Multiple forms of glucose–adenosine triphosphate phosphotransferase in rat mammary gland

Measurements have been made of the total hexokinase activity and of the relative amounts of types I and II hexokinase in rat mammary gland and at different stages of the lactation cycle. The total hexokinase activity increased during lactation, that of type II increasing to a greater extent than that of type I; the type II/type I activity ratio rose from a pregnancy value of about 1 to a mid-lactation value of 3, returning to 1 on involution. The changes in type II hexokinase activity during the lactation cycle parallel the changes in the insulin sensitivity of mammary-gland tissue. A study of the effect of alloxan-diabetes on mammary-gland hexokinase during the mid-lactation period revealed that, although the total glucose-phosphorylating capacity of the mammary gland was almost unchanged, the relative contributions of type I and type II hexokinases altered, decreasing the type II/type I activity ratio to about 1.     

Some properties of rat-liver glucose–adenosine triphosphate phosphotransferases

1. Bacilysin, a peptide which yields l-alanine and l-tyrosine on acid hydrolysis, was produced by a strain of Bacillus subtilis (A 14) in a chemically defined medium containing glucose, ammonium acetate or ammonium chloride, potassium phosphate and other inorganic salts, and ferric citrate. 2. Under the conditions used growth was diphasic. Bacilysin was formed during the second phase of slower growth, and there was little production during the stationary phase. Nevertheless, bacilysin production occurred when protein synthesis was inhibited by chloramphenicol. It thus appears that there is no obligatory coupling of protein synthesis and bacilysin synthesis. 3. When dl-[1-(14)C]alanine was added to a growing culture of B. subtilis, (14)C was incorporated into bacilysin, which contains an N-terminal alanine residue. 4. Under similar conditions virtually no (14)C was incorporated into bacilysin from dl-[2-(14)C]tyrosine, l-[U-(14)C]tyrosine or [1-(14)C]acetate, although these compounds were used by the cell for the biosynthesis of other substances. These results indicate that neither tyrosine nor acetate is a precursor of the fragment of bacilysin which yields tyrosine on hydrolysis with hot 6n-hydrochloric acid. 5. The tyrosine-yielding fragment of bacilysin was labelled with (14)C from [1,6-ring-(14)C(2)]shikimic acid. The biosynthesis of bacilysin thus appears to involve a diversion from the pathway leading to aromatic amino acids at the shikimic acid stage, or a subsequent one.     

Kinetic studies of the reverse reaction catalysed by adenosine triphosphate–creatine phosphotransferase. The inhibition by magnesium ions and adenosine diphosphate

1. Kinetic investigations of the reaction catalysed by ATP–creatine phosphotransferase have been carried out. 2. No firm conclusions could be reached about the reaction of Mg²⁺ at the nucleotide-binding site of the enzyme. The value of the kinetic constant for this reaction depends on the value used for the apparent stability constant of the metal ion–nucleotide complex and, to a smaller extent, on the method of plotting the results. 3. At higher concentrations Mg²⁺ is a non-competitive inhibitor of the enzyme with respect to both MgADP⁻ and phosphocreatine. 4. ADP³⁻ is a competitive inhibitor of the enzyme with respect to MgADP⁻ and a non-competitive inhibitor with respect to phosphocreatine. 5. The concentration of phosphocreatine has little, if any, effect on the kinetic constants for the nucleotide reactants.     

Separation of adenosine diphosphate-adenosine triphosphate–exchange activity from the cerebral microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase

1. A microsomal fraction from ox cerebral cortex catalysed [(14)C]ADP-ATP exchange at a speed similar to that at which it liberated P(i) from ATP in the presence of Na(+), K(+) and Mg(2+). 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na(+), K(+) or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide-cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na(+)-plus-K(+)-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP-ATP-exchange activity does not take part in the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP-ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na(+)-plus-K(+)-stimulated adenosine triphosphatase is indicated.     

Formation of an unusual hybrid in the development of human adenosine 5′-triphosphate–creatine phosphotransferase

1. Starch-gel analysis of extracts from adult human muscle, heart and brain reveals a hybrid creatine kinase with an abnormally high electrophoretic mobility. 2. Hybridization in vitro confirms that the postulated hybrid is formed from a combination of brain- and muscle-type enzyme sub-units. 3. The relative electrophoretic mobility of the hybrid is not affected by changing the starch concentration in the gel or by the buffer system used, or by electrophoresis in thin layers of Sephadex. 4. It is concluded that hybrid formation results in a net increase on the dimeric enzyme of from 4 to 6 negative charges. 5. During development a sharp increase in the rate of production of muscle-type enzyme sub-units occurs in heart at 10–13 weeks' gestation and in muscle at 18–20 weeks' gestation. The latter change is accompanied by a relative decrease in the concentration of brain-type sub-units.     

Activation of brain hexokinase by magnesium ions and by magnesium ion–adenosine triphosphate complex

1. An alternative explanation for the kinetic data obtained by Bachelard (1971) for the brain hexokinase reaction is presented. 2. Apparently sigmoidal saturation curves for MgATP²⁻ based upon Bachelard's (1971) studies can be corrected to hyperbolic curves by use of a stability constant for MgATP²⁻ complex formation. 3. A number of other effects related to the concentration-dependent stability of the MgATP²⁻ complex and to the presence of the inhibitory free uncomplexed ATP⁴⁻ concentration are also explained in terms of a non-allosteric role for either Mg²⁺ or MgATP²⁻ fully consistent with a number of previous reports on this enzyme. 4. A brief discussion of the validity of Hill plots in studies of multisubstrate co-operative enzymes is presented. 5. A simple model is presented that demonstrates how enzymes obeying Michaelis–Menten kinetics can demonstrate sigmoidal velocity responses if the true substrate of the reaction is the metal–substrate complex.     

Allosteric activation of brain hexokinase by magnesium ions and by magnesium-ion–adenosine triphosphate complex

1. The highest blood concentrations of ketone bodies were found at 5 days of age, after which time the concentration fell to reach the adult value by 30 days of age. 2. Both mitochondrial and cytoplasmic hydroxymethylglutaryl-CoA synthase activities were detected, with highest activities being found in the mitochondria at all stages of development. Activity of the mitochondrial enzyme increases rapidly immediately after birth, showing a maximum at 15 days of age, thereafter falling to adult values. The cytoplasmic enzyme, on the other hand, increased steadily in activity after birth to reach a maximum at 40 days of age, after which time activity fell to adult values. 3. Both mitochondrial and cytoplasmic aceto-acetyl-CoA thiolase activities were detected, with the mitochondrial enzyme having considerably higher activities at all stages of development. The developmental patterns for both enzymes were very similar to those for the corresponding hydroxymethylglutaryl-CoA synthases. 4. The activity of heart acetoacetyl-CoA transferase remains constant from late foetal life until the end of the suckling period, after which time there is a gradual threefold increase in activity to reach the adult values. The activity of brain 3-oxo acid CoA-transferase increases steadily after birth, reaching a maximum at 30 days of age, thereafter decreasing to adult values, which are similar to foetal activities. Although at all stages of development the specific activity of the heart enzyme is higher than that of brain, the total enzymic capacity of the brain is higher than that of the heart during the suckling period.     

Coordination of magnesium with adenosine 5′-diphosphate and triphosphate

³¹P NMR chemical shifts of salts of adenosine 5′-triphosphate and diphosphate: ATPH^2?₂2(Me₄N⁺) · H₂O, ATPH^2?₂2 Na⁺ · 3.5 H₂O, ATPH^2?₂Mg²⁺ · 4 H₂O, ATPH^2?₂Ca²⁺ · 2 H₂O, ADPH^2?2(Me₄N⁺) · H₂O and ADPH^2?Mg²⁺ · 4 H₂O have been measured in 0.02 M ²H₂O solutions at 145.7 MHz (22° C) at constant p²H values (8.20 and 6.20). The results are compared with those obtained from salts of adenosine 5′-monophosphate and other simpler phosphomonoesters, e.g. AMP^2?2(Me₄N⁺), AMP^2?Mg²⁺, AMPH^?Me₄N⁺ and (AMPH^?)₂Mg²⁺. It is concluded that the effects exerted by Mg²⁺ and Ca²⁺ on the ³¹P NMR shifts of dipoly- and tripolyphosphates relative to monovalent cations are due mainly to changes in conformation of the polyphosphate chain rather than to purely electronic factors associated with the binding of divalent cations to the phospho-oxyanions. The data are consistent with the existence of the following complexes at p²H 8.20: (MgP_αP_β)ADP^? and (MgP_αP_γ)ATP^2?af (MgP_αP_β)ATP^2?af (MgP_βP_γ)ATP^2? with the latter equilibrium relatively fast in the NMR time scale. Monoprotonation of the terminal phosphate appears to weaken the Mg²⁺-polyphosphate binding, particularly at P_β of MgADPH and at P_β and P_γ of MgATPH^?. The Mg²⁺-polyphosphate binding weakens further at p²H 3.70, i.e. in MgATPH₂. Possible implications of the results in the mechanism of actomyosin Mg²⁺-ATPase in muscle contraction are discussed.     

Potassium ion-stimulated and sodium ion-dependent adenosine diphosphate–adenosine triphosphate exchange activity in a kidney microsomal fraction

1. K(+) did not affect the Mg(2+)-dependent transphosphorylation but markedly increased the Na(+)-stimulated ADP-ATP exchange rate mediated by a microsomal fraction from guinea-pig kidney. 2. Rb(+), Cs(+), NH(4) (+) and Li(+) were equally effective in stimulating the Na(+)-dependent ADP-ATP exchange activity. 3. Treatment of the microsomal fraction with N-ethylmaleimide or increased concentrations of Mg(2+) prevented stimulation of the Na(+)-dependent exchange reaction by K(+). 4. Ouabain (2.5mum) inhibited ATP hydrolysis by 33% but did not decrease the K(+)-stimulated Na(+)-dependent ADP-ATP exchange rate. 5. A possible mechanism for stimulation of exchange activity by K(+) is discussed.     

Inhibition of adenosine 5′-triphosphate–creatine phosphotransferase by substrate–anion complexes. Evidence for the transition-state organization of the catalytic site

1. The substrate combination creatine-MgADP does not significantly protect creatine kinase against inhibition by iodoacetamide in the absence of small anions. 2. Small anions can be divided into three groups according to the way in which they affect creatine kinase: I, acetate reversibly increases enzyme activity in the forward reaction but does not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP; II, planar anions and some halides (HCO(3) (-), HCO(2) (-), NO(3) (-), NO(2) (-), Cl(-), Br(-), F(-)) in the presence of creatine plus MgADP protect the enzyme from inhibition by iodoacetamide; III, tetrahedral anions (SO(4) (2-), HPO(4) (2-), ClO(4) (-), BF(4) (-)) and iodide do not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP but may decrease the protection by class II anions under these conditions. Anions of class II and class III also reversibly inhibit enzyme activity. 3. It is concluded that class II anions form a stable and inactive quaternary enzyme-creatine-MgADP-anion complex and this is responsible for the effect attributed by previous workers to the ternary complex lacking anion. Formation of this complex, particularly in the forward reaction, can lead to markedly non-linear enzyme progress curves. Some previous observations are re-appraised in the light of these findings. 4. From the behaviour of chloride and nitrate ions, and the marked lowering of the K(i) values for creatine and MgADP they produce, it is inferred that planar or monoatomic anions act in the quaternary complex by simulating the transferable phosphoryl group in the transition state (or another intermediate state) of the reaction. 5. It is suggested that, in the course of the reaction, the tetrahedral phosphate-binding site for the transferable phosphoryl group of the substrate (that also binds class II and class III anions) changes into a trigonal bipyramid site (also occupied by class II anions). This strains the phosphoryl group to adopt the transitional sp(3)d hybridized state and must contribute significantly to the low activation energy of the reaction. 6. Catalysis is deduced to proceed by an ;in line' transfer reaction and from the effects of class II anions it is possible to estimate the approximate dimensions of the anionic site in the transition-state complex. 7. The specific protecting effect of an equilibrium mixture of substrates against inhibition by iodoacetamide provides further evidence for the conformational change suggested above as a step in the catalytic process.     

Metabolism of propionate by sheep-liver mitochondria: Effects of α-oxoglutarate, adenosine triphosphate, sodium chloride and potassium chloride

1. A study has been made of the effects of ATP and alpha-oxoglutarate on the rate of metabolism of propionate by whole mitochondria from sheep liver, and by mitochondria disrupted with ultrasonic energy or by freezing and thawing. Whole mitochondria metabolized propionate aerobically; the rate was increased and stabilized by 0.5mm-ATP, and increased at least a further 50% by 1.67mm-alpha-oxoglutarate. 2. Anaerobically, externally added ATP at high concentrations permitted slow consumption of propionate. 3. In the presence of 1.3mm-ATP, but in the absence of alpha-oxoglutarate, there was no significant lag phase in the removal of propionate by whole mitochondria, and the rate declined at concentrations below 2mm. In the additional presence of 1.67mm-alpha-oxoglutarate or -glutamate, propionate was removed at linear rates until the residual propionate concentration was about 0.1mm. 4. Maximum rates of metabolism of propionate by whole mitochondria with 1.3mm-ATP occurred with alkali-metal chloride concentrations of 65-95mm and with K(+)/Na(+) ratios 5-10, both in the presence and absence of alpha-oxoglutarate. 5. With disrupted mitochondria stimulatory effects of alpha-oxoglutarate were obtained only aerobically, only with propionate and not propionyl-CoA as substrate, and only when sufficient mitochondrial structure remained to permit unsupplemented metabolism of propionate to occur. 6. In the presence of ATP and CoA, disrupted mitochondria fixed [2-(14)C]propionate at a rate adequate to explain the rate with whole mitochondria stimulated with ATP and alpha-oxoglutarate. 7. With both whole and partially disrupted mitochondria in the absence of ATP, the rate of metabolism of propionate was inhibited by about 80% by 3.3mm-AMP. The inhibition was partly overcome by alpha-oxoglutarate plus CoA. 8. It is concluded that the ultimate effect of alpha-oxoglutarate was to increase the rate of supply of ATP within the mitochondria. Reasons are given why it is premature to conclude that the extra ATP arose entirely from the oxidation of alpha-oxoglutarate itself.     

The adenosine diphosphate–adenosine triphosphate-exchange reaction of cerebral microsomes and its relation to the sodium ion-stimulated adenosine-triphosphatase reaction

Microsomes from guinea-pig cerebral cortex contain a system capable of exchanging ADP with ATP at rates of about 20mumoles/mg. of protein/hr. The ADP-ATP-exchange reaction requires Mg(2+) for activity. The reaction is not stimulated by Na(+) or K(+) and is not inhibited by ouabain, in contrast with the Na(+)-plus-K(+)-stimulated adenosine triphosphatase. The pH optimum also differs from that of the adenosine triphosphatase. The ADP-ATP-exchange reaction is stimulated two- to three-fold by non-ionic, anionic and cationic detergents, even when these agents are inhibiting the adenosine-triphosphatase reaction. This reaction may represent a component of the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction but is more likely to be due to other enzyme systems present in microsomal subfractions.     

Structure–activity relationships of truncated adenosine derivatives as highly potent and selective human A3 adenosine receptor antagonists

On the basis of potent and selective binding affinity of truncated 4′-thioadenosine derivatives at the human A₃ adenosine receptor (AR), their bioisosteric 4′-oxo derivatives were designed and synthesized from commercially available 2,3-O-isopropylidene-d-erythrono lactone. The derivatives tested in AR binding assays were substituted at the C2 and N⁶ positions. All synthesized nucleosides exhibited potent and selective binding affinity at the human A₃ AR. They were less potent than the corresponding 4′-thio analogues, but showed still selective to other subtypes. The 2-Cl series generally were better than the 2-H series in view of binding affinity and selectivity. Among compounds tested, compound 5d (X = Cl, R = 3-bromobenzyl) showed the highest binding affinity (K_i = 13.0 ± 6.9 nM) at the hA₃ AR with high selectivity (at least 88-fold) in comparison to other AR subtypes. Like the corresponding truncated 4′-thio series, compound 5d antagonized the action of an agonist to inhibit forskolin-stimulated adenylate cyclase in hA₃ AR-expressing CHO cells. Although the 4′-oxo series were less potent than the 4′-thio series, this class of human A₃ AR antagonists is also regarded as another good template for the design of A₃ AR antagonists and for further drug development.     

Properties and reaction with iodoacetamide of adenosine 5′-triphosphate–creatinine phosphotransferase from human skeletal muscle. Further evidence about the role of the essential thiol group in relation to the mechanism of action

1. The purification of creatine kinase from human and monkey skeletal muscle by horizontal electrophoresis on Sephadex blocks is described. 2. The purified enzymes are shown to have similar chemical and kinetic properties to the rabbit muscle enzyme and a common mechanism is inferred. 3. Iodoacetamide has a similar apparent second-order inhibition constant with the human and rabbit enzymes, but the inhibition does not go to completion with the former. This is even more marked with the monkey enzyme, which has more reactive thiol groups, but inhibition is only about 50%. 4. Single substrates have little effect on the inhibition by iodoacetamide, but with the primate enzymes, in contrast with the rabbit enzyme, high concentrations of ADP-Mg(2+) plus creatine convert the essential thiol group from being pH-independent into one with a normal ionization. Low concentrations of ADP-Mg(2+) plus creatine first enhance the rate of inactivation, but cause protection as the reaction proceeds. These results are interpreted to indicate an activation of the thiol group on the subunit to which the substrates bind and a co-operatively induced decrease in the activity of the thiol group on the other subunit which lacks substrates. 5. The effects of a substrate equilibrium mixture on the rate of inhibition are essentially those of ADP-Mg(2+) plus creatine. 6. Since no substrate combination affords significant protection to the thiol group associated with the catalytic site to which the substrates are bound, it is concluded that any mechanism involving the thiol group in a direct participation in the transition-state complex of the catalytic reaction must be abandoned unless the transition state is only a small part of the time taken for one catalytic cycle.     

Use of 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates having poly(ethylene glycol) spacers in kinase-catalyzed phosphorylations

The 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2α), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which K(M) values were determined to be in the 30-200 μM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing protein-protein interactions.     

Brain adenosine 5′-triphosphate–creatine phosphotransferase. Purification, thiol group reactivity and the amino acid sequence around the reactive thiol groups

1. The purification of creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) from ox brain by a method that is quicker, simpler and gives much higher yields than other published procedures is described. 2. Stoicheiometric inhibition studies with iodoacetate showed that the enzyme, like that from muscle, has two reactive thiol groups that are essential for enzyme activity. 3. The amino acid sequence around the essential thiol groups was determined and found to be virtually identical with that in creatine kinases from rabbit and ox muscle, and very similar to that found in arginine kinase; the evolutionary significance of this is discussed. 4. The identification of DNS-amino acids on thin layers of silica gel was found to have, in many cases, distinct advantages over that on polyamide layers.