Active-site labelling of triose phosphate isomerase. The reaction of bromohydroxyacetone phosphate with a unique glutamic acid residue and the migration of the label to tyrosine

Triose phosphate isomerase from chicken muscle reacts stoicheiometrically with the active-site-directed irreversible inhibitor bromohydroxyacetone phosphate with concomitant loss of all catalytic activity. The primary site of attachment has been shown to be a unique glutamic acid residue in the sequence Ala-Tyr-Glu-Pro-Val-Trp. Unless the inhibitor-enzyme bond is stabilized by reduction of the C-2 carbonyl group with borohydride, the phosphate group is lost and the label migrates to the adjacent tyrosine residue. It is suggested that the gamma-carboxylate group of the glutamic acid residue may be the base responsible for primary proton abstraction from substrate in the catalysis. The failure of this reagent specifically to inactivate either muscle or yeast aldolase, and the use of the reagent in preparing isomerase-free glycolytic enzymes, is discussed.     

Phosphorylation of rabbit skeletal muscle myosin in situ

Myosin light chain (P light chain) is phosphorylated by Ca2+ X calmodulin-dependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow-twitch soleus and fast-twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast-twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin-activated ATPase activity, may be related to isometric twitch potentiation.     

Exchange of the actin-bound nucleotide in intact arterial smooth muscle

The actin-bound ADP was separated from cytoplasmic nucleotides by treatment of intact arterial smooth muscle with 50% ethanol. In (32)P-labeled smooth muscle the actin-bound ADP and phosphate readily exchanged with the cytoplasmic [gamma,beta-(32)P]ATP; the specific radioactivity of actin-bound ADP was equal to that of the beta-phosphate of cytoplasmic ATP and the specific radioactivity of actin-bound phosphate was equal to that of the gamma-phosphate of cytoplasmic ATP. In contrast, the exchange of the actin-bound ADP in skeletal muscle was very slow. The presence of cytoplasmic ATP was required for the exchange of the actin-bound ADP and phosphate; if ATP synthesis was inhibited the exchange was also inhibited. The extent of exchange was reduced in muscles contracted by histamine or K(+), as compared with resting muscles. The exchange was also shown in other mammalian smooth muscles, uterus, urinary bladder, and stomach. The data indicate a dynamic state of actin in smooth muscle. The data also suggest that polymerization-depolymerization of actin is part of the contraction-relaxation cycle of smooth muscle.     

Autophosphorylation of glyceraldehydephosphate dehydrogenase and phosphorylation of protein from skeletal muscle microsomes

Glyceraldehydephosphate dehydrogenase purified from rabbit skeletal muscle is auto-phosphorylated with MgATP. Half-maximal phosphorylation is achieved around 0.3 mM. The phosphorylation is Ca2+ independent. The phosphoenzyme complex is labile in alkaline conditions and stable in moderately acid media. The complex is readily hydrolyzed by 0.1 M neutral hydroxylamine, indicating the complex formed is a high-energy acyl phosphate. The phosphorylation is reduced by nicotinamide adenine dinucleotides, reduced form (NADH), glyceraldehyde 3-phosphate, and nicotinamide adenine dinucleotide (NAD+). The enzyme is also dephosphorylated by these metabolites although to a lesser extent by NAD+. Calsequestrin isolated from rabbit skeletal muscle inhibits the phosphorylation of the enzyme. The phosphoenzyme behaves as a kinase catalyzing the phosphorylation of proteins of Mr 80 000 and 72 000 found in the skeletal muscle terminal cisternae/triad preparation. This reaction is enhanced by NADH. The phosphate found in the protein substrate has been shown to be the same phosphate initially involved in the phosphorylation of glyceraldehydephosphate dehydrogenase.     

Unlike effects of denervation on the rate of entry of inorganic phosphate into rat slow and fast muscles.

The increased inorganic phosphate flow, characteristic of denervated gastrocnemius muscle is shown to be present in additional denervated fast muscles, i.e. the plantaris, tibialis anterior and extensor digitorum longus muscles. The response of the soleus, a slow muscle, to denervation is biphasic. After an initial decrease of the phosphate flow at the end of the first postoperative day, there is a secondary rise which has the same general characteristics as the rise observed in fast muscles i.e. an exponential or hyperbolic increase to an asymptotic value reached after thirty days. The denervated fast and slow muscles are not converging to an intermediate metabolic pattern. The changes in phosphate flow induced by denervation are reversible in the soleus as well as in the gastrocnemius muscles.     

Thermodynamic studies of the activation of rabbit muscle lactate dehydrogenase by phosphate.

In an attempt to trace the source of phosphate activation of the enzyme-catalysed pyruvate-lactate interconversion by rabbit muscle lactate dehydrogenase, equilibrium constants were measured to examine the effects of phosphate on interactions pertinent to the enzymic process. Frontal gel-chromatographic studies of the binding of NADH to the enzyme established that the intrinsic association constant is doubled in the presence of 50 mM-phosphate in the buffer (pH 7.4, I0.15). From kinetic studies of the competition between NAD+ and NADH for the coenzyme-binding sites of the enzyme it is concluded that the binding of oxidized nicotinamide nucleotide is also doubled in the presence of 50 mM-phosphate. Competitive-inhibition studies and fluorescence-quenching measurements indicated the lack of a phosphate effect on ternary-complex formation between enzyme-NADH complex and oxamate, a substrate analogue of pyruvate. The equilibrium constant for the interaction between enzyme-NAD+ complex and oxalate, an analogue of lactate, was also shown, by difference spectroscopy, to be insensitive to phosphate concentration. Provided that the effects observed with the substrate analogues mimic those operative in the kinetic situation, the equilibrium constant governing the isomerization of ternary complex is also independent of phosphate concentration. It is concluded that enhanced coenzyme binding is the source of phosphate activation of the rabbit muscle lactate dehydrogenase system.     

Specificity and kinetics of triose phosphate isomerase from chicken muscle

The isolation of crystalline triose phosphate isomerase from chicken breast muscle is described. The values of k(cat.) and K(m) for the reaction in each direction were determined from experiments over wide substrate-concentration ranges, and the reactions were shown to obey simple Michaelis-Menten kinetics. With d-glyceraldehyde 3-phosphate as substrate, k(cat.) is 2.56x10(5)min(-1) and K(m) is 0.47mm; with dihydroxyacetone phosphate as substrate, k(cat.) is 2.59x10(4)min(-1) and K(m) is 0.97mm. The enzyme-catalysed exchange of the methyl hydrogen atoms of the ;virtual substrate' monohydroxyacetone phosphate with solvent (2)H(2)O or (3)H(2)O was shown. This exchange is about 10(4)-fold slower than the corresponding exchange of the C-3 hydrogen of dihydroxyacetone phosphate. The other deoxy substrate, 3-hydroxypropionaldehyde phosphate, was synthesized, but is too unstable in aqueous solution for analogous proton-exchange reactions to be studied.     

Presence of covalently bound energy-rich phosphates in human tracheal smooth muscle myosin

In a preliminary report, the tracheal NaCl-myosin prepared from an old and a young subject was discussed. In the present paper, the total bound phosphate (P) content and its distribution is described in two parallel preparations of human muscle myosin. It was shown that a considerable amount of covalently bound P was present not only in NaCl, but in the fresh preparations of tracheal KCl-myosin. Analysing this phosphate fraction in the alkaline hydrolysate of RNA- and lipid-free preparations of myosin it was confirmed that phosphate was linked to the basic amino acid residues and their hydrolytic derivatives. As the phosphoryl binding sites are partly saturated, the phosphate concentration can be enhanced nearly three-fold compared to the fresh preparation. Phosphate incorporation is an autophosphorylation process depending on the ATP and Mg2+ concentration. Studying the actomyosin fraction in the presence of ATP it was found that its phosphate content can also be increased to a certain degree. It is supposed that the changes in phosphate content of myosin are associated with the formation of crossbridges between the actin and myosin filaments in the processes of muscle contraction and relaxation. The process can be influenced directly and indirectly by some natural factors and drugs resulting in the concentration or relaxation of bronchial muscles.     

Effect of creatine phosphate on the slow inward calcium current,action potential,and the contractile force of frog atrium and ventricle

The effects of creatine phosphate on frog heart muscle contraction have been studied further. It has been shown that after inhibition of mitochondrial oxidative phosphorylation by sodium cyanide the frog heart ventricle contraction can be maintained at a high level by addition of creatine phosphate. The effect of creatine phosphate on the contractile force and action potential is similar for frog heart ventricle and atrium. It has been directly demonstrated by using the voltage-clamp technique that creatine phosphate controls the slow inward calcium current through the surface membrane of frog atrium cells.     

Heterogeneity of metabolic response to muscular exercise in humans. New criteria of invariance defined by in vivo phosphorus-31 NMR spectroscopy

31P NMR spectroscopy at 4.7 T has been used in vivo to follow metabolic changes associated with exercise and subsequent recovery in the forearm flexor digitorum superficialis muscle of 14 healthy volunteers. The muscle content in phosphomonoesters at rest provides an index of glycogenolytic activity. Quantitative linear correlations have been shown to link end-of-exercise acidosis to recovery kinetics of phosphocreatine and phosphocreatine/organic phosphate ratio. These linear relationships constitute new metabolic invariants to be used in the study of myopathies and muscle adaptation to exercise.     

The formation of intravesicular calcium phosphate deposits in microsomes of smooth muscle

Summary The calcium uptake in the microsomial fraction isolated from the smooth muscle of the antrum of the pig stomach is stimulated by phosphate. The microsomial vesicles which are loaded with calcium phosphate can be purified by differential centrifugation. A purification of 36 times in terms of calcium content was reached. Electron microscopy of the freshly prepared material revealed calcium phosphate deposits in the form of needles of crystalline calcium phosphate. This structure differs from that of the deposits which appear in the fragmented sarcoplasmic reticulum of skeletal muscle. Their morphology is that of non-crystalline calcium phosphate. However, on standing these deposits convert slowly into crystalline calcium phosphate. This difference reflects different kinetics of crystallization of the precipitates in the two preparations. After negative staining of the calcium phosphate loaded microsomes of skeletal and of smooth muscle, only few deposits are preserved because a release of calcium occurs as a consequence of the action of the stain and also of the dilution and warming up of the suspension. Smooth muscle microsomes partially purified by loading with calcium phosphate were studied by freeze etching and rotary replication. Membrane fragments displaying subunit intramembrane particles similar to those observed in sarcoplasmic reticulum of skeletal muscle could be identified. However, in the smooth muscle microsomes the intramembrane particles were much less densely packed. Part of these particles could correspond to calcium transport sites.     

Glycogen phosphorylase of skeletal muscles

A review is given on the affinity modification of pyridoxal phosphate and AMP-binding sites as well as on the chemical modification of essential amino acid residues of phosphorylase (histidine residue of the substrate-binding site and cysteine residue of the coenzyme-binding site). The role of allosteric effectors (AMP and glucose-6-phosphate) and functionally important centers of the protein in conformational transitions of rabbit muscle phosphorylase b is discussed. The kinetic properties of rabbit and bovine muscle phosphorylase are compared. Bovine muscle phosphorylase is shown to be a partly phosphorylated form of the enzyme. Some peculiarities of the pH-dependence of kinetic behaviour of the hybrid form of the bovine muscle enzyme are discussed.     

Towards a valid preparation for the in vitro study of energy metabolism in fish muscles

Slices of lateral red muscle and isolated intact Musculus protractor hyoidei of goldfish were examined for their suitability as model systems for the in vitro study of muscular energy metabolism. Slicing of red muscle causes a strong breakdown of ATP and creatine phosphate and a lowering of the adenylate energy charge. Furthermore, slices do not recover when incubated in an oxygenated balanced salt solution, but show a continuous depletion of direct energy reserves. In contrast with red muscle slices, the Musculus protractor hyoidei can be isolated in an intact state, as shown by constancy of creatine phosphate and adenylate levels and stability of the adenylate energy charge during incubation. Therefore, isolated M. protractor hyoidei seems to be promising as a model system for the in vitro study of muscular energy metabolism.     

The Creatine Phosphoryltransfer Reaction in Iodoacetate-Poisoned Muscle

The iodoacetate-nitrogen-poisoned muscle offers the possibility of studying the stoichiometry of the single muscle twitch since metabolic resynthesis by glycolysis and oxidative phosphorylation are blocked, and there remains as an energy source only the creatine phosphoryltransfer system, creatine phosphate reacting with adenosinediphosphate to give the triphosphate and creatine. It is shown, preparatory to a determination of the amount of phosphocreatine split in a single twitch, that iodoacetate does not inhibit creatine phosphoryltransferase at concentrations which block glycolysis. An analysis is developed which assumes that the transferase maintains the creatine phosphoryl transfer reaction in equilibrium following contraction, and further that the creatine phosporyltransfer reaction and the myokinase reaction are isolated in muscle. On the basis of this analysis and the data obtained, an estimate of the equilibrium constant of the creatine phosphoryl reaction in muscle is obtained which agrees with values determined in vitro. Using the estimated equilibrium constant, and the concentrations of creatine, creatine phosphate, and adenosinetriphosphate found, a value for the concentration of free adenosinediphosphate is obtained which is considerably less than that found by direct chemical analysis.     

The creatine phosphate energy shuttle--the molecular asymmetry of a "pool"

The creatine phosphate shuttle energy transfer mechanism was postulated on the basis of the hexokinase acceptor theory of insulin action. It proposes that the movement of chemical energy from the mitochondrion to the myofibril is in the form of creatine phosphate. This occurs because there are isozymes of creatine phosphokinase bound to the inner membrane of the sarcosome and to the A band of the myofibril. These isozymes have been shown to act as transducers of energy from ATP to creatine phosphate at the translocase site and from creatine phosphate back to ATP at the myofibrillar compartment. Calculations show that there is no significant amount of transformation of creatine phosphate to ATP in the intervening space between the mitochondrion and the myofibril so that, essentially, transport between the oxidative sites and the contractile apparatus is through the creatine phosphate shuttle. There is also evidence that another terminus for this shuttle is the microsome so that muscle activity tends to increase energy supply for protein synthesis.     

Effects of denervation on the rate of entry of inorganic phosphate into rat slow and fast muscles: selective inhibition of denervation changes by actinomycin D.

Actinomycin D abolishes the post denervation increase in inorganic phosphate flow observed in the fast gastrocnemius muscle. In the slow soleus muscle, the initial decrease in phosphate flow is unaffected but the secondary rise is suppressed in the same manner as in the fast muscle. These observations put the post denervation increase in inorganic phosphate flow on a par with the development of extrajunctional cholinergic receptors in being the result of the synthesis of new proteins. It has the added advantage of being suitable to quantitative assessment at the whole muscle level.     

The distribution of inorganic phosphate in amphibian muscle

The Na⁺, K⁺, and inorganic phosphate levels of the plasma and sartorius muscle of the toad Bufo marinus were determined. Soaking in normal Ringer brought about the usual cation shifts, but did not alter the level of inorganic phosphate in the cell. Increases in the external phosphate level brought about an increase in the internal phosphate, but the apparent phosphate space of muscle is somewhat smaller than the apparent Cl^- space. Phosphate spaces were compared with inulin spaces and were found to be significantly greater. Alteration of the H⁺ concentration of the high phosphate Ringer did not alter the partition of phosphate across the cell membrane. These results have been found to be consistent with the theory of a three compartment system for muscle, wherein the tissue is assumed to consist of an extracellular phase, and two intracellular phases. The inorganic phosphate of the cell is assumed to be adsorbed onto the "ordered phase," and increments in organic phosphate found on raising the external level are assumed to take place in the "free intracellular phase."     

Postmortem metabolism of short-finned squid muscle (Illex illecebrosus)

Postmortem biochemical changes were examined in the mantle muscle of the short-finned squid (Illex illecebrosus) in relation to the physical events associated with rigor. Unlike mammalian muscle, the major muscle phosphagen is arginine phosphate rather than creatine phosphate. Arginine phosphate levels did not change dramatically during the progress of rigor development. ATP depletion was found to be closely related to glycogen depletion as is often observed in mammalian muscle. The postmortem accumulation of octopine was related to the initial muscle glycogen content at death but a significant lag in its production was observed. The postmortem conversion of glucose to glucose-6-phosphate appeared to be the rate-limiting step in the overall conversion of glycogen to octopine. The intermediates found in the postmortem catabolism of squid muscle ATP were ADP, AMP, IMP Ino and Hx. Unlike most vertebrate fishes, AMP was found to accumulate in squid before conversion to IMP whereas accumulations of IMP and Ino were less than those normally found in vertebrate muscle.     

Mouse lacking NAD+-linked glycerol phosphate dehydrogenase has normal pancreatic beta cell function but abnormal metabolite pattern in skeletal muscle

We surveyed the BALB/cHeA mouse, which lacks cytosolic glycerol phosphate dehydrogenase an enzyme that catalyzes a reaction in the glycerol phosphate shuttle. The other enzyme of this shuttle, mitochondrial glycerol phosphate dehydrogenase, is abundant in skeletal muscle and pancreatic islets suggesting that the shuttle's activity is high in these tissues. Levels of glycerol phosphate (low) and dihydroxyacetone phosphate (high) were very abnormal in nonislet tissue, especially in skeletal muscle. Intermediates situated before the triose phosphates in the glycolysis pathway were increased and those after the triose phosphates were generally low, depending on the tissue. The lactate/pyruvate ratio in muscle was low signifying a low cytosolic NAD/NADH ratio. This suggests that a nonfunctional glycerol phosphate shuttle caused a block in glycolysis at the step catalyzed by glyceraldehyde phosphate dehydrogenase. When exercised, mice were unable to maintain normal ATP levels in skeletal muscle. Blood glucose, serum insulin levels, and pancreatic islet mass were normal. In isolated pancreatic islets insulin release, glucose metabolism and ATP levels were normal, but lactate levels and lactate/pyruvate ratios with a glucose load were slightly abnormal. The BALB/cHeA mouse can maintain NAD/ NADH ratios sufficient to function normally under most conditions, but the redox state is not normal. Glycerol phosphate is apparently formed at a slow rate. Skeletal muscle is severely affected probably because it is dependent on the glycerol phosphate shuttle more than other tissues. It most likely utilizes glycerol phosphate rapidly and, due to the absence of glycerol kinase in muscle, is unable to rapidly form glycerol phosphate from glycerol. Glycerol kinase is also absent in the pancreatic insulin cell, but this cell's function is essentially normal probably because of redundancy of NAD(H) shuttles.     

The effect of sugar phosphates, phosphoenolpyruvate and adenylic acid on muscle, brain and heart creatine kinases]

The effect of adenylic acid, glucose-6-phosphate, fructose-1,6-diphosphate and phosphoenolpyruvate on creatine kinase isoenzymes (brain extract, muscle and heart extracts and purified muscle enzyme) was studied. These effectors, especially phosphoenolpyruvate, are shown to inhibit in different degree the reaction of ATP formation catalysed by creatine kinase from all tissues. The effectors do not inhibit the creatine phosphate synthesis in extracts, but depress purified creatine kinase. The interrelationship of the creatine kinase system and the key glycolytic enzymes (phosphofructokinase, hexokinase, pyruvate kinase) is discussed.