The use of collagen polypeptides in the histochemical demonstration of creatine kinase

Summary The histochemical activity of creatine kinase was studied by incubating sections of rat vastus lateralis muscle in a medium containing collagen polypeptides. There are various advantages of collagen polypeptides over the conventional use of gelatin, particularly its effectiveness in preventing diffusion of creatine kinase during incubation.     

Celloidin as a protective film for improving the histochemical localization of succinate dehydrogenase

Synopsis The effect on the localization of succinate dehydrogenase of coating fresh and frozen-dried cryostat sections of unfixed hamster liver with celloidin was examined. It was found that the protective celloidin film not only leads to a more discrete localization of the enzyme, but is also prevents high losses of nitrogenous materials from sections into the incubation medium, a 30% loss in contrast to the 70% with fresh, non-coated sections. Further, after incubation, the morphology of the coated sections is much better than the uncoated ones.     

A tetrazolium technique for the histochemical localization of ATP: Creatine phosphotransferase

Summary A tetrazolium technique is presented that permits the study of ATP: Creatine phosphotransferase, or creatine kinase, in fixed skeletal muscle tissue sections, within the limits imposed by the properties of the chosen ditetrazole, nitro blue tatrazolium. There is a variation in creatine kinase activity between the muscle fibres. Those with high creatine kinase activity also have high succinate dehydrogenase activity.List of Abbreviations ADP Adenosine-5-diphosphate - ATP adenosine-5-triphosphate - CK creatine kinase - G-6-P glucose-6-phosphate - G-6-P-DH glucose-6-phosphate dehydrogenase - HK hexokinase - NADP nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PMS phenazine methosulphate - SDH succinate dehydrogenase     

An electron microscopic histochemical investigation of the localization of creatine phosphokinase in heart cells

The results of an electron microscopic histochemical investigation performed in the current study indicate that in heart cells creatine phosphokinase is localized:(1) inside mitochondria on the cristae membranes, (2) on the membrane of the sarcoplasmic reticulum, (3) on myofbrils (and in cytoplasm), (4) on the plasma membrane of the cells, (5) on the membrane of the cell nuclei.     

Immunocytochemical localization of creatine kinase M in canine myocardial cells: most creatine kinase M is distributed in the A-band

The localization of creatine kinase (CK) M in canine myocardium was immunocytochemically studied by a direct immunoperoxidase method. Specific antiserum against CK-M was produced in rabbits immunized with canine CK-MM. An anti-CK-M Fab'-horseradish peroxidase conjugate was prepared by the maleimide method. Frozen sections prepared from fixed canine myocardium were stained with the conjugate and observed by light and electron microscopy. In light microscopy of longitudinal sections, CK-M showed a cross-striated pattern consisting of distinct broad and narrow brown bands. Immunoelectron microscopy revealed that the regions of the broad and narrow brown bands corresponded to the A-band and the Z-line, respectively. Most CK-M in the A-band was associated with the thick fibers, and a small amount of CK-M was found in the M-line. These findings suggest that ATP regeneration from the ADP produced by myosin ATPase is related to the participation of this CK associated with the thick fibers rather than that of the M-line-bound CK. Creatine kinase M was also found in the sarcolemmal membrane, the membranes of the sarcoplasmic reticulum, and the mitochondrial outer and inner membranes. This report provides new information for understanding the physiological role of the phosphorylcreatine shuttle in the myocardial energy transport system.     

An improved glyoxylic acid technique for the histochemical localization of catecholamines in brown adipose tissue

Summary A glyoxylic acid method using cryostat sections to demonstrate catecholaminergic fibres of the central nervous system was modified to show the extent of the adrenergic innervation in rat brown adipose tissue. It revealed prominent interlacing fluorescent parenchymal fibres surrounding individual adipocytes. The density of this network of fine fibres was not evident using earlier techniques. The new method also confirmed the dense networks of adrenergic fibres associated with arterial vessels. Its specificity was verified by simultaneously performing radioenzymatic determinations of tissue catecholamine levels and histochemical studies of brown adipose tissue from normal and sympathectomized rats. Chemical sympathectomy with 6-hydroxydopamine resulted in a pronounced decrease in brown adipose tissue and heart catecholamine (noradrenalin and dopamine) levels. Significantly, in brown adipose tissue of sympathectomized animals no fluorescence could be detected in terminal nerves of either the parenchyma or those of vascular smooth muscles. Nevertheless, some intense fluorescence was seen in axon bundles. The findings suggest that catecholamines of the parenchymal innervation form a larger proportion of the total catecholamine content of brown adipose tissue than was previously believed, provide stronger support for direct control of the function of multilocular adipocytes, and also confirm unpublished data reporting considerable dopamine content in brown adipose tissue.     

Regulation of brain-type creatine kinase by AMP-activated protein kinase: Interaction,phosphorylation and ER localization

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.     

Creatine kinase,a histochemical study by the gelatin film-lead precipitation technique

Summary A modification of the method of Hori (1966) has permitted the precise histochemical localization of a portion of the creatine kinase activity in muscle. ATP produced by the action of this enzyme is hydrolysed by adjacent ATPase to inorganic phosphate. This is precipitated as lead phosphate, and then visualised by conversion to brown lead sulphide. The application of the incubation medium in the form of a viscous gelatin film has permitted localization of the lead sulphide in a clearly defined net-like pattern throughout the sarcoplasm. The results of this study are discussed in terms of the role of creatine kinase in the sarcoplasmic reticulum.     

Native mitochondrial creatine kinase forms octameric structures. I. Isolation of two interconvertible mitochondrial creatine kinase forms, dimeric and octameric mitochondrial creatine kinase: characterization, localization, and structure-function relationships

The mitochondrial isoform of creatine kinase (Mi-CK, EC 2.7.3.2) purified to homogeneity from chicken cardiac muscle by the mild and efficient technique described in this article was greater than or equal to 99.5% pure and consisted of greater than or equal to 95% of a distinct, octameric Mi-CK protein species, with a Mr of 364,000 +/- 30,000 and an apparent subunit Mr of 42,000. The remaining 5% were dimeric Mi-CK with an apparent Mr of 86,000 +/- 8,000. Octamerization was not due to covalent linkages or intermolecular disulfide bonding. Upon dilution into buffers of low ionic strength and alkaline pH, octameric Mi-CK slowly dissociated in a time-dependent manner (weeks-months) into dimeric Mi-CK. However, the time scale of dimerization was reduced to minutes by the addition to diluted Mi-CK octamers of a mixture of Mg2+, ADP, creatine and nitrate known to induce a transition-state analogue complex (Milner-White, E.J., and Watts, D. C. (1971) Biochem. J. 122, 727-740). The conversion was fully reversible, and octamers were reformed by simple concentrations of Mi-CK dimer solutions to greater than or equal to 1 mg/ml at near neutral pH and physiological salt concentrations in the absence of adenine nucleotide. After separation of the two Mi-CK species by gel filtration, electron microscopic analysis revealed uniform square-shaped particles with a central negative-stain-filled cavity in the octamer fractions and "banana-shaped" structures in the dimer fractions. Mi-CK was localized inside the mitochondria by immunogold labeling with polyclonal antibodies. A dynamic model of the octamer-dimer equilibrium of Mi-CK and the preferential association of the octameric Mi-CK form with the inner mitochondrial membrane is discussed in the context of regulation of Mi-CK activity, mitochondrial respiration, and the CP shuttle.     

Aggregation of creatine kinase during refolding and chaperonin-mediated folding of creatine kinase

The course of refolding and reactivation of urea-denatured creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) has been studied in the absence and presence of molecular chaperonin GroEL. The enzyme was denatured in Tris--HCl buffer containing 6 M urea for 1 h. In the refolding studies, the denatured enzyme was diluted 60-fold into the same buffer containing GroEL or not for activity, turbidity, fluorescence measurements and polyacrylamide gel electrophoresis. The results show that the reactivation process is dependent of creatine kinase concentration in the concentration range 2.5--4 microM. The levels of activity recovery decrease with increasing enzyme concentration because of the formation of wrong aggregates. The molecular chaperonin GroEL can bind the refolding intermediate of creatine kinase and thus prevent the formation of wrong aggregates. This intermediate is an inactive dimeric form that is in a conformation resembling the 'molten globule' state.     

Subcellular localization of creatine kinase in Torpedo electrocytes: association with acetylcholine receptor-rich membranes

Creatine kinase (CK, EC 2.7.3.2) has recently been identified as the intermediate isoelectric point species (pl 6.5-6.8) of the Mr 40,000-43,000 nonreceptor, peripheral v-proteins in Torpedo marmorata acetylcholine receptor-rich membranes (Barrantes, F. J., G. Mieskes, and T. Wallimann, 1983, Proc. Natl. Acad. Sci. USA, 80: 5440-5444). In the present study, this finding is substantiated at the cellular and subcellular level of the T. marmorata electric organ by immunofluorescence and by protein A-gold labeling of either ultrathin cryosections of electrocytes or purified receptor-membrane vesicles that use subunit-specific anti-chicken creatine kinase antibodies. The muscle form of the kinase, on the one hand, is present throughout the entire T. marmorata electrocyte except in the nuclei. The brain form of the kinase, on the other hand, is predominantly located on the ventral, innervated face of the electrocyte, where it is closely associated with both surfaces of the postsynaptic membrane, and secondarily in the synaptic vesicles at the presynaptic terminal. Labeling of the noninnervated dorsal membrane is observed at the invaginated sac system. In the case of purified acetylcholine receptor-rich membranes, antibodies specific for chicken B-CK label only one face of the isolated vesicles. No immunoreaction is observed with anti-chicken M-CK antibodies. A discussion follows on the possible implications of these localizations of creatine kinase in connection with the function of the acetylcholine receptor at the postsynaptic membrane, the Na/K ATPase at the dorsal electrocyte membrane, and the ATP-dependent transmitter release at the nerve ending.     

Origin of the genes for the isoforms of creatine kinase

Creatine kinase (CK) is a member of a family of phosphoryl transfer enzymes called phosphagen (guanidino) kinases which play a central role in cellular energy homeostasis. There are three CK isoform gene groups, each coding for proteins targeted to different intracellular compartments--cytoplasmic (CytCK), mitochondrial (MtCK) and flagellar (FlgCK). The former two CKs are either dimeric or octameric while FlgCKs are contiguous trimers consisting of three fused, complete CK domains. Conventional wisdom supports the view that CKs evolved from a cytoplasmic, monomeric ancestral protein closely related to a phosphagen kinase homologue, arginine kinase (AK). Recently, it has been shown that a demosponge (Phylum Porifera) expresses a true MtCK and two dimeric, protoflagellar CKs (protoflgCK) with great similarity to FlgCKs. To further probe the early evolution of CK, we have obtained additional sequences for Mt- and protoflgCKs from two more demosponges and from three hexactinellid (glass) sponges as well as an MtCK sequence from a basal metazoan cnidarian. Phylogenetic analyses using Maximum Likelihood (ML) of these new CK sequences with other CKs and phosphagen kinases yielded a consensus tree containing an assemblage of MtCKs and a supercluster consisting of protoflg-, Flg- and CytCKs. The MtCKs appear basal in the tree topology consistent with prior results. Within the protoflg-, Flg- and CytCK supercluster, the protoflgCKs appear to be allied to the domains of the FlgCKs, although the support is not robust. PCR amplification of genomic DNA and sequencing of the genes for Mt- and protoflgCK from the demosponge Suberites fuscus showed that the sponge MtCK shares four-five common intron:exon boundaries with invertebrate, protochordate and vertebrate MtCKs supporting a common ancestry and the extreme conservation of intron:exon organization in MtCK genes. The protoflgCK gene organization was highly divergent in relation to other CK genes but shares a common intron:exon boundary with domain 2 of the gene for the FlgCK from the tunicate Ciona intestinalis, providing support for the linkage of the protoflgCKs with the FlgCKs. Our results show that the two, major CK gene lineages are present in arguably the oldest, extant metazoan group, the hexactinellid sponges, indicating that these two genes are ancient and confirming prior work that the MtCK gene is likely basal and ancestral.     

A coupled peroxidatic oxidation technique for the histochemical localization of monoamine oxidase A and B and benzylamine oxidase

Summary A coupled peroxidatic oxidation technique is presented which employs benzylamine and tyramine as substrates and clorgyline, deprenyl, phenelzine and pargyline as specific inhibitors. Using this technique with frozen sections of human term placenta and rat liver, the histochemical localization of monoamine oxidase A and B and benzylamine oxidase has been demonstrated.     

Velocity of the creatine kinase reaction in the neonatal rabbit heart: role of mitochondrial creatine kinase

To examine the role of changes in the distribution of the creatine kinase (CK) isoenzymes [BB, MB, MM, and mitochondrial CK (mito-CK)] on the creatine kinase reaction velocity in the intact heart, we measured the creatine kinase reaction velocity and substrate concentrations in hearts from neonatal rabbits at different stages of development. Between 3 and 18 days postpartum, total creatine kinase activity did not change, but the isoenzyme distribution and total creatine content changed. Hearts containing 0, 4, or 9% mito-CK activity were studied at three levels of cardiac performance: KCl arrest and Langendorff and isovolumic beating. The creatine kinase reaction velocity in the direction of MgATP production was measured with 31P magnetization transfer under steady-state conditions. Substrate concentrations were measured with 31P NMR (ATP and creatine phosphate) and conventional biochemical analysis (creatine) or estimated (ADP) by assuming creatine kinase equilibrium. The rate of ATP synthesis by oxidative phosphorylation was estimated with oxygen consumption measurements. These results define three relationships. First, the creatine kinase reaction velocity increased as mito-CK activity increased, suggesting that isoenzyme localization can alter reaction velocity. Second, the reaction velocity increased as the rate of ATP synthesis increased. Third, as predicted by the rate equation, reaction velocity increased with the 3-fold increase in creatine and creatine phosphate contents that occurred during development.