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In sperm of the sea urchin Strongylocentrotus purpuratus, a functional phosphocreatine shuttle, that requires the existence of mitochondrial and cytosolic creatine kinase (CK) isoforms in distinct locations, is essential for sperm motility. S. purpuratus sperm have an unusually large, 145 kDa CK isoform, present exclusively in the sperm tail (TCK), that is enriched in flagellum membrane preparations. Purified TCK contains two very similar proteins, designated TCKI and TCKII, of which only TCKII associates readily with liposomes and detergent micelles in vitro. Here we demonstrate by gas chromatography/mass spectrometry combined with selective ion monitoring that ions diagnostic for the presence of myristoylglycine in proteins are found in TCKII, but not TCKI. By contrast, TCKI, but not TCKII, served in vitro as a substrate for recombinant, polyhistidine-tagged N-myristoyltransferase and was myristoylated to high stoichiometries (0.58 +/- 0.14 pmol of myristate/pmol of TCK), in the presence of myristoyl-CoA, on glycine in amide linkage. In vitro myristoylated TCKI associated with phosphatidylcholine (PC)/phosphatidylserine (PS) (75:25) liposomes and Triton X-100 detergent micelles in gel filtration assays and with PC/PS liposomes in a centrifugation assay in the same manner as did TCKII. In gel filtration experiments, TCKI required at least 25-fold higher PC/PS liposome concentrations than TCKII to obtain 50% association. A partition coefficient of 0.8 x 10(5) M(-1) was determined for TCKII with PC/PS (75:25) liposomes in the centrifugation assay. Thus, myristoylated and nonmyristoylated forms of TCK exist side-by-side in the sea urchin flagellum, and myristoylation is essential for efficient liposome association of TCK.

Different isoforms of creatine kinase, an important enzyme of vertebrate energy metabolism, were localized in bovine photoreceptor cells, with particular emphasis on the identification and quantification of the brain-type isoform within the outer segment compartment. Using immunofluorescence and immunoelectron microscopy, brain-type creatine kinase was shown to be present in bovine photoreceptor cell outer and inner segments. The presence of this isoenzyme in rod outer segments was additionally confirmed by immunoblotting and immunolabeling of isolated rod outer segments. The content of creatine kinase in rod outer segments was quantified by measuring creatine kinase activity after membrane disruption with detergent. The ATP regeneration potential provided by the creatine kinase in isolated, washed bovine rod outer segments was 1.2 +/- (0.4) i.u. mg-1 rhodopsin. This value was calculated to be at least an order of magnitude larger than that necessary to replenish the energy required for cGMP resynthesis in rod outer segments, and high enough to regenerate the entire ATP pool of rod outer segments within the time span of a photic cycle. A mitochondrial creatine kinase isoenzyme was located within the ellipsoid portions of bovine rod and cone inner segments by immunofluorescence microscopy and, using immunogold staining, was specifically localized in the mitochondria clustered within bovine rod and cone inner segments. These results suggest that vertebrate photoreceptor cells contain a functional phosphocreatine circuit. Outer segment creatine kinase may play an important role in phototransduction by providing energy for the visual cycle, maintaining high local ATP/ADP ratios and consuming protons produced by enzymes located in the outer segment.

TCK, the flagellar creatine kinase (ATP:creatine N-phosphotransferase) of sperm from the sea urchin Strongylocentrotus purpuratus is a membrane-associated lipophilic protein involved in energy transport. The cDNA derived protein sequence contains a consensus site sufficient for the covalent attachment of myristate. To examine whether TCK was myristoylated, mouse fibroblast Swiss 3T3 and baby hamster kidney cell lines were transfected with a cDNA encoding the entire TCK protein linked to a metallothionein promotor. TCK expression was induced by zinc and paralleled by incorporation of [3H]myristic acid derived label into the protein. 3H Label incorporated into TCK was resistant to hydroxylamine treatment. The 3H-labeled material released from TCK by acid methanolysis eluted from a C18 reverse phase high pressure liquid chromatography column at the positions of myristic acid and methylmyristate. Thus, TCK expressed in transfected mammalian cell lines contains authentic myristic acid, covalently attached through amide linkage. [3H]Myristoyl TCK comigrated on two-dimensional gels with the purified lipophilic isoform TCK II from sea urchins. Furthermore, like TCK II, [3H]myristoyl TCK associated with phospholipid liposomes, suggesting that myristoylation may mediate the observed membrane association of TCK. Myristoylation of sea urchin sperm flagellar creatine kinase may play a role in confining this enzyme to the flagellum during spermatogenesis.

The flagellar creatine kinase (TCK) of Strongylocentrotus purpuratus sperm is both a principal component of sperm tail membrane preparations and a cytosolic enzyme. An improved purification scheme identified three pools of TCK, termed TCK I, TCK II, and TCK III. TCK I and II were essentially homogeneous protein preparations, while TCK III was heavily contaminated with other flagellar proteins, predominantly guanylate cyclase, and alpha- and beta-tubulin. The three TCK species are roughly present in a 1:10:1 ratio as assessed by activity measurements. TCK I and II are similar proteins as shown by two-dimensional gel electrophoresis, partial proteolytic fragmentation, and cellulose polyacetate electrophoresis and have the same pH-dependent specific activity. However, they are functionally distinct with respect to their capacity to associate with lipids. TCK II associated readily with phospholipid liposomes and detergent micelles, while TCK I did not. Association of TCK II was as a protein monomer with an apparent Kd of approximately 1-2 mM at a 10(4):1 lipid or detergent to protein ratio. Whereas the Kd estimates were pH independent, the rate of association increased 2-3-fold between pH 6.5 and 8. The data are consistent with membrane-association of TCK II being a two-step process, involving a pH-dependent, intramolecular, TCK-specific step and a charge-facilitated, but pH-independent, membrane association step. Membrane association of TCK may, together with microtubule association (Tombes, R.M., Farr, A., and Shapiro, B.M. (1988) Exp. Cell Res. 178, 307-317) represent a mechanism required for specific accumulation of the enzyme within the flagellum.

In addition to the two monomer subunits of chicken brain-type creatine kinase (B-CK, EC, 2.7.3.2), termed Bb (basic) and Ba (acidic), another subspecies called Bb* was identified by chromatofocussing in the presence of 8 M urea (Quest et al., ). The latter low abundance protein species, isolated from tissue extracts, comigrated on 2D-gels with three minor species (Bb1-3), initially identified in immunoprecipitated, [35S]methionine labeled in vitro translation products of cDNA coding for the basic monomer Bb. During in vitro translation experiments in the presence of [32P]-gamma-ATP, Bb1-3 were labeled while phosphatase treatment eliminated these minor species. It is concluded that Bb* is identical to Bb1-3 and represents phosphorylated derivatives of Bb. B-CK dimer populations from different tissues were separated by ion-exchange chromatography and the Km values of the resulting fractions were determined under phospho-creatine (CP)-limiting conditions. In fractions containing only Bb and Bb* two kinetically different enzyme species were detected (Km values for CP = 1.6 mM and 0.8 mM), while fractions containing B-CK dimers composed of the major Ba and Bb monomers, but no Bb*, were homogeneous in this respect (Km for CP = 1.6 mM). Phosphorylation of Bb to yield Bb* is concluded to reduce the Km of B-CK dimers for CP by about 50%. This Km shift is within the range of CP concentrations found in tissues expressing the B-CK isoform and may therefore be of physiological relevance.

Brain-type creatine kinase B-CK (EC 2.7.3.2) was purified from several chicken tissues, e.g. cardiac muscle, brain, gizzard and retina. Two major monomeric chicken B-CK subunits, designated Bb (basic) and Ba (acidic), which differ in isoelectric point, were separated by chromatofocusing in the presence of 8 M urea on a MonoP column. The two subunits were shown by peptide mapping, amino acid analysis and partial sequencing, as well as by immunological criteria, to be distinct B-CK polypeptides. The N-terminal sequence of 30 amino acid residues of Bb correspond entirely to data derived from a B-CK c-DNA clone termed H4 [(1986) Nucleic Acids Res. 14, 1449-1463], whereas the N-terminus of the acidic Ba species was blocked. Native dimeric B-CK isoenzymes obtained from these tissues were separated by ion exchange chromatography on a MonoQ column yielding two B-CK dimer populations, type-I and type-II B-CK, varying in relative proportions. Quantitation of the CK activity peak ratios of these two populations revealed the existence of a tissue-specific, post-translational mechanism regulating B-CK dimerization in neural tissues. Tissue-specific dimerization of the two distinct B-CK monomer species may represent a means of specifying the intracellular distribution of the dimeric B-CK subspecies.

A method for the purification of brain-type creatine kinase (B-CK) from several tissues of the chicken, e.g., brain, retina, gizzard and heart was developed involving (1) an affinity chromatography step on Sepharose Blue from which B-CK was specifically eluted by ADP and (2) a subsequent anion exchange chromatography step on a fast protein liquid chromatography Mono-Q column. Two distinct peaks with B-CK activity, both purified to greater than or equal to 99% homogeneity and displaying specific enzyme activities of 300-400 mumol CP/min/mg 1t pH 7.0 and 25 degrees C, were eluted by a salt gradient at a plateau of 150 mmol/l NaCl. The ratio of the two B-CK peaks varied in a tissue-dependent manner, indicating that in chicken the dimerization of native BB-CK from the two major B-CK subunit species is tissue-specific and nonrandom in neural tissues. The fast, efficient and convenient method for the purification of B-CK at small or large scale, operating at yields of 50-70%, makes the purification of this rather labile enzyme from small amounts of tissues possible and greatly facilitates the subsequent characterization of both major and minor dimeric BB-CK subspecies present in these different tissues.