Optimal ATP Concentration Research Articles

Partial purification and ouabain sensitivity of lubrol-extracted sodium-potassium transport adenosine triphosphatases from brain and cardiac tissues

Lubrol extracts containing (Na + K)-activated adenosine triphosphatase of specific activity 70–100 μmoles P l/mg of protein/hr were prepared from rat brain microsomal fractions. The enzyme was stable in 0.32 M sucrose containing 1 mM EDTA at pH 6.9 and had properties, regarding optimal concentrations of ATP, Na and K required for maximal activation, similar to those of particulate preparations of the rat brain enzyme. The apparent K m for the enzyme was 5.0 × 10 −4 M and the optimal pH was 6.8. Sucrose density gradient separation further purified the (Na + K)-activated adenosine triphosphatase, but the resulting preparation was not homogeneous, despite high specific activity (400 μmoles P i/mg of protein/hr), and contained at least five protein staining bands on disc gel electrophoresis. Lubrol treatment was effective in extracting the enzyme from cardiac tissue of both rat and guinea pig and appeared to increase the sensitivity of the enzyme to ouabain. Enzymes of cerebral origin from the two species were of equal sensitivity to ouabain. The enzyme extracted by Lubrol treatment from guinea pig heart microsomes was 23 times more sensitive to ouabain than a similar preparation from rat heart.

Hormone action at the membrane level. I. Properties of adenyl cyclase in isolated plasma membranes of rat liver

Membarne bound adenyl cyclase in isolated rat liver plasma membranes exhibits a requirement for Mg 2+ and has optimal activity near pH 8.0. The optimal concentration of ATP is approx. 0.3 mM at a Mg 2+ concentration of 1.0 mM. Ca 2+ at a concentration range of 0.03–0.3 mM stimulates the basal activity of this enzyme. The enzyme activity is abolished by 1 min heating at 100°. Detergents such as Triton X-100 and sodium dodecyl sulfate alter the membrane structure and slightly enhance the enzyme actively, whereas sodium deoxycholate slightly inhibits the enzyme. Glucagon and epinephrine stimulate whereas insulin inhibits adenyl cyclase activity. When studied under identical conditions on the same membrane preparation the glucagon stimulation is seen earlier and at a lover hormone concentration (1 · 10 −6 M) than is the epinephrine stimulation which requires 10 −5 M hormone. The inhibition by insulin is seen at 1 · 10 −5 M or greater. The stimulation by glucagon is inhibited by 1 · 10 −4–1 · 10 −5 M Ca 2+ but the stimulation by epinephrine is enhanced by 1 · 10 −4–1 · 10 −5 M Ca 2+. Moreover, epinephrine enhances, whereas glucagon inhibits the binding of Ca 2+ to the membrane. Studies on the combined effects of the hormones show that the stimulation by glucagon plus epinephrine is not additive and that insulin antogonizes the glucagon stimulation of adenyl cyclase. Na + inhibits the basal activity of adenyl cyclase but K + stimulates the enzyme. F − inhibits the enzyme. These results point to a complex interplay of hormones and metal ions with the membrane bound adenyl cyclase.

Reversal of Ethionine‐Induced Inhibition of Elongation of Avena Coleoptiles by Purines, Adenosine, and Glutamic Acid

AbstractEthionine‐induced inhibition of elongation of Avena coleoptile segments has been measured in water and in indol‐3yl‐acetic acid. In the presence of 10 mM L‐ethionine the inhibition amounts to about 70 %. It has been shown previously that the addition of adenosine triphosphate effectively counteracts this inhibition; optimal ATP concentrations are between 0.25 and 0.5 mM. Adenine, adenine sulfate, adenosine, guanine, S‐adenosylmethionine, and glutamic acid have now been shown to act similarly to ATP in reversing the ethionine‐induced inhibition of elongation.

Generation of reducing power in chemosynthesis V. The mechanism of pyridine nucleotide reduction by nitrite in the chemoautotroph Nitrobacter agilis

1. Cell-free extracts from Nitrobacter catalyzed an energy-dependent reduction of NAD + by NO 2 −; the process could be driven either at the expense of added ATP or by the energy generated from NO 2 − oxidation. 2. The reduction of cytochrome c by NO 2 − was observed to be an energydependent reaction which involved reversal of electron transfer from cytochrome a 1. The subsequent energy-linked reduction of the flavoproteins and pyridine nucleotides occurred concomitantly with the oxidation of cytochrome c. 3. The reduction of each mole of NAD + by NO 2 − required the utilization of approx. 5 moles of ATP. These observations are in harmony with the calculated energetics of the overall reverse electron flow process which involves a free energy gap of some 35 kcal. 4. Under conditions when ATP was limiting, the energy-linked reduction of 1 molecule of NAD + by nitrite required the concomitant oxidation of 2 molecules of cytochrome c. However, when the process was driven by an optimal ATP concentration (0.7 mM), the rate of NAD + reduction was about 1.5-fold compared to the rate of cytochrome c oxidation. 5. The process of energy-linked reversal of electron transfer in Nitrobacter was markedly sensitive to the inhibitors of the flavoprotein systems as well as to antimycin A or 2- n-heptyl-4-hydroxyquinoline N-oxide. In addition, CN − was observed to be a potent inhibitor. The uncouplers of oxidative phosphorylation caused a strong inhibition of the ATP-linked reverse electron transfer, and the energy transfer from ATP to drive reverse electron flow was also inhibited by oligomycin.

Effects of Carnosine and Anserine on Muscle Adenosine Triphosphatases

Natural dipeptides of skeletal muscles, carnosine (β-alanylhistidine) and anserine (β-alanyl-1-methylhistidine), have been demonstrated to be myosin ATPase activators, the latter being much more effective than the former under identical experimental conditions. At low ionic strengths of K+, Mg++, and Ca++ the activating effect of carnosine was marked at optimal concentration of ATP, and at levels of ATP in which there was apparent substrate inhibition the effect was very pronounced. The effect of carnosine appears to be specific rather than an ionic strength effect. The lowest concentrations of carnosine and anserine found to be activating were comparable to concentrations found in tissue.

K +-dependent phosphatase activity observed in the presence of both adenosine triphosphate and Na +

1. 1.Addition of both ATP and Na + caused a remarkable increase in the activity of the K +-dependent phosphatase at low concentrations of K +, although addition of either ATP or Na + alone was inhibitory. The apparent K m for K + decreased from 2 mM to p.2 mM on addition of both substances. The υ max was depressed by them. 2. 2.CTP, ITP, and ADP showed effects similar to ATP in the presence of Na +. The optimal concentrations of ATP and ADP were observed to be 10 −4 M and 2 · 10 −4 M, but that of CTP and ITP was 10 −3 M. AMP, 3′,5′-cyclic AMP, CDP, UTP, and GTP had no significant stimulatory effect. 3. 3.Replacement of Na + with other monovalent cations, such as Rb +, NH 4 +, Cs +, or Li +, caused the stimulatory effect of ATP to disappear. 4. 4.The K +-dependent phosphatase became heat labile on addition of K +, but was stabilized by further addition of Na + and ATP. 5. 5.Ouabain inhibited the K +-dependent phosphatase activity more in the presence of ATP and Na + than in their absence. 6. 6.The K +-dependent phosphatase activity was less sensitive to treatment with F − in the presence of ATP and Na + than in their absence. 7. 7.From these results it was concluded that the affinity of the K +-dependent phosphatase for K + was increased by addition of ATP and Na +, and the properties came closer to those of the (Na +-K +)-ATPase.

Micro-colorimetric determination of adenosinetriphosphatase activity in freeze-dried sections of rat diaphragm muscle.

Abstract A technique is described for determination of ATPase activity in 3–30 μg samples dissected from freeze-dried sections of rat diaphragm muscle. Tissue samples are incubated at room temperature with optimal concentrations of ATP (10 mM) and MgSO4 (5 mM) in 0·1 M tris/HCl buffer at pH 7·4. Inorganic phosphate is measured spectrophotometrically. Mean activity for 20 rats was 0·22 ± 0·06 (s.d.) mole-Pi/kg/wet tissue/15 min. Results did not depend on plane of section or section thickness. 5 mM Mg2+, 10 mM Ca2+ and 1 mM 2, 4-dinitrophenol produced maximal ATPase activation and inhibition was obtained with p-chloromercuribenzoic acid (pI503·9) but not chlorpromazine (0·01–0·1 mM). There was no significant evidence of Na+ plus K+ activated ATPase or inhibition with ouabain (5 mM). ATPase activity was uncharacterized and relative contributions of specified muscle ATPase systems were unknown. As an insoluble enzyme system was involved and photomicrographs showed characteristic muscle features in freeze-dried sections, ATPase activity appeared due to enzymic function in situ. Results are discussed in relation to mutual availability of enzymic sites and reagents.

Fatty acid esterifying enzymes in rat adipose tissue homogenates.

Some characteristics of enzymes in homogenates of rat adipose tissue concerned with esterification of fatty acids have been investigated. Acyl-CoA thiokinase is the most active of the enzymes studied, with optimal concentrations of palmitate and ATP being 2 mM and 25 mM respectively. The thiokinase has a pH optimum between 8 and 10 and reacts with fatty acids ranging in chain length from C4 to C22, with the greatest activity towards palmitate. About 50% of the activity is found in the 100 000 × g supernatant. Acyl-CoA : α-glycerolphosphate acyltransferase and acyl-CoA deacylase have pH optima between 7 and 8. Albumin at a concentration of 1% activates the former, while the latter is inhibited by concentrations of albumin greater than 0.5%. Deacylase activity is found almost entirely in the 100 000 × g supernatant. Both the acyl-CoA : α-glycerolphosphate acyltransferase and the acyl-CoA : diglyceride acyltransferase lose much of their activity upon fractionation. The pH optimum of the acyl-CoA : diglyceride acyltransferase ranges from pH 7 to 9, while that of the phosphatidc acid phosphatase is 6. The latter enzyme is distributed equally between particulate and soluble portions of the homogenate. When these enzymes are assayed in homogenates from fed and fasted animals, a significant alteration is found only in the level of acyl-CoA deacylase, which is decreased. The properties of these enzymes can be related to the extent and type of esterification in homogenates from fed and fasted animals.

Energy requirements in the incorporation of choline by isolated rat liver mitochondria.

Optimal concentrations of ATP, CTP and ADP for the incorporation of 14C‐labelled choline into phospholipid by isolated rat liver mitochondria were 0.7 mM, 0.25 mM and 1.0 mM respectively. Higher concentrations markedly depressed the incorporation. Substitution of CTP for other nucleotide triphosphates led to lower rates of incorporation.Inhibitors of electron transport and of oxidative phosphorylation inhibited the respiration‐supported but not the ATP‐supported incorporation of choline. It is concluded that high energy intermediates of oxidative phosphorylation are not obligatory for the incorporation process.

Reversal of Hydroxyproline-Induced Inhibition of Elongation of Avena Coleoptiles

Hydroxy-l-proline-induced inhibition of elongation of Avena coleoptile segments was measured in water and in indole-3-acetic acid. This inhibition was completely reversed by l-proline.Time-sequence experiments revealed that some time had to elapse, or some elongation had to occur, prior to the onset of hydroxyproline inhibition. The presence of sucrose supported the rate of auxin-induced elongation throughout a 24-hour period, and enhanced the effectiveness of hydroxyproline as an inhibitor of elongation.The application of adenosine triphosphate effectively counteracted the hydroxy-proline-induced inhibition of elongation; completely in water, and partially in indole-3-acetic acid. Optimal ATP concentrations were between 0.25 and 0.75 mm. Similarly, guanine-HCL, l-glutamic acid, and l-ornithine-HCL were capable of reversing the hydroxyproline inhibition. Other compounds that were tested but which proved to be less effective are tabulated.It is suggested that hydroxyproline may exert its inhibitory effect on the protein and/or RNA synthesis necessary for elongation of the oat coleoptile by interference with the metabolism of adenosine triphosphate or other high energy compounds.

The in vitro release of protein from Escherichia coli ribosomes

The in vitro release of newly formed protein from the ribosome to the hot trichloroacetic acid precipitable supernatant fraction requires prelabeled ribosomes, ribosomal-free supernatant fraction, ATP, and probably Mg2+. The optimal ATP concentration is six-fold higher than that required for overall amino acid incorporation. Puromycin and deoxyribonuclease stimulate the release of radioactive protein from ribosomes both in the presence and absence of ATP, while chloramphenicol is neither stimulatory nor inhibitory. At least half the radioactivity released from prelabeled ribosomes is not precipitable by trichloroacetic acid. The amount of this material increases with time. ATP is required to effect the release of this trichloroacetic acid non-precipitable component.

The in vitro release of protein from Escherichia coli ribosomes

The in vitro release of newly formed protein from the ribosome to the hot trichloroacetic acid precipitable supernatant fraction requires prelabeled ribosomes, ribosomal-free supernatant fraction, ATP, and probably Mg 2+. The optimal ATP concentration is six-fold higher than that required for overall amino acid incorporation. Puromycin and deoxyribonuclease stimulate the release of radioactive protein from ribosomes both in the presence and absence of ATP, while chloramphenicol is neither stimulatory nor inhibitory. At least half the radioactivity released from prelabeled ribosomes is not precipitable by trichloroacetic acid. The amount of this material increases with time. ATP is required to effect the release of this trichloroacetic acid non-precipitable component.

Studies on the function of intracellular ribonucleases. III. The relationship of the ribonuclease activity of rat liver microsomes to their biological activity.

Attempts have been made to prepare rat liver microsomes and ribosomes free of RNase activity. Washing of microsomes with a large number of reagents, as well as preparation of microsomes by homogenizing the liver in the presence of a variety of reagents chosen to remove or inhibit RNase activity, failed to abolish completely the enzyme activity. However, when rat liver was homogenized in the presence of optimal concentrations of ATP the microsomes subsequently obtained showed no RNase activity. The composition of such microsomes was compared to controls prepared without the use of ATP. Preparation of microsomes with the use of ATP apparently repressed but did not remove the RNase activity for, when such microsomes were treated with 1 per cent deoxycholate to obtain ribosomes, the latter exhibited normal RNase activity. A possible explanation for these results based on several experiments is given. The incorporation of C(14) of L-leucine-C(14) into control and ATP-treated microsomes was measured. Repression of RNase activity by use of ATP or with RNase inhibitor, significantly reduced the incorporation. As a result of these and other experiments it is tentatively concluded that an alkaline RNase is a normal constituent of rat liver ribosomes and plays a role in the biological activity of these particles.