共查询到20条相似文献,搜索用时 15 毫秒
1.
G.J. Kemp D.N. Manners J.F. Clark M.E. Bastin G.K. Radda 《Molecular and cellular biochemistry》1997,174(1-2):29-32
We describe a model of mitochondrial regulation in vivo which takes account of spatial diffusion of high-energy (ATP and phosphocreatine) and low-energy metabolites (ADP and creatine), their interconversion by creatine kinase (which is not assumed to be at equilibrium), and possible functional 'coupling' between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on relationships between oxidative ATP synthesis rate and spatially-averaged metabolite concentrations. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, to a degree which depends on the degree of coupling. At high creatine kinase activity, the fraction of flow carried by ATP is small. Lowering the creatine kinase activity raises this fraction, especially when there is little functional coupling. All metabolites show small spatial gradients, more so at low cytosolic creatine kinase activity, and unless there is near-complete coupling, so does net creatine kinase flux. During workjump transitions, spatial-average responses exhibit near-exponential kinetics as expected, while concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. (Mol Cell Biochem 174: 29–32, 1997) 相似文献
2.
The paper reviews the current evidence on the role of thyroid hormones in regulating the creatine kinase energy transfer system at multiple structures in cardiac cells. 1) Thyroid hormones modulate the overall synthesis of phosphocreatine (PCr) by increasing the rate of mitochondrial oxidative phosphorylation. 2) Thyroid hormones regulate the total activity of creatine kinase and its isoenzyme distribution. In comparison with normal thyroid state (euthyroidism), hypothyroidism is characterized by decreased total creatine kinase activity owing to diminished fraction of creatine kinase. On the other hand, hyperthyroidism, while causing no change in total creatine kinase activity, leads to increased fractions of neonatal isoforms of creatine kinase, and, in case of prolonged hyperthyroidism, to decreased fraction of mitochondrial creatine kinase. The latter change is associated with partial uncoupling between mitochondrial creatine kinase and adenine nucleotide translocase reflected by decreased PCr/O ratio. 3) Hyperthyroidism leads to increased passive sarcolemmal permeability due to which the leakage of creatine along its concentration gradient occurs. As a result of (i) increased sarcolemmal permeability for creatine, (ii) uncoupling of mitochondrial PCr synthesis, and (iii) increased energy utilization rate the steady state intracellular PCr content decreases under hyperthyroidism which, in turn, increases the myocardial susceptibility to hypoxic damage. Thyroid state also modulates the protective effects of exogenous PCr on energetically depleted myocardium. 相似文献
3.
The exchange scheme of high energy phosphate transport in a whole heart relies on a system of CK functioning in different ways. This suggests that the CKs are able to act both like a shuttle and like a buffer for the energy transfer. The challenge is to understand how these two functions are balanced in the CK system. One key of this balance is the knowledge of the local concentrations of the ADP nucleotide. These concentrations cannot be directly measured, but they may be derived by computation. In the present report we introduce the known properties of the enzymes catalyzing the exchange of high energy phosphate into the model of flux pathways derived from NMR experiments to compute both the maximum activity of each enzyme and the local concentrations of all the substrates. We show that the ADP distribution must be heterogeneous for the system to work. Its concentration is 50% higher in the vicinity of ATPase sites and 50% lower in the intermembrane space of the mitochondria than in the cytosol. Another result of this analysis is that the apparent large unbalance of the CKmito pathway is imposed by the adenosine nucleotide transferase fluxes. This analysis proves that it is possible to deduce the local concentrations of a substrate by combining data originating from NMR, biochemistry and enzymology into a common model. 相似文献
4.
5.
Soboll Sibylle Conrad Annette Hebisch Siegbert 《Molecular and cellular biochemistry》1994,133(1):105-113
The influence of mitochondrial creatine kinase on subcellular high energy systems has been investigated using isolated rat heart mitochondria, mitoplasts and intact heart and skeletal muscle tissue.In isolated mitochondria, the creatine kinase is functionally coupled to oxidative phosphorylation at active respiratory chain, so that it catalyses the formation of creatine phosphate against its thermodynamic equilibrium. Therefore the mass action ratio is shifted from the equilibrium ratio to lower values. At inhibited respiration, it is close to the equilibrium value, irrespective of the mechanism of the inhibition. The same results were obtained for mitoplasts under conditions where the mitochondrial creatine kinase is still associated with the inner membrane.In intact tissue increasing amounts of creatine phosphate are found in the mitochondrial compartment when respiration and/or muscle work are increased. It is suggested that at high rates of oxidative phosphorylation creatine phosphate is accumulated in the intermembrane space due to the high activity of mitochondrial creatine kinase and the restricted permeability of reactants into the extramitochondrial space. A certain amount of this creatine phosphate leaks into the mitochondrial matrix.This leak is confirmed in isolated rat heart mitochondria where creatine phosphate is taken up when it is generated by the mitochondrial creatine kinase reaction. At inhibited creatine kinase, external creatine phosphate is not taken up. Likewise, mitoplasts only take up creatine phosphate when creatine kinase is still associated with the inner membrane. Both findings indicate that uptake is dependent on the functional active creatine kinase coupled to oxidative phosphorylation.Creatine phosphate uptake into mitochondria is inhibited with carboxyatractyloside. This suggests a possible role of the mitochondrial adenine nucleotide translocase in creatine phosphate uptake.Taken together, our findings are in agreement with the proposal that creatine kinase operates in the intermembrane space as a functional unit with the adenine nucleotide translocase in the inner membrane for optimal transfer of energy from the electron transport chain to extramitochondrial ATP-consuming reactions. 相似文献
6.
The synthesis of creatine phosphate (CP) by mitochondrial creatine kinase during oxidative phosphorylation was terminated when the mass action ratio of the creatine kinase reaction = [ADP]·[CP][ATP]·[Cr] became equal to the apparent equilibrium constant (K
eq
app) of this reaction. Subsequent excess of over the K
eq
app was due to an increase in the ADP concentration in the medium. A comparable increase in the ADP concentration also occurred in the absence of creatine (Cr) in the incubation medium. Increase in the ADP concentration was shown to be associated with a decrease in the rate of oxidative phosphorylation and with a relative increase in the ATPase activity of mitochondria during the incubation. A low concentration of ADP (<30 M) and relatively high concentrations (1-6 mM) of other components of the creatine kinase reaction prevented the detection of the reverse reaction within 10 min after exceeded the K
eq
app, but the reverse reaction became evident on more prolonged incubation. The reverse reaction was accompanied by a further increase in . Low ADP concentration in the medium was also responsible for the lack of an immediate conversion of the excess creatine phosphate added although > K
eq
app. The findings are concluded to be in contradiction with the concept of microcompartment formation between mitochondrial creatine kinase and adenine nucleotide translocase. 相似文献
7.
Aliev Mayis K. van Dorsten F.A. Nederhoff M.G. van Echteld C.J.A. Veksler Vladimir Nicolay Klaas Saks Valdur A. 《Molecular and cellular biochemistry》1998,184(1-2):209-229
A mathematical model of the compartmentalized energy transfer in cardiac cells is described and used for interpretation of novel experimental data obtained by using phosphorus NMR for determination of the energy fluxes in the isolated hearts of transgenic mice with knocked out creatine kinase isoenzymes. These experiments were designed to study the meaning and importance of compartmentation of creatine kinase isoenzymes in the cells in vivo. The model was constructed to describe quantitatively the processes of energy production, transfer, utilization, and feedback between these processes. It describes the production of ATP in mitochondrial matrix space by ATP synthase, use of this ATP for phosphocreatine production in the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocation, diffusional exchange of metabolites in the cytoplasmic space, and use of phosphocreatine for resynthesis of ATP in the myoplasmic creatine kinase reaction. It accounts also for the recently discovered phenomenon of restricted diffusion of adenine nucleotides through mitochondrial outer membrane porin pores (VDAC). Practically all parameters of the model were determined experimentally. The analysis of energy fluxes between different cellular compartments shows that in all cellular compartments of working heart cells the creatine kinase reaction is far from equilibrium in the systolic phase of the contraction cycle and approaches equilibrium only in cytoplasm and only in the end-diastolic phase of the contraction cycle.Experimental determination of the relationship between energy fluxes by a 31P-NMR saturation transfer method and workload in isolated and perfused heart of transgenic mice deficient in MM isoenzyme of the creatine kinase, MM -/- showed that in the hearts from wild mice, containing all creatine kinase isoenzymes, the energy fluxes determined increased 3-4 times with elevation of the workload. By contrast, in the hearts in which only the mitochondrial creatine kinase was active, the energy fluxes became practically independent of the workload in spite of the preservation of 26% of normal creatine kinase activity. These results cannot be explained on the basis of the conventional near-equilibrium theory of creatine kinase in the cells, which excludes any difference between creatine kinase isoenzymes. However, these apparently paradoxical experimental results are quantitatively described by a mathematical model of the compartmentalized energy transfer based on the steady state kinetics of coupled creatine kinase reactions, compartmentation of creatine kinase isoenzymes in the cells, and the kinetics of ATP production and utilization reactions. The use of this model shows that: (1) in the wild type heart cells a major part of energy is transported out of mitochondria via phosphocreatine, which is used for complete regeneration of ATP locally in the myofibrils - this is the quantitative estimate for PCr pathway; (2) however, in the absence of MM-creatine kinase in the myofibrils in transgenic mice the contraction results in a very rapid rise of ADP in cytoplasmic space, that reverses the mitochondrial creatine kinase reaction in the direction of ATP production. In this way, because of increasing concentrations of cytoplasmic ADP, mitochondrial creatine kinase is switched off functionally due to the absence of its counterpart in PCr pathway, MM-creatine kinase. This may explain why the creatine kinase flux becomes practically independent from the workload in the hearts of transgenic mouse without MM-CK. Thus, the analysis of the results of studies of hearts of creatine kinase-deficient transgenic mice, based on the use of a mathematical model of compartmentalized energy transfer, show that in the PCr pathway of intracellular energy transport two isoenzymes of creatine kinase always function in a coordinated manner out of equilibrium, in the steady state, and disturbances in functioning of one of them inevitably result in the disturbances of the other component of the PCr pathway. In the latter case, energy is transferred from mitochondria to myofibrils by alternative metabolic pathways, probably involving adenylate kinase or other systems. 相似文献
8.
Tiivel T Kadaya L Kuznetsov A Käämbre T Peet N Sikk P Braun U Ventura-Clapier R Saks V Seppet EK 《Molecular and cellular biochemistry》2000,208(1-2):119-128
In saponin-skinned muscle fibers from adult rat heart and m. soleus the apparent affinity of the mitochondrial oxidative phosphorylation system for ADP (Km = 200-400 M) is much lower than in isolated mitochondria (Km = 10-20 M). This suggests a limited permeability of the outer mitochondrial membrane (OMM) to adenine nucleotides in slow-twitch muscle cells. We have studied the postnatal changes in the affinity of mitochondrial respiration for ADP, in relation to morphological alterations and expression of mitochondrial creatine kinase (mi-CK) in rat heart in vivo. Analysis of respiration of skinned fibers revealed a gradual decrease in the apparent affinity of mitochondria to ADP throughout 6 weeks post partum that indicates the development of mechanism which increasingly limits the access of ADP to mitochondria. The expression of mi-CK started between the 1st and 2nd weeks and reached the adult levels after 6 weeks. This process was associated with increases in creatine-activated respiration and affinity of oxidative phosphorylation to ADP thus reflecting the progressive coupling of mi-CK to adenine nucleotide translocase. Laser confocal microscopy revealed significant changes in rearrangement of mitochondria in cardiac cells: while the mitochondria of variable shape and size appeared to be random-clustered in the cardiomyocytes of 1 day old rat, they formed a fine network between the myofibrils by the age of 3 weeks. These results allow to conclude that in early period of development, i.e. within 2-3 weeks, the diffusion of ADP to mitochondria becomes progressively restricted, that appears to be related to significant structural rearrangements such as formation of the mitochondrial network. Later (after 3 weeks) the control shifts to mi-CK, which by coupling to adenine nucleotide translocase, allows to maximally activate the processes of oxidative phosphorylation despite limited access of ADP through the OMM. 相似文献
9.
Bernard Font Denise Eichenberger Denise Goldschmidt Christian Vial 《Molecular and cellular biochemistry》1987,78(2):131-140
Summary Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes.The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers.Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase.Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu++-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization.In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.Abbreviations AK
Adenylate Kinase (EC 2.7.4.3)
- CKm
Mitochondrial Isoenzyme of Creatine Kinase (EC 2.7.3.2)
- DMS
Dimethyl Suberimidate
- DTT
Dithiothreitol
- EGS
Ethylene Glycol bis (succinimidyl succinate)
- EGTA
Ethylene Glycol bis (aminoethyl ether)
- N,N,N,N
Tetraacetic acid
- G6P
Glucose 6 Phosphate, Hepes - N-2 Hydroxyethyl Piperazine N-2 Ethane Sulfonic Acid
- HK
Hexokinase (EC 2.7.1.1)
- MABI
methyl 4-Azido Benzoimidate
- NaPi
Sodium Phosphate
- SANPAH
N-Succinimidyl 6(4 azido 2 nitrophenylamino) Hexanoate
- SDS
Sodium Dodecyl Sulfate (sodium lauryl sulfate)
- Tris
Tris (hydroxymethyl) Aminomethane 相似文献
10.
Lourdes Guerrero-Ontiveros Maria Wallimann Theo 《Molecular and cellular biochemistry》1998,184(1-2):427-437
Interest in creatine (Cr) as a nutritional supplement and ergogenic aid for athletes has surged over recent years. After cellular uptake, Cr is phosphorylated to phosphocreatine (PCr) by the creatine kinase (CK) reaction using ATP. At subcellular sites with high energy requirements, e.g. at the myofibrillar apparatus during muscle contraction, CK catalyzes the transphosphorylation of PCr to ADP to regenerate ATP, thus preventing a depletion of ATP levels. PCr is thus available as an immediate energy source, serving not only as an energy buffer but also as an energy transport vehicle. Ingestion of creatine increases intramuscular Cr, as well as PCr concentrations, and leads to exercise enhancement, especially in sprint performance. Additional benefits of Cr supplementation have also been noticed for high-intensity long-endurance tasks, e.g. shortening of recovery periods after physical exercise.The present article summarizes recent findings on the influence of Cr supplementation on energy metabolism, and introduces the Cr transporter protein (CreaT), responsible for uptake of Cr into cells, as one of the key-players for the multi-faceted regulation of cellular Cr homeostasis. Furthermore, it is suggested that patients with disturbances in Cr metabolism or with different neuro-muscular diseases may benefit from Cr supplementation as an adjuvant therapy to relieve or delay the onset of symptoms. Although it is still unclear how Cr biosynthesis and transport are regulated in health and disease, so far there are no reports of harmful side effects of Cr loading in humans. However, in this study, we report that chronic Cr supplementation in rats down-regulates in vivo the expression of the CreaT. In addition, we describe the presence of CreaT isoforms most likely generated by alternative splicing. 相似文献
11.
Compartmentation of ATP synthesis and utilization in smooth muscle: roles of aerobic glycolysis and creatine kinase 总被引:5,自引:0,他引:5
Ishida Y. Riesinger I. Wallimann T. Paul R. J. 《Molecular and cellular biochemistry》1994,133(1):39-50
The phosphocreatine content of smooth muscle is of similar magnitude to ATP. Thus the function of the creatine kinase system in this tissue cannot simply be regarded as an energy buffer. Thus an understanding of its role in smooth muscle behavior can point to CK function in other systems. From our perspective CK function in smooth muscle is one example of a more general phenomenon, that of the co-localization of ATP synthesis and utilization. In an interesting and analogous fashion distinct glycolytic cascades are also localized in regions of the cell with specialized energy requirements. Similar to CK, glycolytic enzymes are known to be localized on thin filaments, sarcoplasmic reticulum and plasma membrane. In this chapter we will describe the relations between glycolysis and smooth muscle function and compare and contrast to that of the CK system. Our goal is to more fully understand the significance of the compartmentation of distinct pathways for ATP synthesis with specific functions in smooth muscle. This organization of metabolism and function seen most clearly in smooth muscle is likely representative of many other cell types. 相似文献
12.
Schlattner Uwe Forstner Michael Eder Michael Stachowiak Olaf Fritz-Wolf Karin Wallimann Theo 《Molecular and cellular biochemistry》1998,184(1-2):125-140
Mitochondrial creatine kinase (Mi-CK) is a central enzyme in energy metabolism of tissues with high and fluctuating energy requirements. In this review, recent progress in the functional and structural characterization of Mi-CK is summarized with special emphasis on the solved X-ray structure of chicken Mib-CK octamer (Fritz-Wolf et al., Nature 381, 341-345, 1996). The new results are discussed in a historical context and related to the characteristics of CK isoforms as known from a large number of biophysical and biochemical studies. Finally, two hypothetical functional aspects of the Mi-CK structure are proposed: (i) putative membrane binding motifs at the top and bottom faces of the octamer and (ii) a possible functional role of the central 20 Å channel. 相似文献
13.
Sacnicte Ramírez Ríos Frédéric Lamarche Cécile Cottet-Rousselle Anna Klaus Roland Tuerk Ramon Thali Yolanda Auchli René Brunisholz Dietbert Neumann Luc Barret Malgorzata Tokarska-Schlattner Uwe Schlattner 《BBA》2014
AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca2+-pumping. 相似文献
14.
Kemp Graham J. Manners David N. Clark Joseph F. Bastin Mark E. Radda George K. 《Molecular and cellular biochemistry》1998,184(1-2):249-289
After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional coupling between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters and that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible coupling are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when or is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when or is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless = 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the adenine nucleotide flux at the ends of the model into creatine flux in the middle; the overall net flux is small, but only zero if = . A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model. 相似文献
15.
The main purpose of this study was to investigate the effect of free radicals and experimental diabetes on cytosolic creatine kinase activity in rat heart, muscle and brain. Hydrogen peroxide decreased creatine kinase activity in a dose dependent manner which was reversed by catalase. Xanthine/xanthine oxidase, which produces superoxide anion, lowered the creatine kinase activity in the same manner whose effect was protected by superoxide dismutase. N-acetylcysteine and dithiothreitol also significantly ameliorated the effect of Xanthine/xanthine oxidase and hydrogen peroxide. Experimental diabetes of twenty-one days (induced by alloxan), also caused a similar decrease in the activity of creatine kinase. This led us to the conclusion that the decrease in creatine kinase activity during diabetes could be due to the production of reactive oxygen species. The free radical effect could be on the sulfhydryl groups of the enzyme at the active sites, since addition of sulfhydryl groups like N-acetylcysteine and dithiothreitol showed a significant reversal effect. 相似文献
16.
Hiroshi Hashimoto Ritsuko Nishi Masaaki Umeda Hirofumi Uchimiya Atsushi Kato 《Plant molecular biology》1993,22(1):163-164
We isolated a rice cDNA clone which encodes an open reading frame of 382 amino acids. Its deduced amino acid sequence corresponds to an ATP/ADP translocator protein. Its homology with a maize ATP/ADP translocator was 83.9% in nucleotide sequence, and 90.2% of the amino acid level. Expression of this gene is regulated by such external stresses as salinity and low temperature. 相似文献
17.
Spermatogenesis-related change in the synthesis of the creatine kinase B-type and M-type isoforms in human spermatozoa 总被引:6,自引:0,他引:6
We have demonstrated earlier that the per sperm creatine-N-phosphotransferase (CK) activity was increased in oligospermic vs. normospermic men. The increased sperm CK activity is related to higher concentrations of cellular CK, which may indicate a defect of cytoplasmic extrusion during spermatogenesis. In the present work, we examined whether in spermatozoa, similar to muscle, there is a change in the synthesis of B-CK and M-CK isoforms during cellular differentiation. In 109 normospermic and 50 oligospermic specimens (sperm concentrations 60.6 +/- 3.7 vs. 8.8 +/- 1.3 million sperm/ml; all values expressed as mean +/- SEM), the relative concentrations of the M-CK isoform (M-CK/M-CK + B-CK) were 27.2% +/- 2.1% vs. 6.7% +/- 0.9% (P less than 0.001). The per sperm CK activities showed comparable differences (0.21 +/- 0.02 vs. 0.89 +/- 0.1 CK IU/100 million sperm; P less than 0.001) in the two groups, and there was a close correlation between per sperm CK activities and M-CK concentrations (R = 0.69, P less than 0.001, N = 159). This indicates that the loss of cytoplasm and the commencement of M-CK isoform synthesis are related events during the last phase of spermatogenesis, also that the incidence of spermatozoa with incomplete cellular maturation is higher in oligospermic specimens. In characterizing the M-CK, we found that sperm (unlike muscle tissue) lack the MB hybrid of CK dimers. However, in the presence of muscle M-CK, the muscle-sperm MB-CK hybrid has formed. Thus in sperm and muscle the M-CK isoforms are structurally different, whereas the B-CKs are apparently homologous.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
Hornemann T Kempa S Himmel M Hayess K Fürst DO Wallimann T 《Journal of molecular biology》2003,332(4):877-887
Muscle-type creatine kinase (MM-CK) is a member of the CK isoenzyme family with key functions in cellular energetics. MM-CK interacts in an isoform-specific manner with the M-band of sarcomeric muscle, where it serves as an efficient intramyofibrillar ATP-regenerating system for the actin-activated myosin ATPase located nearby on both sides of the M-band. Four MM-CK-specific and highly conserved lysine residues are thought to be responsible for the interaction of MM-CK with the M-band. A yeast two-hybrid screen led to the identification of MM-CK as a binding partner of a central portion of myomesin (My7-8). An interaction was observed with domains six to eight of the closely related M-protein but not with several other Ig-like domains, including an M-band domain, of titin. The observed interactions were corroborated and characterised in detail by surface plasmon resonance spectroscopy (BiaCore). In both cases, they were CK isoform-specific and the MM-CK-specific lysine residues (K8. K24, K104 and K115) are involved in this interaction. At pH 6.8, the dissociation constants for the myomesin/MM-CK and the M-protein/MM-CK binding were in the range of 50-100 nM and around 1 microM, respectively. The binding showed pronounced pH-dependence and indicates a dynamic association/dissociation behaviour, which most likely depends on the energy state of the muscle. Our data propose a simple model for the regulation of this dynamic interaction. 相似文献
19.
Genetic and molecular analysis of nonrandom dimer assembly of the creatine kinase isozymes of fishes
Species within many families of actinopterygian bony fishes (class Osteichthyes) have a two-banded allelic isozyme phenotype in individuals heterozygous at the creatine kinase A locus. This two-banded pattern is formed by the presence of the two homodimeric isozymes and the absence of the expected heterodimer. Sharks and amphibians have retained the ability to form all three allelic isozymes in individuals which are heterozygous. Reversible denaturation procedures were able to assemble the different allelic CK-A subunits within a species to form CK-A2 heterodimers. Furthermore, heterodimers were formed from different CK-A subunits from highly divergent species after this in vitro molecular hybridization process. It is concluded from these studies that the polypeptidebinding sites of creatine kinase are structurally conservative in most fishes and that the absence of a heterodimer in heterozygous individuals is not due to a structural incompatibility between the different A subunit types or to an instability of the heterodimer during electrophoresis. A temporal and/or spatial isolation of allelic CK-A subunit synthesis and assembly, within differentiated skeletal muscle, appears to have evolved in the actinopterygian bony fishes.This research was supported by NSF Grant PCM76-08383 to G. S. W. and by a NIH Cell and Molecular Biology Traineeship to S. D. F. 相似文献