Auto-ubiquitination of ubiquitin-activating enzymes from chicken breast muscle.

A soluble ubiquitin-depleted fraction from chicken skeletal muscle (fraction II), when incubated at neutral pH for several hours with 125I-ubiquitin and ATP, formed small amounts of a ubiquitin derivative (Mr 115,000) of the ubiquitin-activating enzyme E1 as well as certain similarly modified E2 species (Mr 37,000, 34,000 and 24,000). Treatment of such mixtures with NaOH during the incubations, even at early times, greatly enhanced the appearance of these entities; up to two-thirds of the thiolesters of ubiquitin bound to these proteins before alkali treatment were thus converted. The bonds involved had properties compatible with their being peptidic in nature, suggesting that auto-ubiquitination had occurred in each case. The protease inhibitor and alkylating agent tosyl-lysylchloromethane ('TLCK'), when preincubated at 50 microM with fraction II for 2 h at 37 degrees C before the addition of 125I-ubiquitin and ATP, promoted the subsequent auto-ubiquitination of E1 and inhibited its adenylate-forming and thiolester-transferring activities. The findings have a bearing on the physiological substrate- and site-specificity of ubiquitin-conjugating reactions.     

Preparation and properties of creatine kinase from the breast muscle of normal and dystrophic chicken (Gallus domesticus)

1. The purification of creatine kinase from normal and genetically dystrophic chicken breast muscle is described. Enzyme recovery was significantly lower from dystrophic muscle. 2. Both enzymes had the same number of reactive and total thiol groups and had similar specific activities and similar amino acid compositions. 3. No significant differences were observed in sedimentation, electrophoretic or kinetic properties. 4. Peptide ;maps' showed no significant differences, and electrophoresis of partial acid hydrolysates of the labelled enzymes suggested that corresponding amino acid sequences around all the thiol groups were very similar. 5. The enzymes showed identical temperature stabilities. 6. No significant differences between the enzymes from normal and dystrophic muscle were observed.     

Decreased mitochondrial creatine kinase activity in dystrophic chicken breast muscle alters creatine-linked respiratory coupling

Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT (p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36-44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 mM creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20-50 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent KD, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered.     

Muscle cell cultures from chicken breast muscle have increased specific activities of creatine kinase when incubated at 41 degrees C compared with 37 degrees C

Chicken muscle cell cultures were incubated at 41 degrees C, the physiological chicken body temperature, and compared with cultures incubated at 37 degrees C, the typical cell culture incubation temperature. The cultures incubated at 41 degrees C show not only an increase in creatine kinase (CK)-specific activity but also a marked increase in the percentage of adult muscle CK isozyme (MM-CK) in 7-day muscle cultures. Muscle cell cultures incubated in the presence of cytosine arabinoside (ara-C), a cell proliferation inhibitor, do not have the mononucleated cell overgrowth seen at 41 degrees C and thus exhibit a further increase in creatine kinase-specific activity compared with cultures incubated at 41 degrees C in the absence of ara-C. These results suggest that muscle cell cultures incubated at 41 degrees C are more highly differentiated than those incubated at 37 degrees C.     

Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.     

Content and synthesis of glycolytic enzymes and creatine kinase in skeletal muscles and normal and dystrophic chickens

A number of workers have reported that avian muscular dystrophy causes alterations in the levels of certain enzyme activities in "fast-twitch" muscle fibers but has little effect on enzyme activities in "slow-twitch" muscle fibers. In the present work, the effects of this disease on the content and relative rates of synthesis of a number of glycolytic enzymes and the skeletal muscle-specific MM isoenzyme of creatine kinase in chicken muscles was investigated. It was shown that (i) the approximate 50% reductions in steady-state concentrations of three glycolytic enzymes (aldolase, enolase, and glyceraldehyde-3-P dehydrogenase) in dystrophic breast (fast-twitch) muscle result predominantly from decreases in relative rates of synthesis, rather than accelerations in relative rates of degradation, of these proteins in the diseased tissue; (ii) in contrast to the situation with the glycolytic enzymes, muscular dystrophy has only minor effects (25% or less) on the content and relative rate of synthesis of MM creatine kinase in breast muscle fibers; (iii) the muscular dystrophy-associated alterations in content and synthesis of the glycolytic enzymes in breast muscle fibers become apparent only during postembryonic maturation of this tissue; and (iv) as expected, muscular dystrophy has no significant effect on the content or relative rates of synthesis of glycolytic enzymes in slow-twitch lateral adductor muscles of the chicken. These results are discussed in terms of the apparent similarities between the effects of muscular dystrophy and surgical denervation on the protein synthetic programs expressed by mature fast-twitch muscle fibers.     

The amino acid sequence of the peptide containing the thiol group of creatine kinase from normal and dystrophic chicken breast muscle. Comparison of some of the immunological properties of the antibodies developed in rabbits against these enzymes

The major (14)C-labelled peptides from creatine kinase from normal and dystrophic chicken muscle obtained by carboxymethylating the reactive thiol groups with iodo[2-(14)C]acetic acid and digestion with trypsin were purified by ion-exchange chromatography on Dowex-50 (X2) and by paper electrophoresis. The chromatographic characteristics of the (14)C-labelled peptides, their electrophoretic mobilities at pH6.5, and their amino acid compositions were identical for the two enzymes. The sequence of amino acids around the essential thiol groups of creatine kinase from normal and dystrophic chicken muscle was shown to be Ile-Leu-Thr-CmCys-Pro-Ser-Asn-Leu-Gly-Thr-Gly-Leu-Arg (CmCys, carboxymethylcysteine). This sequence is almost identical with that for the creatine kinases in human and ox muscle and bovine brain and is very similar to that of arginine kinase from lobster muscle. Antibodies to the enzymes were raised in rabbits and their reaction with the creatine kinase from normal and dystrophic muscles in interfacial, immunodiffusion and immunoelectrophoretic experiments was studied. The cross-reaction between normal muscle creatine kinase and antisera against the dystrophic muscle enzyme (or vice versa) observed by immunodiffusion and by immunoelectrophoretic experiments further suggests that the enzymes from normal and dystrophic chicken muscle are similar in structure. The results of the present study, the identical amino acid sequence of the peptides containing the reactive thiol group from both the normal and dystrophic chicken muscle enzymes and the immunological similarities of the two enzymes are in accord with the similarity of the two enzymes observed by Roy et al. (1970).     

Partial purification of a ribonucleic acid cAMP-independent protein kinase from embryonic chicken muscle

A cAMP-indepedent protein kinase (P38 kinase) from embryonic chicken muscle with ability to phosphorylate a 38,000 molecular weight polypeptide and to bind to RNAs has been further characterized. An approximately 2000-fold purification of this enzyme was achieved by a combination of affinity and ion-exchange chromatography. Our studies indicate that this protein kinase can not phosphorylate the small subunit of rabbit reticulocyte initiation factor eIF-2 in the presence of its normal endogenous substrate, nor is it activated over a wide range of concentrations of double-stranded RNA. This P38 kinase is, therefore, distinct from the hemin-regulated translational inhibitor of protein synthesis in rabbit reticulocytes and from the interferon-induced protein kinase identified In several systems.     

Three-step preparation and purification of phosphorus-33-labeled creatine phosphate of high specific activity

Rabbit heart mitochondria were used as a source of enzymes for the synthesis of phosphoruslabeled creatine phosphate. This method is based on the coupled reaction between mitochondrial oxidative phosphorylation and mitochondrial-bound creatine kinase. It is possible to convert more than 90% of the inorganic phosphate (P_i) to creatine phosphate. The method used only small amounts of adenine nucleotides which led to a product with only slight nucleotide contamination. This could be removed by activated charcoal extraction. For further purification, a method for the removal of residual P_i is described.     

Crystallization and preliminary X-ray analysis of two different forms of mitochondrial creatine kinase from chicken cardiac muscle

Crystals of mitochondrial creatine kinase isolated from chicken heart were grown by precipitation with polyethylene glycol 1000. The enzyme has been crystallized in the absence and presence of ATP in two different space groups. Crystals are tetragonal, with space group P42(1)2, a = b = 171 A, c = 150 A in the absence of ATP; and P422, a = b = 101 A, c = 114.4 A in the presence of ATP. We suggest that there is one octamer (346 kDa) per asymmetric unit without ATP and one dimer (86 kDa) per asymmetric unit with ATP. Using synchrotron radiation, the octameric form diffracts to at least 3 A resolution.     

Properties of creatine kinase from skeletal muscle mitochondria

Creatine kinase from pigeon breast muscle was obtained in a homogeneous (as evidenced from polyacrylamide gel SDS electrophoresis) state. The molecular mass of the enzyme monomer is 43,000. Ultracentrifugation in a sucrose density gradient and gel filtration revealed that the enzyme is present in solution as a mixture of two major forms, i.e., octamer and dimer, which differ in their activity. The decrease of ionic strength from 0.25 to 0.02 results in reversible dissociation of the octameric form. A temperature rise from 5 degrees to 20 degrees C or the nature of monovalent anions (e.g., Cl-, CH3COO-, NO3-) and cations (K+, Na+) present in the medium do not influence the distribution of oligomeric forms. At pH 6.0 the major form is represented by the octamer; its dissociation is caused by an increase of pH. The octamer dissociation occurs in a mixture of substrates of the creatine kinase reaction in the presence of Mg2+; no such dissociation is observed in the absence of Mg2+ and in the presence of each of the reaction substrates. The non-interacting pair of substrates--ADP and creatine--causes the dissociation of the octamer in the presence of nitrate ions but not acetate. It is concluded that the dissociating effect of substrates is due to the conformational changes of subunits during catalysis. At physiological concentrations of nucleotide substrates the degree of octamer dissociation depends on the ratio of creatine phosphate and creatine concentrations, as well as on the presence of chlorine and phosphate ions. A qualitative estimation of the rate of pH- and substrate-dependent dissociation of creatine kinase octamer revealed that under the given experimental conditions the pH-dependent dissociation is completed within hours, whereas the substrate-dependent one--within seconds or minutes. According to its properties, mitochondrial creatine kinase from pigeon breast muscle is close to its bovine heart counterpart; the observed differences were found to be quantitative.     

Differences in properties of creatine kinase BB from chicken tissues

BB-creatine phosphokinases (CPK-BB, EC 2.7.3.2) were isolated and purified from the chicken brain, heart and gizzard with specific activities 250-300 IU/mg and equal electrophoretic mobilities. Certain differences in thermal stability, activation energy, Km and optimal temperature of the CPK reaction are shown. The amino acid analysis has also revealed different content of certain amino acids in CPK-BB molecules. The obtained results confirm the existence of tissue-specific heterogeneity in CPK-BB which may be significant for functioning of the phosphocreatine-creatine phosphokinase system in different types of tissues.     

Purification and crystallization of creatine kinase from rabbit skeletal muscle

Crystallization is the primary rate-limiting step in protein structure determination. It has been our experience over approximately 10 years that crystals are obtained in about 20% of the proteins attempted and that only about 10% of these crystals are sufficiently well ordered to permit atomic resolution structure analysis. In attempts to overcome this limitation, we have investigated the effect on crystallization of microheterogeneity in a protein regarded as pure by conventional criteria. Creatine kinase was purified from rabbit skeletal muscle and crystallized from methylpentanediol. The protein appeared to be nearly pure judging by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high specific activity. The crystals that were obtained were of poor quality, and an extensive survey of precipitants, crystallization conditions, and additives failed to discover conditions from which usable crystals could be obtained. The enzyme was then subjected to a series of further purification steps. After each purification step, the quality of the crystals obtained under almost identical conditions improved. The final purification step was flat-bed isoelectric focusing. Crystals grown from focused creatine kinase are well ordered and diffract to approximately 3-A resolution.     

Effects of acrylamide on creatine kinase from rabbit muscle

The mechanism of inhibition of creatine kinase (CK) by acrylamide (Acr) has been examined (in vitro). Within the concentration range of 0 to 1 M, Acr markedly inhibited CK and depleted the protein thiols. Both inactivation and thiol depletion were time- and Acr concentration-dependent. Addition of dithiothreitol (DTT) did not reactivate CK inactivated by Acr. However, CK with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) pre-blocked thiols can be reactivated by DTT after incubation with Acr. The transition-state analogue also had a significant protective effect on CK against Acr inhibition. We conclude that thiol alkylation is a critical event in inactivation of CK by Acr. Furthermore, Acr binding to CK changed its surface charge, which may be the same effect for the toxicity of Acr towards other proteins.