Phosphorylation Of Creatine Research Articles

β-Guanidinopropionate and phosphorylated β-guanidinopropionate as substrates for creatine kinase

Rabbit muscle creatine kinase catalyzes transphosphorylation between ATP and β-guanidinopropionate at pH 9 and 23°C with a K m for β-guanidinopropionate of 50 m m and a V max of 0.21 μmole/min/mg of creatine kinase. The comparable values for the phosphorylation of creatine are 16.7 for K m and 75.1 for V max. In the reverse direction at pH 7.05 and 23°C, phosphorylated β-guanidinopropionate has a K m of 4.9 and a V max of 0.22 in comparison to a K m of 2.2 and a V max of 231 for phosphocreatine.

Synthesis of glucose 6-phosphate by a 1,3-diphosphoglycerate-glucose phosphotransferase activity of liver microsomes

1. 1. Glucose 6-phosphate can be synthesized directly from 1,3-diphosphoglycerate and glucose in a reaction catalyzed by a liver and kidney microsomal enzyme whose properties resemble those of glucose-6-phosphatase (EC 3.1.3.9) with its accompanying inorganic pyrophosphate-glucose phosphotransferase activity. 2. 2. Specifically labeled radioactive 1,3-[1- 32P]diphosphoglycerate has been prepared and used to show that it is exclusively the acid anhydride phosphate on C-1 of that compound that is transferred to glucose. 3. 3. The possibilities that, in the system used, the glucose 6-phosphate synthesis occurs indirectly from 1,3-diphosphoglycerate by way of 3-phosphoglycerate kinase-catalyzed ATP formation or by way of phosphoglucomutase phosphorylation have been eliminated. 4. 4. Enzymatically catalyzed hydrolysis of 1,3-diphosphoglycerate occurs at 30°C and pH 6.0 in addition to the appreciable spontaneously occurring hydrolysis of the compound. 5. 5. The enzyme activity, like that of glucose-6-phosphatase and related enzyme activities, is greatly increased by pretreatment of the microsomes at about pH 10 or by detergent pretreatment. 6. 6. In activated preparations the pH optimum of the phosphotransferase reaction was 5.2–5.3 and the K m = 1.4 mM for 1,3-diphosphoglycerate and 0.08 M for glucose. 7. 7. Under comparable conditions, 1,3-diphosphoglycerate was 30–50% as effective a donor as PP i in the synthesis of sugar phosphates. Other sugars and sugar alcohols, as well as glycerol can be enzymatically phosphorylated by 1,3-diphosphoglycerate, the relative extent of phosphorylation of each acceptor compound being approximately the same as is observed with PP i as phosphate donor. The enzyme was unable to catalyze the phosphorylation of creatine with either PP i or 1,3-diphosphoglycerate as donor. 8. 8. PP i glucose (glycerol) phosphotransferase was found to be unable to catalyze the introduction of a second phosphate group into glycerol 3-phosphate or glucose 1-phosphate.

Creatine Kinase of Rat Heart Mitochondria: COUPLING OF CREATINE PHOSPHORYLATION TO ELECTRON TRANSPORT

Abstract Intact mitochondria from human, beef, and rat heart, as well as rat brain and rat skeletal and smooth muscle, contain considerable creatine kinase activity; those from rat or rabbit liver, kidney, and testis contain essentially none. In rat heart mitochondria creatine kinase activity is far higher than the activity of adenylate kinase and nucleoside diphosphokinase. The heart mitochondrial creatine kinase is specific for ATP and ADP as phosphate donor and acceptor. The optimum pH is 6.7 in the direction of ATP formation and 8.0 in the direction of phosphocreatine formation. The mitochondrial isoenzyme of creatine kinase is not present in the same compartment as the intermembrane marker enzyme adenylate kinase, but may be released in soluble form by exposure to sodium acetate or sodium phosphate medium at pH 7.4. The phosphorylation of creatine by intact mitochondria takes place outside the atractyloside-sensitive ADP-ATP carrier system of the inner membrane and thus requires external ATP. When creatine in the concentration range known to be present in intact heart is added to rat heart mitochondria neither the initial State 4 or State 3 rate of respiration (succinate) is affected; however, there is a large increase in the State 4 rate following addition of ADP, whose magnitude increases as the creatine concentration is increased. In the presence of physiological concentrations of creatine and ADP rat heart mitochondria respire at a constant high rate equal to 75 to 85% of their maximal State 3 rate; under these conditions phosphocreatine is the end product of oxidative phosphorylation. The Km values for rat heart mitochondria are 35 µm for ADP, 0.72 mm for phosphocreatine, 100 µm for ATP, and 6 mm for creatine. These and other properties suggest that in addition to the ∼P storage function usually postulated for it, the phosphocreatine-creatine kinase system serves to smooth out or buffer the rate of respiration of heart mitochondria at a high and constant rate, as well as to channel ∼P to the contractile system via the mitochondrial and sarcoplasmic isoenzymes of creatine kinase.

Studies with a reconstituted muscle glycolytic system. The rate and extent of creatine phosphorylation by anaerobic glycolysis.

A mixture of purified muscle glycolytic enzymes was reconstituted and the mixture shown to behave in a fashion analogous to that occurring in vivo. Glycolysis leads to ATP production in muscle and results in the phosphorylation of creatine. The extent of this phosphorylation by anaerobic glycolysis was shown to depend to a small extent on the relative proportions of available P(i) and creatine initially, but more importantly on the first step in glycolysis, in this case the enzyme phosphorylase. With less than 0.1% of the phosphorylase in the a form, only about one-third of the creatine was phosphorylated in 30min, whereas with 4% or more of phosphorylase a, 90% of the creatine was phosphorylated within this time. Inclusion of an adenosine triphosphatase decreased the steady-state concentration of phosphocreatine in the system. Calculations of the theoretical concentrations of ADP and AMP showed that phosphorylase b was almost inactive even in the presence of 9mum-AMP, because of ATP inhibition. With phosphorylase a present, glycolysis was able to continue at least until the calculated concentration of MgADP(-) was only 7mum, and AMP in the sub-mumolar range. The relation of these values to measured concentrations of nucleotides and to phosphorylase a percentages in intact muscle is discussed.

Attempts to find creatine phosphokinase and 5'-nucleotidase activity in canine prostatic fluid.

An attempt was made to establish the presence of creatine phosphokinase and 5′-nucleotidase activities in canine prostatic secretion. Pooled prostatic fluid and purified enzyme fractions were utilized as sources of enzyme. Transferase activity was followed by phosphorylation of creatine and dephosphorylation of phosphocreatine, and 5′-nucleotidase activity by hydrolysis of ade-nosine-5′-phosphate. No creatine phosphokinase activity could be detected in the sources of enzyme but a small amount of 5′-nucleo-tidase activity may have been observed. The latter could not be distinguished from prostatic acid phosphatase by purification procedures or inhibition with tartrate and sodium fluoride.

Die bestimmung der serum-kreatinphosphokinase im optischen test

Various test arrangements for measuring the serum creatine phosphokinase in the optic test were compared with each other. It was found that in the determination based on the “forward reaction” (phosphorylation of creatine) a set-up elaborated by the authors, at pH 7.6 presented the slightest proneness to disturbances. Only with one of the four arrangements at pH 9 (the pH optimum of the “forward reaction”) were activities measured which were about 20% higher than those measured at pH 7.6. With the other set-ups, patently lower values were found than at pH 7.6. In the determination based on the “reverse reaction” (phosphorylation of ADP) only in the presence of AMP could an adequately linear proportionality between serum quantity and reaction rate be achieved. Although in the determination based on the “reverse reaction” activities were measured which were 3 to 4 times as high as those measured in the forward reaction, the considerably higher cost of the reagents as well as the greater susceptibility to disturbances are an impediment to a general clinical application of this test set-up.

Participation of adenine nucleotides in the proposed direct pathway of biosynthesis of N-phosphorylcreatine.

EVIDENCE obtained in this laboratory1,2 suggested that the phosphorylation of creatine by a phosphoryl donor, catalysed by a soluble enzyme system from rat muscle, might not require adenosine diphosphate (ADP). Morrison and Doherty3 reach the opposite conclusion. Results obtained independently in this laboratory, by procedures already described1,2, lead us to agree with their conclusion.

The role of DPN in the enzymic phosphorylation of creatine

The role of DPN in the enzymic phosphorylation of creatine

Enzymic phosphorylation of creatine by 1:3-diphosphoglyceric acid.

Enzymic phosphorylation of creatine by 1:3-diphosphoglyceric acid.