The temperature dependence of creatine kinase fluxes in the rat heart

A ³¹P nuclear magnetic resonance saturation transfer method was used to measure the temperature dependence of creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts. A decrease in temperature from 37 to 4°C lowered the observed steady-state fluxes by about 80%. These data were used in conjunction with calculated changes in substrate concentrations with temperature to estimate the activation energy for creatine kinase in situ. The apparent activation energy of 42 kJ/mol agrees reasonably well with the range of literature values for the enzyme in vitro. This demonstrates that the reaction is not diffusion-limited in situ and that extraction and dilution of the enzyme for study in vitro does not alter fundamental kinetic properties of the enzyme exhibited in the intact tissue.     

Occurrence of creatine kinase activity in human erythrocyte membrane

Some evidences for creatine kinase activity in normal human erythrocyte membrane were presented. The creatine kinase was indicated to be a constituent of the integral proteins of erythrocyte membrane or to be tightly bound to the membrane, and was contrasted to the results obtained with adenylate kinase. Isoenzyme distribution of the erythrocyte creatine kinase by electrophoresis was identical to MM-creatine kinase from rabbit muscle.     

The role of Arg-96 in Danio rerio creatine kinase in substrate recognition and active center configuration

In creatine kinases (CKs), the amino acid residue-96 is a strictly conserved arginine. This residue is not directly associated with substrate binding, but it is located close to the binding site of the substrate creatine. On the other hand, the residue-96 is known to be involved in expression in the substrate specificity of various other phosphagen (guanidino) kinases, since each enzyme has a specific residue at this position: arginine kinase (Tyr), glycocyamine kinase (Ile), taurocyamine kinase (His) and lombricine kinase (Lys). To gain a greater understanding of the role of residue-96 in CKs, we replaced this residue in zebra fish Danio rerio cytoplasmic CK with other 19 amino acids, and expressed these constructs in Escherichia coli. All the twenty recombinant enzymes, including the wild-type, were obtained as soluble form, and their activities were determined in the forward direction. Compared with the activity of wild-type, the R96K mutant showed significant activity (8.3% to the wild-type), but 10 mutants (R96Y, A, S, E, H, T, F, C, V and N) showed a weak activity (0.056–1.0%). In the remaining mutants (R96Q, G, M, P, L, W, D and I), the activity was less than 0.05%. Our mutagenesis studies indicated that Arg-96 in Danio CK can be substituted for partially by Lys, but other replacements caused remarkable loss of activity. From careful inspection of the crystal structures (transition state analog complex (TSAC) and open state) of Torpedo cytoplasmic CK, we found that the side chain of R96 forms hydrogen bonds with A339 and D340 only in the TSAC structure. Based on the assumption that CKs consist of four dynamic domains (domains 1–3, and fixed domain), the above hydrogen bonds act to link putative domains 1 and 3 in TSAC structure. We suggest that residue-96 in CK and equivalent residues in other phosphagen kinases, which are structurally similar, have dual roles: (1) one involves in distinguishing guanidino substrates, and (2) the other plays a key role in organizing the hydrogen-bond network around residue-96 which offers an appropriate active center for the high catalytic turnover. The mode of development of the network appears to be unique each phosphagen kinase, reflecting evolution of each enzyme.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart: P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by ³¹P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg²⁺ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 μmol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

A P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart: 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by ³¹P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in $\text{phosphocreatine}\text{creatine}$ ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg²⁺ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 μmol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in $\text{[}\text{phosphocreatine}\text{]}\text{[}\text{creatine}\text{]}$ ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

Subcellular localization and substrate specificity of dolichol kinase from rat liver

When purified subcellular fractions were prepared from rat liver and assayed for dolichol kinase activity using pig liver dolichol as a substrate, the microsomes were found to contain the highest specific activity and greater than 75% of the total actvity. With regard to substrate specificity, the microsomal enzyme showed a marked preference for saturation of the α-isoprene: dolichol-16 and -19 were 2.5-fold more active than the corresponding polyprenols. For a given class of prenol, the 16 and 19 isoprenologs exhibited similar activity, whereas the 11 isoprenolog appeared less active. The enzyme was twice as active against the naturally occurring polyprenol-16 (α-cis-isoprene) compared to synthetic α-trans-polyprenol-16. Taken together, the data indicate that the α-isoprene specificity follows the order: saturated>cis>trans. In addition, all-trans-2,3-dihydrosolanesol was not a substrate, suggesting that at least one cis isoprene residue is required.     

Creatine kinase kinetics,ATP turnover,and cardiac performance in hearts depleted of creatine with the substrate analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% of the creatine substrate analogue β-guanidinopropionic acid for 6–10 weeks. ³¹P-NMR investigation of isolated, glucose-perfused working hearts showed a 90% reduction in [phosphocreatine] from 22.2 to 2.5 μmol/g dry wt in guanidinopropionic acid-fed animals but no change in [P_i], [ATP], or intracellular pH. The unidirectional exchange flux in the creatine kinase reaction (direction phosphocreatine → ATP) was measured by saturation transfer NMR in hearts working against a perfusion pressure of 70 cm of water. This exchange was 10 μmol/g dry wt per s in control hearts and decreased 4-fold to 2.5–2.8 μmol/g dry wt per s in hearts from guanidinopropionic acid-fed animals. Oxygen consumption and cardiac performance were measured in parallel experiments at two perfusion pressures, 70 and 140 cm. No significant differences were observed in oxygen uptake or in any of the performance criteria between hearts from control and guanidinopropionic acid-fed rats at either workload. Assuming an ADP:O ratio of 3, the oxygen consumption measurements correspond to ATP turnover rates of 4.2–7.8 μmol/g dry per s. These rates are 1.5–3-times greater than the rate of the phosphocreatine → ATP exchange in hearts from guanidinopropionic acid-fed rats. These data suggest that phosphocreatine cannot be an obligate intermediate of energy transduction in the heart.     

Tissue- and cell-specific distribution of creatine kinase B: A new and highly specific monoclonal antibody for use in immunohistochemistry

A synthetic 17-mer peptide corresponding to an unique sequence in the amino-terminal region of human creatine kinase B was used to raise a new and highly B-subunit-specific monoclonal antibody, CK-BYK/21E10. We show here that the monoclonal antibody is suitable for immunohistochemistry of unfixed frozen sections as well as formaldehyde- or Bouin-fixed, paraffin-embedded sections of human, rabbit, and mouse tissues. Moreover, in the study of cell- and tissue-specific distribution patterns, parallel Western blot analysis and immunoelectron microscopy is possible using this antibody. Our analyses demonstrate that creatine kinase B expression is restricted to a specific subset of cell types in various tissues. In brain, the B-subunit was found only in neurocytes, but not in glia cells. High expression was also observed in inner segments of photoreceptor cells and the outer plexiform layer of the retina, in the parietal cells of the stomach and in gut enterocytes, gallbladder and epithelial cells of the urogenital system. The possible roles of the creatine kinase/phosphocreatine-ATP system in these tissues are discussed.     

Measurement of the exchange rates of rapidly exchanging amide protons: Application to the study of calmodulin and its complex with a myosin light chain kinase fragment

Summary A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in¹H–¹⁵N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys⁷⁵ through Thr⁷⁹ located in the central helix of calmodulin, and for the C-terminal residues Ser¹⁴⁷ and Lys¹⁴⁸. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr¹¹⁰ through Glu¹¹⁴.Istituto Guido Donegani, Novara, Italy