Creatine 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])... 

Description of Method. Creatine is an organic acid found in muscle tissue that supplies energy for muscle contractions. One of its metabolic products is creatinine, which is excreted in urine. Because the concentration of creatinine in urine and serum is an important indication of renal function, rapid methods for its analysis are clinically important. In this method the rate of reaction between creatinine and picrate in an alkaline medium is used to determine the concentration of creatinine in urine. Under the conditions of the analysis, the reaction is first-order in picrate, creatinine, and hydroxide. 

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 ... 

Enzymes, measured in clinical laboratories, for which kits are available include y-glutamyl transferase (GGT), alanine transferase [9000-86-6] (ALT), aldolase, a-amylase [9000-90-2] aspartate aminotransferase [9000-97-9], creatine kinase and its isoenzymes, galactose-l-phosphate uridyl transferase, Hpase, malate dehydrogenase [9001 -64-3], 5 -nucleotidase, phosphohexose isomerase, and pymvate kinase [9001-59-6]. One example is the measurement of aspartate aminotransferase, where the reaction is followed by monitoring the loss of NADH ... 

Detoxifica.tlon. Detoxification systems in the human body often involve reactions that utilize sulfur-containing compounds. For example, reactions in which sulfate esters of potentially toxic compounds are formed, rendering these less toxic or nontoxic, are common as are acetylation reactions involving acetyl—SCoA (45). Another important compound is. Vadenosylmethionine [29908-03-0] (SAM), the active form of methionine. SAM acts as a methylating agent, eg, in detoxification reactions such as the methylation of pyridine derivatives, and in the formation of choline (qv), creatine [60-27-5] carnitine [461-06-3] and epinephrine [329-65-7] (50). 

This reaction is strongly exergonic and AG° at 37°C is —42.8 kj/mol. Physiological concentrations of phosphocreatine, creatine, and inorganic phosphate are normally between 1 mMand 10 mM. Assuming 1 mMconcentrations and using Equation (3.12), the AG for the hydrolysis of phosphocreatine is... 

The overall direction of the reaction will be determined by the relative concentrations of ATP, ADP, Cr, and CrP and the equilibrium constant for the reaction. The enzyme can be considered to have two sites for substrate (or product) binding an adenine nucleotide site, where ATP or ADP binds, and a creatine site, where Cr or CrP is bound. In such a mechanism, ATP and ADP compete for binding at their unique site, while Cr and CrP compete at the specific Cr-, CrP-binding site. Note that no modified enzyme form (E ), such as an E-PO4 intermediate, appears here. The reaction is characterized by rapid and reversible binary ES complex formation, followed by addition of the remaining substrate, and the rate-determining reaction taking place within the ternary complex. 

ATP stores in muscle are augmented or supplemented by stores of phosphocreatine. During periods of contraction, phosphocre-atine is hydrolyzed to drive the synthesis of needed ATP in the creatine kinase reaction ... 

The middle panel shows the fall in PCr concentration in the muscle and the simultaneous increase in Pj released daring ATP degradation and resynthesis via the creatine kinase reaction ... 

The lower panel shows the decreasing concentration of ATP, to about 60% of resting levels, and the simultaneous equimolar increase in IMP. The fall in ATP started when most of the PCr store was utilized, resulting in a decreased rate of ADP phosphorylation via the creatine kinase reaction. The resultant accumulation of ADP stimulates adenylate kinase activity and subsequently IMP is formed via the AMP deaminase reaction ... 

In our laboratory, we have focused our attention on the syntheses of various analogs of creatine, 17, a substrate for the enzyme creatine kinase from rabbit muscle (2). The reaction catalyzed by this enzyme is... 

Some Kinetic Parameters for Analogs of Creatine in the Creatine Kinase Reaction (71)... 

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 ... 

Ca2+aq reacts very rapidly with adp3- and with atp4-(236,710). Rate constants for onward reaction of [M(adp)] and [M(atp)]2, M — Ca2+, Mg24, or Mn2+, with creatine phosphotransferase are similar - 1. 7 x 106, 5.3 x 106, 7.4 x l(r M 4s 1 respectively (at 284 K) for the adp complexes, and indeed are similar to the value of 23 x 106M-1s-1 for the reaction of adp itself with creatine phosphotransferase (711). Kinetic parameters and binding constants have been established for interaction of the antiparallel form of the G-quadruplex d(T4G4T4) with Ca2+aq and for the subsequent Ca2+-promoted antiparallel parallel equilibrium (cf. Section IV.C above) (607). 

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],... 

Many assays have been described in which the initial product forms the substrate of an intermediary reaction involving auxiliary enzymes. The assay of creatine kinase (EC 2.13.2), for example, involves hexokinase (EC 2.7.1.1) as the auxiliary enzyme and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as the indicator enzyme ... 

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. 

One of the earlier recorded observations indicating that people s taste reactions for a particular chemical substance may not by any means be uniform was made with respect to creatine, which was found to be quite tasteless to some individuals but bitter and biting to others.34 About the same time it was found that individuals vary in their ability to taste phenylthiocarbamide (PTC) and related compounds.35 To most it is either violently bitter or completely tasteless. A small minority however, assign to it various other tastes.36,37,38... 

One might suppose on the basis of the amount of attention PTC has received that it is quite a unique substance in its ability to elicit different responses from different individuals. Actually this is not the case at all since wide interindividual differences in taste threshold and taste reactions can be observed with almost anything that can be tasted. Hundred-fold variations in taste thresholds are very common (even when small groups are studied) with respect to substances like sodium or potassium chlorides or hydrochloric acid.41 Saccharine, quinine, cascara, and mannose are among the substances, in addition to creatine mentioned above, for which individuals are known to show highly diverse taste reactions.42 Richter found some children who could not taste 20 per cent sugar solutions.43... 

The first observed product is the hydroxo-N-bound phosphoramidate complex, although there are almost certainly other intermediates. Both ester hydrolysis (to nitrophenolate ion) and transfer of a phosphate residue from O to N occur. An acceleration of at least 10 fold can be assessed for both processes, compared with the reaction of the uncoordinated ester with NH3 or 0H ion. The O to N transfer is a general biochemical occurrence, e.g. creatine kinase uses and creatine to transform ATP to ADP and form creatine... 

In addition to the processes described above, there still remains one further process which, at least in some cells or tissues, is required prior to the utilisation of ATP in the cytosol that is, the transport of energy within the cytosol, via a shuttle. The transport of ATP out and ADP into the mitochondrion, via the translocase, results in a high ATP/ ADP concentration ratio in the cytosol. However, a high ratio means that the actual concentration of ADP in the cytosol is low, which could result in slow diffusion of ADP from a site of ATP utilisation back to the inner mitochondrial membrane. If sufficiently slow, it could limit the rate of ATP generation. To overcome this, a process exists that transports energy within the cytosol, not by diffusion of ATP and ADP, but by the diffusion of phosphocreatine and creatine, a process known as the phosphocreatine/creatine shuttle. The reactions involved in the shuttle in muscle help to explain the significance of the process. They are ... 

The coenzyme tetrahydrofolate (THF) is the main agent by which Ci fragments are transferred in the metabolism. THF can bind this type of group in various oxidation states and pass it on (see p. 108). In addition, there is activated methyl, in the form of S-adenosyl methionine (SAM). SAM is involved in many methylation reactions—e. g., in creatine synthesis (see p. 336), the conversion of norepinephrine into epinephrine (see p. 352), the inactivation of norepinephrine by methylation of a phenolic OH group (see p. 316), and in the formation of the active form of the cytostatic drug 6-mercaptopurine (see p. 402). 

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). 

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