Alterations in the creatine kinase system in the myocardium of cardiomyopathic hamsters

Immunochemical and biochemical methods were used to assess quantitatively the changes in the heart creatine kinase system in the myopathic Syrian hamsters, line CHF I46. Cardiomyopathy in I75-200 day old animals was characterized by decreased content of mitochondria and lower total creatine kinase activity. In isolated mitochondria only the creatine kinase activity was decreased while cytochromes aa3 content and respiration rate were unchanged. The share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to I8% and that of BB form was elevated from 5% in control to 20%, at unchanged relative level of MM. Immunoassay showed decreased amount of the mitochondrial creatine kinase in the tissue and its decreased ratio to cytochromes aa3. The results show altered expression of creatine kinase isoenzymes in cardiomyopathy.     

Cardiac phosphocreatine deficiency induced by GPA during postnatal development in rat

The effect of chronic administration of -guanidinopropionic acid (GPA) on the protein profiling, energy metabolism and right ventricular (RV) function was studied in the rat heart during the weaning and adolescence period. GPA was given in tap water (1–1.5%) using pair drink controls. The feeding of animals with GPA solution for a six week period resulted in elevation of heart to body weight ratio due to body growth retardation. GPA accumulated in the myocardium up to 67.37 ± 5.3 moles.g dry weight and the tissue content of total creatine, phosphocreatine and ATP was significantly decreased to 15%, 9% and 65% of control values respectively. Total activity of creatine kinase (CK) was not changed, but the proportion of mitochondrial (Mi) CK isoenzyme was decreased; the percentage of MB isoenzyme of CK was significantly higher. GPA treatment resulted in an elevation of the content of cardiac collagenous proteins and decrease of non-collagenous proteins in the heart; in parallel, a decrease of the collagen I to collagen III ratio was detected. The function of the RV was assessed using an isolated perfused heart with RV performing pressure-volume work. As compared to pair-drink controls, RV function was significantly impaired the GPA group: at any given right atrial filling pressure, the RV systolic pressure and the rate of pressure development were decreased by almost a factor of two. Elevation of the RV diastolic pressure with increasing pulmonary artery diastolic pressure was also significantly steeper in the GPA group which also showed decrease of cardiac output, especially at high outflow resistance. It may be assumed that chronic administration of GPA deeply influenced metabolic parameters, protein profiles and contractile function of the developing heart. On the other hand, concentrations of glucose, total lipids and triglycerides in blood plasma were not affected. All these data confirm the concept that the CK system is of central importance both for heart function and for the regulation of normal growth of cardiac myocytes.     

Variant minihelix RNAs reveal sequence-specific recognition of the helical tRNA(Ser) acceptor stem by E.coli seryl-tRNA synthetase.

Aminoacylation rate determinations for a series of variant RNA minihelix substrates revealed that Escherichia coli seryl-tRNA synthetase (SerRS) recognizes the 1--72 through 5--68 base pairs of the E.coli tRNA(Ser) acceptor stem with the major recognition elements clustered between positions 2--71 and 4--69. The rank order of effects of canonical base pair substitutions at each position on kcat/Km was used to assess the involvement of major groove functional groups in recognition. Conclusions based on the biochemical data are largely consistent with the interactions revealed by the refined structure of the homologous Thermus thermophilus tRNA(Ser)-SerRS complex that Cusack and colleagues report in the accompanying paper. Disruption of an end-on hydrophobic interaction between the major groove C5(H) of pyrimidine 69 and an aromatic side chain of SerRS is shown to significantly decrease kcat/Km of a minihelix substrate. This type of interaction provides a means by which proteins can recognize the binary information of 'degenerate' sequences, such as the purine-pyrimidine base pairs of tRNA(Ser). The 3--70 base pair is shown to contribute to recognition by SerRS even though it is not contacted specifically by the protein. The latter effect derives from the organization of the specific contacts that SerRS makes with the neighboring 2--71 and 4--69 acceptor stem base pairs.     

Metabolic compartmentation and substrate channelling in muscle cells

The published experimental data and existing concepts of cellular regulation of respiration are analyzed. Conventional, simplified considerations of regulatory mechanism by cytoplasmic ADP according to Michaelis-Menten kinetics or by derived parameters such as phosphate potential etc. do not explain relationships between oxygen consumption, workload and metabolic state of the cell. On the other hand, there are abundant data in literature showing microheterogeneity of cytoplasmic space in muscle cells, in particular with respect to ATP (and ADP) due to the structural organization of cell interior, existence of multienzyme complexes and structured water phase. Also very recent experimental data show that the intracellular diffusion of ADP is retarded in cardiomyocytes because of very low permeability of the mitochondrial outer membrane for adenine nucleotidesin vivo. Most probably, permeability of the outer mitochondrial membrane porin channels is controlled in the cellsin vivo by some intracellular factors which may be connected to cytoskeleton and lost during mitochondrial isolation. All these numerous data show convincingly that cellular metabolism cannot be understood if cell interior is considered as homogenous solution, and it is necessary to use the theories of organized metabolic systems and substrate-product channelling in multienzyme systems to understand metabolic regulation of respiration. One of these systems is the creatine kinase system, which channels high energy phosphates from mitochondria to sites of energy utilization. It is proposed that in muscle cells feed-back signal between contraction and mitochondrial respiration may be conducted by metabolic wave (propagation of oscillations of local concentration of ADP and creatine) through cytoplasmic equilibrium creatine and adenylate kinases and is amplified by coupled creatine kinase reaction in mitochondria. Mitochondrial creatine kinase has experimentally been shown to be a powerful amplifier of regulatory action of weak ADP fluxes due to its coupling to adenine nucleotide translocase. This phenomenon is also carefully analyzed.It is easier to explain biochemistry in terms of transport than it is to explain transport in terms of biochemistry. P. Mitchell The Ninth Sir Hans Krebs Lecture, Dresden, July 2, 1978.     

Dissociation of photoreceptors from whole heads of the fruit fly,Drosophila melanogaster

Photoreceptor cells that were mostly free of extracellular material and suitable for most electrophysiological study procedures were dissociated from whole heads of the fruit fly, Drosophila melanogaster, by a simple smash technique employing gentle chopping by a razor blade through Parafilm sheets. A variety of commonly available proteolytic and glycolytic digestion enzymes were tested as additions to the basic dissociation procedure described. With the aid of Nomarski interference contrast optics, periodic acid-Schiff staining, and fluorescent labeling and microscopy methods, it was determined that proteolytic enzymatic digestion does little to enhance the dissociation procedure, and instead, often damages the cells that one is attempting to recover. Unexpectedly, certain glycolytic enzymes, when added to the basic procedure, appear to enhance the recovery of intact viable Drosophila photoreceptors that are stripped of most extracellular material. Based on these results, a hypothesis concerning the biochemical nature of the extracellular matrix of the Drosophila retina is proposed. Drosophila photoreceptors are an interesting model system for the study of invertebrate phototransduction and photoreceptor cell biology because of their many well-characterized mutant strains. The technique described here should produce clean viable photoreceptors or ommatidia that respond to light, and that are suitable for patch clamping or cell culture.     

In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP

Relative diffusivities of ADP and creatine in cardiomyocytes were studied. The isolated rat cardiomyocytes were lysed with saponin (40 micrograms/ml) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenase. In these cardiomyocytes mitochondria were used as 'enzymatic probes' to determine the average local concentration of substrates exerting acceptor control of respiration--ADP or creatine (the latter activates respiration via mitochondrial creatine kinase reaction)--when their concentrations in the surrounding medium were changed. The kinetic parameters for ADP and creatine in control of respiration of saponin-treated cardiomyocytes were compared with those determined in isolated mitochondria and skinned cardiac fibers. The apparent Km for creatine (at 0.2 mM ATP) was very close and in a range of 6.0-6.9 mM in all systems studied, showing the absence of diffusion difficulties for this substrate. On the contrary, the apparent Km for ADP increased from 18 +/- 1 microM for isolated mitochondria to 250 +/- 59 microM for cardiomyocytes with the lysed sarcolemma and to 264 +/- 57 microM for skinned fibers. This elevation of Km was not eliminated by inhibition of myokinase with diadenosine pentaphosphate. When 25 mM creatine was present, the apparent Km for ADP decreased to 36 +/- 6 microM. These data are taken to indicate specific restrictions of diffusion of ADP most probably due to its interaction with intermediate binding sites in cardiomyocytes. The important role of phosphocreatine-creatine kinase system of energy transport is to overcome the restrictions in regulation of energy fluxes due to decreased diffusivity of ADP.     

Studies of mitochondrial respiration in muscle cells in situ: Use and misuse of experimental evidence in mathematical modelling

Applications of permeabilized cell and skinned fiber techniques in combination with methods of mathematical modelling for studies of mitochondrial function in the cell are critically evaluated. Mathematical models may be useful tools for explaining biological phenomena, but only if they are selected by fitting the computing results with real experimental data. Confocal microscopy has been used in experiments with permeabilized cardiomyocytes and myocardial fibers to determine the maximal diffusion distance from medium to the core of cells, which is shown not to exceed 8-10 microm. This is a principal index for correctly explaining high apparent Km for exogenous ADP (200-300 microM) in regulation of mitochondrial respiration in oxidative muscle cells in situ. The best fitting of the results of in silico studies may be achieved by using of the compartmentalized energy transfer model. From these results, it may be concluded that in cardiac muscle cells the mitochondria and ATPases are organized into intracellular energetic units (ICEUs) separated from the bulk phase of cytoplasm by some barriers which limit the diffusion of adenine nucleotides. In contrast, alternative models based on the concept of the cell as homogenous system do not explain the observed experimental phenomena and have led to misleading conclusions. The various sources of experimental and conceptual errors are analyzed.