmol·g^?1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM·s^?1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM·s^?1). The rate of PGPA resynthesis during recovery (0.018 mM·s^?1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM·s^?1). The creatine kinase flux (creatine phosphate → ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM·s^?1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.     

On the Purification and some Properties of 5′-Adenylic Acid-Deaminating Enzyme from Microsporum audouini

From culture broth of Microsporum audouini, 5′-adenylic acid-deaminating enzyme has been purified to about 600-fold. The pH optimum was found to be 5.0 in acetate, 5.5 in succinate, 5.7 in citrate buffer. Velocity constant was 1.83×10^?1 per minute. The optimal temperature was 40°C and activation energy was 15,000 calories. Michaelis-Menten constant was 6×10^?4 m. This enzyme preparation removes amino groups of 5′- AMP, ADP and ATP quickly, of adenosine, 3′-AMP, 5′-deoxyAMP and NAD slowly, but adenine, 2,6-diaminopurine, 2′-AMP and NADP were not deaminated. The enzyme activity was inhibited with F^?, pCMB, Fe^{+ + +}, Cu^{+ +} and Zn^{+ +}     

Glutamine synthetase from Salmonella typhimurium: manganese(II), substrate, and inhibitor interaction with the unadenylylated enzyme.

Unadenylylated glutamine synthetase (EC 6.3.1.2) was isolated and purified to homogeneity from Salmonella typhimurium. The enzyme molecule is a symmetrical aggregate of 12 subunits arranged in two hexagonal layers, as is evident from electron micrographs. The subunit molecular weight of the enzyme was found to be approximately 50,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate when compared to Escherichia coli glutamine synthetase and other protein standards. A long tube of glutamine synthetase was formed as a single-stranded coil resulting from incubation of the enzyme in a low ionic strength buffer. A study of Mn(II) binding to the unadenylylated enzyme at 25 °C was conducted as a function of pH. At pH 7.1 two classes of metal ion sites per subunit were found with K_D values of 3.7 × 10^?6 and 1.7 × 10^?4m, while at pH 6.8 these values were 1.1 × 10^?5 and 1.0 × 10^?4m, respectively. Only one set of binding sites was observed at pH 6.2 with a K_D value of 1.0 × 10^?4m. The metal ion binding sites were further investigated by monitoring proton relaxation rates (prr) and the epr spectrum of enzyme-bound Mn(II). The longitudinal prr of water protons at pH 7.1 indicate that protons interacting with enzyme-Mn(II) at the “tight” site (K_D = 3.7 × 10^?6) are de-enhanced (?_b1 = 0.42) and result from water protons beyond the inner coordination sphere. The second Mn(II) site has a value of ?_b2 = 35 for the binary enhancement, suggesting that this site probably has two to three rapidly exchanging water molecules in its coordination sphere. The epr spectrum of enzyme-bound Mn(II) at the “tight” site is isotropic and is dramatically sharpened by adding the substrate analog methionine sulfoximine. Subsequent addition of ATP or the ATP analog, AMP-PCP (adenylyl methylene diphosphate) produced anisotropic spectra that were similar, suggesting that both ATP and AMP-PCP bind similarly on the enzyme surface. However, a marked change in the Mn(II) environment from anisotropic to near cubic results from the addition of ADP to the quaternary enzyme-Mn(II)-sulfoximine- (AMP-PCP) complex, indicating that ADP displaces AMP-PCP. No change in the anisotropic spectrum due to the enzyme-Mn(II)-sulfoximine-ATP complex is seen by the addition of ADP. This experimental result supports the experimental findings of Ronzio and Meister [Proc. Nat. Acad. Sci. USA59, 164 (1968)], who established that ATP phosphorylates methionine sulfoximine, thereby producing an inactive enzyme. The allosteric effectors, AMP and Trp, have little effect on the epr spectrum of the complex formed from Mn(II), enzyme, sulfoximine, and ADP, suggesting the absence of direct coordination of AMP or Trp to the bound Mn(II). The prr and epr results reported herein with glutamine synthetase from S. typhimurium when compared to those seen for the enzyme from E. coli [Villafranca et al., Biochemistry15, 544 (1976)] demonstrate some similarities but also many substantial differences between the enzymes from these two bacterial sources.     

Tracer kinetic analysis of incorporation of glucose and adenine by yeast cells

A kinetic model of adenine and glucose incorporation into log phase yeast cells has been developed, and experimental tests of certain predictions of the model validate it. The cellular pool of purine nucleotides is 6 × 10^?3 μmoles per 10⁷ cells, the turnover time of this pool is 21.8 minutes, and the rate of incorporation into nucleic acids is 4.86 × 10^?2 μmoles per hour per 10⁷ cells. Corresponding figures for glucose are given. The model should be useful in other kinetic studies and the method of applying it is explained.     

Phospholipase A2 from Trypanosoma congolense: Characterization and haematological properties

Phospholipase A₂ was isolated from Trypanosoma congolense and purified to electrophoretic homogeneity. The enzyme appeared to exist in a dimeric form with subunit molecular weights of 16 500 and 18 000. It had a pH optimum of 6·8. Kinetic analysis with different substrates, showed that the enzyme had exceptional specificity for 1,2,dimyristoyl-sn-phosphatidylcholine and 1,2,dioleoyl-sn-phosphatidylcholine with K_m values of 1·85 × 10^?3 M and 2·12 × 10^?3 M respectively. The Arrhenius plot was linear with an activation energy of 5·8 kcal mol^?1. Inhibition studies with parahydroxymercuribenzoate and tri-butyltinoxide were positive thus implicating a thiol group at the catalytic site of the enzyme. The enzyme was stable to heat treatment and possessed haemolytic and anticoagulating properties.