Changes in phosphofructokinase activity upon exposure of muscles and muscle extracts to injurious agents

A study has been made on changes of outflux, extractability and activity of phosphofructokinase (PPK) under the action of heating, and of urea on the frog's skeletal muscles and on their extracts. Under the action of heating on muscles, the decrease of PPK activity (to 35%) is first revealed 34--36 degress C, when decrease of excitability and the contracture are not yet detected, and the extractability of the total water-soluble protein does not change. At the start of contracture, and at the decrease of excitability (at 38 degrees C) PPK in the muscle loses its activity. The thermolability of PPK is the greatest one compared to all the enzymes investigated before. The data on the high thermolability of PPK are discussed in terms of the regulating role of PPK in glycolysis. The PPK activity of extracts of muscles altered by urea, during the action not accompanied by the death of muscles, does not change. At the irreversible disappearance of muscle excitability, PPK is inactivated. PPK in the cell is more stable to injuring agents than PPK in isolated state.     

Changes in lactate dehydrogenase and glucose-6-phosphate dehydrogenase activity in the course of muscle alteration]

Changes of outflux, extractability and activity of lactate dehydrogenase (LDG) and glucose-6-phosphate dehydrogenase (G-6PhDG) of muscles under the action of heating (at 32--44 degrees for 15 min) and urea (1 M during 10 min, 30 min, 2 hr. and 9 hr.) on the skeletal muscles of R. temporaria L. were studied. Under the thermal action not accompanied by contracture and fall of the excitability (32--36 degrees), the increase of outflux of LDG out of muscles into surrounding solutions is observed. G-6-PhDG in the external medium under any heating action was not revealed. Extractibility of LDG and G-6-PhDG did not change. Under the thermal action accompanied by the fall of excitability and by the contracture, along with the prolong increase of outflux of LDG, a decrease of extractability of LDG takes place. The decrease of G-6-PhDG is set at 42 degrees. Under the alteration of muscles by urea in the period of the temporary fall of excitability and contracture (10 and 30 min) an increase of the outflux of LDG out of muscles is observed. G-6PhDG in the surrounding medium was not revealed up to 9 hr. of incubation of muscle. In the period of the recovery of the excitability and relaxation of muscles (2hr.) the outflux of LDG approaches the control level. During the temporary loss and recovery of excitability, the extractability of LDG and G-6-PhDG does not change. In the period of irreversible contracture and loss of the excitability (6--10 hr.) a sharp increase of outflux of LDG out of muscles takes place. The extractability of the examined enzymes, especially of LDG, decreases.     

Nature of the changes in the activity of water-soluble enzymes in exposure of muscles to harmful agents. IV. The study of hexokinase extractability from intact and altered muscles

A possibility of hexokinase binding with actomyosin in skeletal muscles of Rana temporaria L., and the effect of thermal alteration (15 min at 36, 37, 38, 40 and 42 degrees C) on the binding were studied. Solutions of KCl (0.075 M and 0.15 M) extract more hexokinase from intact and altered muscles than does an non-electrolyte medium. Hexokinase freely dissolved in hyaloplasm is extracted in non-electrolyte medium. Hexokinase bound with structural components of the muscle cell is extracted upon the increase in ionic force of the extractant. The solubilizing effect of electrolytes on hexokinase is higher in alterated muscles than in the intact muscles indicating the increase in hexokinase binding under thermal alteration. Actomysin isolated from muscles reveals hexokinase activity. In reprecipitated actomyosin, the larger part of its hexokinase remains in actomyosin gel, the level of hexokinase activity not depending on the number of reprecipitation procedures or on the volume of washing solution. Hexokinase in actomyosin gel is less stable to the thermal action than in water supernatant of muscle extract. This may be due to the increase in hexokinase binding with actomiosin whose sorption activity increases under the thermal denaturation.     

Nature of the changes in the activity of water-soluble enzymes as affected by noxious agents acting on the muscles. I. A study of the extractability of phosphofructokinase from intact and altered muscles

Binding of phosphofructokinase (PPK) in intact and thermally altered (15 minutes at 38 degrees C) skeletal muscles of frogs (Rana temporaria) the the extractability of PPK from muscles was studied. PPK activity in actomyosin was also studied. Inhibiting effect of electrolytes (KCl, NaCl, CaCl2, MgCl2) on PPK in muscle extract does not allow to use them for the decision of the question of the interprotein interactions of PPK. 5 mM Na2-EDTA extracts additional PPK from homogenates of intact and altered muscles in comparison with PPK extracted in the media without Na2-EDTA (for intact muscles and altered muscles--509 and 729%). Under alteration of muscle, the binding of PPK increases. Among the structural components of muscle which bind PPK, proteins of actomyosin complex have been found.     

Denaturation of thymidylate synthetase from amethopterin-resistant Lactobacillus casei.

The effects of various concentrations of urea and guanidine hydrochloride on enzyme activity and on subunit association were determined. Incubation of thymidylate synthetase with buffered solutions of 3M to 3.5M guanidine hydrochloride or 5 M to 6 M urea resulted in the loss of about 90% of the enzyme activity. Under these denaturing conditions a red shift of the fluorescence emission maximum from 340 nm to 351 nm was observed together with a significant decrease in the relative fluorescence intensity of the protein. Studies at both 4 degrees C and 25 degrees C indicated that the enzyme was in the dimer form in 2 M guanidine hydrochloride but was dissociated into monomers in concentrations of this denaturant of 3 M and above. Although only monomeric species were evident at 4 degrees C in 6 M urea, at 25 25 degrees C this denaturant caused protein aggregation which increased with decreasing phosphate buffer concentration. Enzyme (5 mg/ml) in 0.5 M potassium phosphate buffer, pH 6.8, containing 4 M guanidine hydrochloride gave a minimum S20, w value of 1.22S at 25 degrees C. Sedimentation behavior of the native enzyme in the range of 5 to 20 mg/ml was only slightly concentration-dependent (4.28 S to 4.86 S) but extensive aggregation occurred above 20 mg/ml.     

The nature of changes in the activity of water-soluble enzymes during the action of injurious agents on the muscles. V. A study of the extractability of lactate dehydrogenase from intact and altered muscles

A study was made of lactate dehydrogenase (LDH) extractability from intact and thermally injured muscles of Rana temporaria L. in the 0.15 M KCl solution and in the non-electrolyte medium. A 15 minute incubation of intact muscles and those treated with heat at 38, 42, 44 and 46 degrees C in the 0.15 M KCl solution led to a much higher extraction of LDH than in the non-electrolyte medium. Following heating at 38 degrees C, causing irreversible injury of muscles, the LDH extractability in the non-electrolyte medium is seen to fall, whereas in the 0.15 M KCl solution it remained at the same level as after LDH extraction from intact muscles. The decrease in LDH extractability in non-electrolyte medium from thermally injured muscles may be due to the increase in LDH binding with thermolabile structural components of muscle. One of the components of muscle cell, known to bind LDH, is actomyosin, since it is isolated from muscles together with LDH, which cannot be removed by actomyosin reprecipitation or by increasing the volume of washing solution.     

Heat-induced aggregation of XRCC5 (Ku80) in nontolerant and thermotolerant cells.

XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5 degrees C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37 degrees C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.     

The possible existence of a heat-stable alkaline proteinase (HAP) in rat skeletal muscle

1. A unique caseinolytic activity was found in the crude extract from chicken and rat skeletal muscle. Hardly any activity was detected at physiological assay temperatures at pH 8.0 but did well at around 60 degrees C. 2. The activity partially purified from rat skeletal muscle showed optimum pH at around 8.0 at 60 degrees C. It hardly hydrolyzed casein below 50 degrees C, but in the presence of 5 M urea it showed relatively high activity at 30 degrees C. The activity was completely stable at 50 degrees C for 1 hr. 3. The activity seems to be contained in a high mol. wt (450,000) protein from the elution volume and is due to cysteine proteinase from the effect of inhibitors. 4. The above properties agreed with those of the heat-stable alkaline proteinase (HAP) of fish purified homogeneously by electrophoresis. This seems to suggest that HAP may also exist in rat skeletal muscle.     

Ribonuclease Rs from Rhizopus stolonifer: lowering of optimum temperature in the presence of urea

RNase Rs showed an approx. 2-fold increase in its activity when incubated in the presence of 2 M urea at 37 degrees C. The increase in its activity, in the presence of urea, was comparable to the activity at its optimum temperature, i.e. 45 degrees C. Compared to the native enzyme at 37 degrees C, the K(m) and V(max) of RNase Rs at 45 degrees C and in the presence of 2 M urea at 37 degrees C showed an increase while k(cat)/K(m) decreased. Arrhenius plots in the presence and absence of urea showed a decrease in the activation energy in the presence of urea. Though there was no change in the secondary structure of the protein in the presence of urea, minor changes were observed in the tertiary structure. Hence, the increase in the activity of RNase Rs, in the presence of 2 M urea at 37 degrees C, is due to the lowering of the activation energy as a result of changes in the microenvironment of the active site.     

Effect of temperature and pH on the ribulose-1,5-diphosphate carboxylase activity of the thermophilic hydrogen bacterium, Pseudomonas thermophila

The activity of ribulose 1,5-diphosphate (RDP) carboxylase was found in the soluble fraction of the cytoplasm from sonicated Pseudomonas thermophila K-2 cells. The enzyme is relatively thermolabile and completely loses its activity at 80 degrees C. The activity of RDP carboxylase at 60 degrees C increases by 40% during the first 10 min of heating in the presence of Mg2+ ions, bicarbonate and dithiothreitol, and again decreases if the enzyme is heated over 20 min. The optimum temperature of the enzyme is 50--55 degrees C. The specific activity of the enzyme in fresh preparations under these conditions reaches 0.22 unit per 1 mg of protein in the extract. The calculated value of the activation energy for RDP carboxylase is 6.4 kcal-mole-1, but 11.6 kcal-mole-1 in frozen preparations. The optimal pH is 7.0--7.3 depending on the buffer. The temperature optimum for the enzyme action does not depend on pH within the range of 7.3 to 8.8. Therefore, RDP carboxylase of Ps. thermophila K-2 differs from RDP carboxylases of mesophilic cultures studied earlier by a higher susceptibility to a decrease in temperature (the enzyme activity is negligible at 30 degrees C), by a lower value of the activation energy at suboptimal temperatures, and by a lower pH optimum of the enzyme action.     

Rapidly reversible enzyme inhibition in a temperature-sensitive mammalian cell mutant lacks thermotolerance

The temperature-sensitive (ts) Chinese hamster ovary (CHO) cell mutant tsH1 contains a thermolabile leucyl-tRNA synthetase. Upon incubation at the nonpermissive temperature of 39.5 degrees C, the enzyme became reversibly inhibited over a period of minutes, and the cells lost viability over a period of many hours. However, killing of tsH1 by acute heating at 45 degrees C was identical to that of wild-type (SC) cells. In addition, the heat-induced inhibition of protein synthesis was similar for both cell types, as measured after acute heating at 45 degrees C. Furthermore, both killing and inhibition of protein synthesis showed thermotolerance in both cell types. In contrast to the effects at 45 degrees C, at 39.5 degrees C, neither the inhibition of leucyl-tRNA synthetase activity nor the killing of tsH1 expressed thermotolerance. Also, treatment of tsH1 at 39.5 degrees C did not induce thermotolerance to killing at 45 degrees C. The inhibition of leucyl-tRNA synthetase activity in tsH1 at 39.5 degrees C was further distinguished from the 45 degrees C-induced inhibition of protein synthesis in SC cells by a much more rapid reversal of the inhibition of leucyl-tRNA synthetase activity. Also, the rate of reversal of the inhibition of protein synthesis by 45 degrees C in SC cells was decreased by increased heat dose. Such was not true for the 39.5 degrees C inhibition of leucyl-tRNA synthetase activity in tsH1. The data indicate that there exist two distinct types of thermal inhibition--one slowly reversible type which was observed during and after heating at 45 degrees C and both induced and expressed thermotolerance, and a second, rapidly reversible type, which was evident only during heating of tsH1 at 39.5 degrees C and neither induced nor expressed thermotolerance.     

The properties of the extracellular alkaline phosphatase of the causative agent of melioidosis]

After growing P. pseudomallei VPA on solid medium extracellular alkaline phosphatase with a molecular weight of 93,000 AMU was isolated, and practically purified from the extract of this medium by precipitation with ammonium sulfate, subsequent gel chromatography and concentration on membrane filters. The optimum conditions for enzymatic reaction were found to be pH 9.0 and a temperature of 50 degrees C. The enzyme was resistant to freezing and to heating at a temperature of up 60 degrees C for 30 minutes, as well as to the action of pH 3.0-10.5, but became completely inactivated after heating at 90 degrees C for 10 minutes and incubation at pH 2.0 for 20 hours.     

Effect of streptozotocin-induced diabetes on the turnover of hexokinase II in the rat

Skeletal muscle hexokinase II activity and turnover rates were measured in the normal and streptozotocin-induced diabetic rat. Enzyme activity decreases in the diabetic animal relative to the normal rat; however, the specific activity of hexokinase II is essentially the same for the two conditions. No alteration is observed in the relative rate of hexokinase II synthesis in the normal or diabetic rats, but there is a 3-fold increase in the rate of hexokinase II degradation in the latter group of animals. These results suggest that the primary cause of the well-established decrease in hexokinase II activity in skeletal muscle of the diabetic is an increase in the rate of enzyme degradation.     

Urea and methylamine effects on rabbit muscle phosphofructokinase. Catalytic stability and aggregation state as a function of pH and temperature

The effects of urea and several methylamine solutes on the catalytic stability and aggregation properties of rabbit muscle phosphofructokinase were assessed at physiologically realistic concentrations of the solutes under several pH and temperature regimes. The loss of catalytic activity observed under conditions of pH-induced cold lability was significantly reduced in the presence of trimethylamine-N-oxide, N-trimethylglycine and N-methylglycine (order of decreasing effectiveness). The concentration-dependent methylamine stabilization of the enzyme, seen with as little as 50 mM trimethylamine-N-oxide, was accompanied by increased aggregation of the enzyme to molecular weights greater than the tetramer (polytetramer) as solute concentration was raised to 400 mM. At pH 6.5-6.7 and 25 degrees C, concentrations of urea greater than 25 mM promoted a time-dependent inactivation of the enzyme which was enhanced at lower temperatures. The urea sensitivity of the enzyme exhibited with 0.8 M urea for 1 h at pH 8.0 did not result in measurable inactivation. The fluorescence emission wavelength maximum of the enzyme was shifted to longer wavelengths and the fluorescence intensity was increased as pH was lowered to 7.0, suggesting the occurrence of a protein conformation change as specific amino acid residues of the tetramer became protonated. Measurements of enzyme light scattering indicated that perturbation by urea was correlated with tetramer dissociation, which was irreversible by dialysis at 25 degrees C. The urea and methylamine influences on phosphofructokinase activity and structure were not counteracting. The synergistic interactions among pH, temperature, and solutes observed with phosphofructokinase are compared to effects on other associating-dissociating protein systems in order to evaluate possible mechanisms of action of these low molecular weight solutes.     

Hexokinase activity from eggs of the sea urchin Arbacia punctulata

1. The hexokinase activity of homogenates of eggs and embryos of the sea urchin Arbacia punctulata has been measured. Expressed as micrograms glucose consumed at 20°C., per hour per milligram of protein the following values were obtained: unfertilized eggs, 67; fertilized eggs, 72; 24 hour plutei, 94; 48 hour plutei, 226. The concentration of the enzyme in the eggs is small and may be calculated to be about 0.001 per cent of the dry weight of unfertilized eggs. 2. The hexokinase activity of the egg homogenate was virtually all recovered in the supernatant fraction when the homogenate was centrifuged at 20,000 x g for 30 minutes and was found to have the following properties: The concentrations for half maximal hexokinase activity with various substrates were, approximately: Glucose, 0,00003 M; fructose, 0.00075; mannose, 0.00007; 2-desoxyglucose, 0.00025. The relative rates of phosphorylation of various sugars by the supernate fraction when saturated with substrate were, approximately: Glucose, 1.0; mannose, 1.2; fructose, 1.8; 2-desoxyglucose, 2.0; glucosamine, 0.6. Adenosinediphosphate and glucose-6-phosphate inhibited the enzyme. No evidence for more than one hexokinase in the Arbacia extracts was found.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea.

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

The activation of urease

1. It has been shown that the activity of solutions of twice recrystallized urease is reversibly increased by moderate heating and reversibly decreased by storage in the cold, even in the frozen state. 2. Crude extracts of jack bean meal containing potent urease undergo this same type of reversible activation by heating and inactivation by cooling. Dilution has the same potentiating effect on the activity as moderate heating. As much as a fivefold increase in activity can be obtained when a sample previously inactivated by storage for 24 hours at -10 degrees C. is heated for 5 minutes at 60 degrees C. 3. Solutions of crystalline urease protected by serum albumin and preserved in the cold give a constant "potential" activity over a period of more than 30 days if heated 5 minutes at 60 degrees C. before assay. 4. The data presented have been interpreted to mean that an association between urease molecules (or between urease and other proteins) might occur, resulting in inactivation of the enzyme which would be reversed on dissociation. 5. It has been postulated that the same forces are responsible for the reversible inactivation brought about by standing at temperatures above or below the freezing point.     

Heat denaturation enhanced immunoglobulin production stimulating activity of lysozyme from hen egg white

Lysozyme from hen egg white was identified as an immunoglobulin production stimulating factor (IPSF) that enhances immunoglobulin production by hybridomas and lymphocytes. The IPSF activity of lysozyme was facilitated by heat treatment. The heat treatment of lysozyme at 83 degrees C for 30 min activated its specific IPSF effect 30.0-fold compared with that of native lysozyme. The IPSF activity of lysozyme heat-treated at 83 degrees C in 4 M urea solution was enhanced 8.4-fold than that of native lysozyme. However, lysozyme that was not heated in 4 M urea solution completely lost its IPSF activity. This means that the IPSF activity of this enzyme in 4 M urea was reactivated by thermal treatment. Moreover, coexistence of 0.5 mM 2-mercaptoethanol (2-ME) during heating in 4 M urea solution extremely enhanced the IPSF activity up to 77.8-fold. The uptake of lysozyme by hybridoma cells was enhanced by heat denaturation in 4 M urea. The hydrophobicity of lysozyme was extremely increased by heat-treatment in 2-ME containing urea solution. It is expected from these findings that the increase in the hydrophobicity caused the enhancement of incorporation of lysozyme into target cells, and resulted in the acceleration of IgM production.     

High molecular weight heat-stable alkaline proteinase from white croaker and chum salmon muscle: comparison of the activating effects by heating and urea

Effects of heating and urea on the heat-stable alkaline proteinase from white croaker and chum salmon muscle were compared in order to know the regulating mechanism of the proteinase. Chum salmon proteinase required a higher temperature for activity and was more heat-stable than white croaker proteinase. In the presence of 5M urea, the activity was observed to some degree at 37 degrees C only in white croaker proteinase, while both proteinases lost their activities at usual assay temperature around 60 degrees C. These results suggest that the stability of the regulatory and catalytic subunits of the proteinases is somewhat different among fish species.     

Characterization of an alkaline proteinase of fish muscle

1. The alkaline proteinase showing pH optimum 8.0 from white croaker (Sciaena schlegeli) skeletal muscle was purified electrophoretically homogeneously (2000-fold) using a combination of DEAE-cellulose chromatography, hydroxylapatite chromatography and Ultrogel AcA 34 gel filtration. 2. It was stable for 1 hr at 50 degrees C. The molecular weight of the enzyme was estimated to be 430,000 by gel filtration, with the enzyme composed of four kinds of subunits, the chain molecular weights of which were 45,000, 48,000, 51,000 and 57,000. 3. From the effects of inhibitors, the enzyme was identified as cysteine proteinase. ATP and Cu2+ inhibited the activity 50% at 10 mM and 70% at 0.1 mM, respectively. 4. Thus the enzyme was characterized as a high molecular weight, heat-stable, alkaline cysteine proteinase (HAP). 5. The enzyme showed hardly any activity below 50 degrees C but considerable activity at around 60 degrees C against myofibrils, digesting myosin heavy chain, actin and tropomyosin. With the addition of 5 M urea the enzyme hydrolyzed myofibrils well at around 30 degrees C.