Creatine phosphate reactions

In muscle, arginine is also involved as a precursor of creatine and creatine phosphate (Reaction 4 below). 

Clinical Analysis. A wide range of clinically important substances can be detected and quantitated using chemiluminescence or bioluminescence methods. Coupled enzyme assay protocols permit the measurement of kinase, dehydrogenase, and oxidases or the substrates of these enzymes as exemplified by reactions of glucose, creatine phosphate, and bile acid in the following ... 

Figure 31-3. Arginine, ornithine, and proline metabolism. Reactions with solid arrows all occur in mammalian tissues. Putrescine and spermine synthesis occurs in both mammals and bacteria. Arginine phosphate of invertebrate muscle functions as a phosphagen analogous to creatine phosphate of mammalian muscle (see Figure 31-6).

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

Energy may be transferred from creatine phosphate to ADP by way of the following reaction ... 

The same reaction was recently proposed to detect creatine kinase (CK), an enzyme of high clinical significance in relation to the investigation of skeletal muscle disease and the diagnosis of myocardial infarct or cerebrovascular accidents. As ATP is a reaction product obtained from the reaction of ADP with creatine phosphate catalyzed by CK, this enzyme can be indirectly measured by the CL intensity read from the subsequent reaction of ATP with luciferin. Using the technique of electrophoretically mediated microanalysis (EMMA), it is possible to detect the enzyme using nanoliter volumes of biological sample with an improved speed and simplicity with respect to a conventional colorimetric method [100],... 

Under standard conditions, this reaction would be unfavourable but physiological conditions during recovery phase after exercise are such as to allow creatine phosphate formation to occur. 

Muscle-specific auxiliary reactions for ATP synthesis exist in order to provide additional ATP in case of emergency. Creatine phosphate (see B) acts as a buffer for the ATP level. Another ATP-supplying reaction is catalyzed by adenylate kinase [1] (see also p.72). This disproportionates two molecules of ADP into ATP and AMP. The AMP is deaminated into IMP in a subsequent reaction [2] in order to shift the balance of the reversible reaction [1 ] in the direction of ATP formation. 

In resting muscle, creatine phosphate forms due to the high level of ATP. If there is a risk of a severe drop in the ATP level during contraction, the level can be maintained for a short time by synthesis of ATP from creatine phosphate and ADP. In a nonenzymatic reaction [6], small amounts of creatine and creatine phosphate cyclize constantly to form creatinine, which can no longer be phosphorylated and is therefore excreted with the urine (see p. 324). 

This muscle phosphotransferase (EC 2.132) catalyzes the reversible rephosphorylation of ADP to form ATP (/.c., T eq = [ATP][Creatine]/([Creatine phosphate] [ADP]) = 30). In resting muscle, creatine phosphate is synthesized at the expense of abundant stores of ATP intracellular creatine phosphate stores often reach 50-60 mM. If ATP is suddenly depleted by muscle contraction, its product ADP is immediately converted back into ATP by the reverse of the creatine kinase reaction. Depending on the pH at which the enzyme is studied, the kinetic reaction can be either rapid equilibrium random or rapid equilibrium ordered. A-Ethylglycocyamine can also act as a substrate. 

Occasionally, one can maintain initial rate conditions by using a coupled reaction system to regenerate one of the limiting substrates. For example, to regenerate ATP in a phosphotransferase reaction, one can use creatine phosphate and creatine kinase acetylphosphate and acetate kinase or phosphoenolpyruvate and pyruvate kinase. 

Phosphocreatine (Fig. 13-5), also called creatine phosphate, serves as a ready source of phosphoryl groups for the quick synthesis of ATP from ADP. The phosphocreatine (PCr) concentration in skeletal muscle is approximately 30 nra, nearly ten times the concentration of ATP, and in other tissues such as smooth muscle, brain, and kidney [PCr] is 5 to 10 mM. The enzyme creatine kinase catalyzes the reversible reaction... 

Lawson, J.W.R. Veech, R.L. (1979). Effects of pH and free Mg2 on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions. J. Biol. Chem. 254, 6528-6537. 

In the standard state, the equilibrium for this reaction lies far to the left in other words, the reaction is unfavored. However, in the standard state, all the reactants and products are at one molar concentration. In other words, the ratio of ATP to ADP concentrations would be 1. In an actively metabolizing state, the ratio of ATP to ADP is as much as 50 or 100 to 1—this means that the formation of Cr P will occur to a reasonable level. Creatine phosphate forms a reservoir for high-energy phosphate in the same way that water can be pumped upstream to a reservoir and released for use later on. 

An example of a random type of reaction is creatine + ATP creatine phosphate + ADP, which is catalyzed by creatine kinase (see Chapter 20). In this case, creatine and ATP are bound to the active site in either sequence, and after the transfer of the phosphate group of the bound ATP to the bound creatine both products are released in either sequence. 

Nevertheless, there is enough creatine phosphate to sustain only a few seconds of intensive work. Another system available for ATP generation is the myokinase reaction ... 

S-30 fraction from wheat germ—commercial (Fisher catalog L-4440) or freshly prepared (see Appendix) supplemented with 125 mM ATP (potassium salt), 2.5 mM GTP (potassium salt), 100 mM creatine phosphate, and 0.5 mg/ml creatine Reaction buffer (300 mM HEPES, 30 mM dithiothreitol [pH 7.1])... 

In addition to the enzyme the reaction mixture contained Hepes buffer, IMP, GTP, MgCl, and creatine phosphate and phosphocreatine kinase (a regeneration system for GTP). The reaction was initiated by adding aspartic acid. Samples were removed at intervals, and the reactions were terminated by direct injection onto the HPLC column. Figure 9.113 shows chromatograms of samples removed at 0,5, and 10 minutes of incubation. The disappearance of IMP and GTP and the appearance of GTP and sAMP can be noted. 

Both creatine and creatine phosphate undergo a nonenzymic reaction to yield creatinine, which is metaboUcaUy useless and is excreted in the urine. Because the formation of creatinine is a nonenzymic reaction, the rate at which it is formed, and the amount excreted each day, depends mainly on muscle mass, and is therefore relatively constant from day to day in any one individual. This is commonly exploited in clinical chemistry urinary metabolites are commonly expressedper mole of creatinine, and the excretion of creatinine is measured to assess the completeness of a 24-hour urine collection. There is normally Utffe or no excretion of creatine in urine significant amounts are only excreted when there is breakdown of muscle tissue. 

Colorimetric assay (H7) is by incubation of only 0.1 ml of serum at 37° C and pH 7.4 with Tris/HCl-buffered ADP and creatine phosphate in the presence of Mg+ + ions and cysteine to maintain enzyme action after 30 minutes the reaction is stopped by addition of p-chloromercuriben-zoate, the mixture deproteinized by Ba(OH)2/ZnS04, and the creatine content of the supernatant determined colorimetrically by the a-naph-thol/diacetyl method using a 60-minute development at 37°C for creatine chromogen. Controls are prepared simultaneously by the same procedure, without incubation, by replacing ADP with water and adding p-chloromercuribenzoate first, and standards in the same way by using a pure creatine solution instead of water. 

A-30. Glycine is involved in the synthesis of haemoglobin, creatine phosphate, purines, glutathione, proteins, phospholipids, bile acids and detoxification reactions. 

Creatinine results from the irreversible, nonenzymatic dehydration and loss of phosphate from phosphocreatine (Fig. 1). Creatinine is used as an indicator of skeletal muscle mass because it is a by-product of the creatine kinase reaction and it is one of the most widely used clinical markers to assess renal function. Urine levels of creatinine are good indicators of the glomerular filtration rate of the kidneys (i.e., the amount of fluid filtered per unit time). 

ATP phosphorylates creatine to form creatine phosphate in a reaction catalyzed by creatine kinase (also known as creatine phosphokinase). 

The amount of ATP in muscle suffices to sustain contractile activity for less than a second. Creatine phosphate in vertebrate muscle serves as a reservoir of high-potential phosphoryl groups that can be readily transferred to ATP. Indeed, we use creatine phosphate to regenerate ATP from ADP every time that we exercise strenuously. This reaction is catalyzed by creatine kinase. 

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