Sea urchin sperm creatine kinase: The flagellar isozyme is a microtubule-associated protein

Sea urchin sperm contain two isozymes of creatine kinase (CrK) in the sperm head and tail, as termini of a phosphocreatine shuttle to transport energy. The head isozyme is located at the mitochondrion. By using an antibody prepared against denatured flagellar CrK, we now show that the tail isozyme exists along the entire flagellum. This unusual CrK isozyme, of Mr 145 kDa, is a component of the flagellar axoneme as indicated by electron microscopic immunolocalization and cell fractionation. Flagellar CrK specifically reassociated with extracted sperm axonemes as well as with in vitro polymerized sea urchin egg microtubules. Neither sperm mitochondrial CrK nor mammalian muscle CrK bound to axonemes under similar conditions. Thus, although the two sperm isozymes have similar kinetic properties, they differ in affinity for microtubules, a characteristic that may determine the regional differentiation needed for establishing a phosphocreatine shuttle.     

Enzyme termini of a phosphocreatine shuttle. Purification and characterization of two creatine kinase isozymes from sea urchin sperm

Two isozymes of creatine kinase have been purified from sperm of the sea urchin, Strongylocentrotus purpuratus. One isozyme was purified from the sperm flagellum, and the other from the head. Both require nonionic detergent for extraction from sperm. The flagellar isozyme is a monomeric species with an Mr of 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126,000 from sucrose density gradient and gel filtration analyses. Creatine kinase from sperm heads was localized to the mitochondrion by an antibody raised against mouse muscle creatine kinase. This purified mitochondrial isozyme is multimeric, with an Mr of 47,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but 240,000 for the native enzyme. Peptide mapping indicates that the two isozymes are not related. The following kinetic characteristics were observed for the purified flagellar and mitochondrial isozymes, respectively. In the direction of ATP formation, at pH 6.6 and 25 degrees C, specific activities were 235 and 180 units/mg; pH optima were 6.7 and 6.9 and Michaelis constants were 0.13 and 0.055 mM for ADP and 5.8 and 2.7 mM for phosphocreatine. In the direction of phosphocreatine formation, at pH 7.5 and 25 degrees C, specific activities were 29 and 47 units/mg; pH optima were 7.5 and 7.7 and Michaelis constants were 0.89 and 0.31 mM for ATP and 39 and 62 mM for creatine. These unique isozymes constitute the termini of the phosphocreatine shuttle of sea urchin sperm that is responsible for energy transport from the mitochondrion to the distal flagellum (Tombes, R. M., and Shapiro, B. M. (1985) Cell 41, 325-334; Tombes, R. M., Brokaw, C. J., and Shapiro, B. M. (1987) Biophys. J., 52, 75-86).     

Creatine kinase-dependent energy transport in sea urchin spermatozoa. Flagellar wave attenuation and theoretical analysis of high energy phosphate diffusion.

The significance of a phosphocreatine (PCr) shuttle in the energy transport of motile spermatozoa (Tombes, R. M., and B. M. Shapiro, 1985, Cell, 41:325-334) has been tested by a quantitative analysis of motility. Computer-assisted analysis of stroboscopic photomicrographs of live sea urchin spermatozoa whose creatine kinase has been specifically inhibited by fluorodinitrobenzene reveals that motility is impaired due to a progressive damping of bending waves as they propagate along the flagellum. This lesion, which has been defined as attenuation and can be quantified, is repaired when these spermatozoa are demembranated and reactivated to swim with ATP. The implication that attenuation is due to the inhibition of energy transport via a PCr shuttle resulting in the decrease of ATP and accumulation of inhibitory levels of ADP distally has been supported by calculating sperm PCr and ATP levels resulting from diffusion along the flagellum. The specific alterations of motility seen with creatine kinase inhibition and their reversal with ATP are as expected from the model and provide strong support for the PCr shuttle in high energy phosphate transport.     

A sea urchin sperm flagellar adenylate kinase with triplicated catalytic domains

The mitochondrion of sea urchin sperm is located at the base of the sperm head, and the flagellum extends from the mitochondrion for approximately 40 microM. These sperm have two known flagellar, non-mitochondrial, enzymatic systems to rephosphorylate ADP. The first involves the phosphocreatine shuttle, where flagellar creatine kinase (Sp-CK) uses phosphocreatine to rephosphorylate ADP. The second system, studied in this report, is adenylate kinase (Sp-AK), which uses 2 ADP to make ATP + AMP. Cloning of Sp-AK shows that, like Sp-CK, Sp-AK has three catalytic domains. Sp-AK localizes along the entire flagellum, and most of it is tightly bound to the axoneme. Sp-AK activity and flagellar motility were studied using demembranated sperm. The specific Sp-AK inhibitor Ap5A blocks enzyme activity with an IC50 of 0.41 microM. In 1 mm ADP, flagella reactivate motility in 5 min; 1 microM Ap5A completely inhibits this reactivation. No inhibition of motility occurs in Ap5A when 1 mm ATP is added to the reactivation buffer. The pH optimum for Sp-AK is 7.7, an internal pH at which sperm are fully motile. The pH optimum for Sp-CK is 6.7, an internal pH at which sperm are immotile. In isolated, detergent-permeabilized flagella, assayed at pH 7.6, the Km for Sp-AK is 0.32 mm and the Vmax is 2.80 microM ATP formed/min/mg of protein. When assayed at pH 7.6, the Sp-CK Km is 0.25 mm and the Vmax 5.25. At the measured in vivo concentrations of ADP of 114 microM, at pH 7.6, the axonemal Sp-AK could contribute approximately 31%, and Sp-CK 69%, of the total non-mitochondrial ATP synthesis associated with the demembranated axoneme. Thus, Sp-AK could contribute substantially to ATP synthesis utilized for motility. Alternatively, Sp-AK could function in the removal of ADP, which is a potent inhibitor of dynein ATPase.     

Creatine ethyl ester: a new substrate for creatine kinase

The creatine kinase/phosphocreatine system plays a key role in cell energy buffering and transport, particularly in cells with high or fluctuating energy requirements, like neurons, i.e. it participates in the energetic metabolism of the brain. Creatine depletion causes several nervous system diseases, alleviated by phosphagen supplementation. Often, the supplementation contains both creatine and creatine ethyl ester, known to improve the effect of creatine through an unknown mechanism. In this work we showed that purified creatine kinase is able to phosphorilate the creatine ethyl ester. The K(m) and V(max) values, as well as temperature and pH optima were determined. Conversion of the creatine ethyl ester into its phosphorylated derivative, sheds light on the role of the creatine ethyl ester as an energy source in supplementation for selected individuals.     

Ultrastructure of the sperm of blue sprat, Spratelloides gracilis; Teleostei, Clupeiformes, Clupeidae

The general sperm structure of the investigated clupeoids possess an oliviform head with a distinct deep nuclear fossa, a midpiece with one mitochondrion and a posterior flagellum. They are characterized by two apomorphies: an annular or C-shaped mitochondrion and ITDs (intratubular differentiation) in the A tubules of the axonemal doublets. The fine structure of the spermatozoa of blue sprat, Spratelloides gracilis, was investigated to see if resulting data conformed to the current hypotheses of the taxonomy of the Clupeidae. The mature sperm is characterized by the following features: (1) the nucleus is oliviform; its prominent deep nuclear fossa encloses the initial portion of flagellum, (2) a proximal centriole has not been identified, (3) a single spherical mitochondrion is located laterally in relation to the flagellum and (4) no cytoplasmic canal is present. Our blue sprat-spermatozoan morphology data suggest that the blue sprat has Clupeomorpha affinities, while indicating no close affinity with Elopomorpha. Clupeoid sperm exhibit morphological variations with the nucleus and mitochondrion being particularly variable. This study provides useful systematic characters to the existing knowledge of comparative spermatology and may provide additional clue to Euteleost phylogeny.     

An ultracytochemical study of the respiratory potency, integrity, and fate of the sea urchin sperm mitochondria during early embryogenesis

Cytochrome oxidase activity via cytochrome c, as demonstrated by the diaminobenzidine procedure, has been employed in this electron microscope cytochemical study to determine the respiratory potency, integrity and fate of the Arbacia sperm mitochondrion at fertilization and during early embryogenesis. The sperm mitochondrion remained intact and was intensely positive for cytochrome oxidase activity both during and after penetration into the egg. The mitochondrion remained highly reactive throughout zygote formation, up to the eight-cell stage. The sperm mitochondrion formed many projections and buds in the cytoplasm of immature oocytes, monospermic and polyspermic eggs, and in blastomeres. At all stages of early embryogenesis, close juxtaposition and structural contact were observed between the highly reactive sperm mitochondrion and the less reactive egg mitochondria. The results suggest that following fertilization the mitochondrion of the sea urchin spermatozoon retains some degree of metabolic autonomy within the ooplasm. The structural integrity of the paternal mitochondrion is maintained along with a functional respiratory enzyme system (cytochrome c-a3). The hypothesis that the fertilizing sperm mitochondrion may have some relevance to sea urchin development is discussed.     

Comparison of the two different phosphagen systems in the lugworm Arenicola marina

The different phosphagen systems in the lugworm Arenicola marina, the phosphotaurocyamine/taurocyamine kinase system of the body wall and the phosphocreatine/creatine kinase system of the spermatozoa, have been investigated to answer the question whether the change reflects different functional modes of these phosphagen systems. Enzyme analyses have shown that in contrast to the body wall taurocyamine kinase, creatine kinase of spermatozoa exists in at least two different forms which are compartmented in the mitochondria (creatine kinase I) and in the flagellum (creatine kinase II). Creatine kinase I is strongly attached to cell structures which require detergents and high phosphate concentrations for solubilization. The affinities of taurocyamine kinase and creatine kinase for all substrates are very similar except the extremely high K _m for creatine of both creatine kinase I and II. The level of creatine in spermatozoa is fivefold higher than taurocyamine in the body wall at similar phosphorylation potential (ATP/ADO_free) and ATP-buffer capacity (phosphagen/ATP), reflecting the higher equilibrium constants of the creatine kinase reaction compared to that of the taurocyamine kinase reaction (Ellington 1989). The high creatine concentration gives the phosphocreatine/creatine kinase system an advantage over the phosphotaurocyamine/taurocyamine kinase system for transport of energyrich phosphate at high phosphorylation potential by increasing the radial diffusion flux. The maximum diffusive flux of free ADP in spermatozoa is three orders of magnitude below the respiratory ATP production while the creatine flux would allow an unlimited energy transport over the long diffusion distance. In lugworm body wall, however, the low ATP turnover and the low diffusion distances between mitochondria and myosin-ATPases do not require a phosphagen shuttle.Abbreviations ADP _free cytoplasmic adenosine diphosphate - Ap ₅ A P₁, P₅-di(adenosine-5-) pentaphosphate - AK arginine kinase - CK creatine kinase (EC 2.7.3.2) - DTT dithiothreitol - GAPDH glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) - HOADH 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) - IEP isoelectric point - MIM mitochondria isolating medium - P _i-free cytoplasmic inorganic phosphate - (P)Arg (phospho)arginine - (P)Cr (phospho)creatine - (P)Tc (phospho)taurocyamine - SEM scanning electron microscopy - TK taurocyamine kinase - TEM transmission electron microscopy     

Morphologically Specialized Sperm from the Ovary of Isometra vivipara (Echinodermata — Crinoidea)

The morphology of the sperm of Isometra vivipara is markedly different from the spherical-headed sperm typical of crinoids in general. The sperm head of Isometra has a flattened, oval shape. In addition to a nucleus, the head includes a lateral acrosome on one flat surface and a lateral mitochondrion on the opposite flat surface. From the latter surface of the sperm head, a tail flagellum arises adjacent to the mitochondrion. The discussion considers the relationships between reproductive habits and sperm morphology in Isometra and other crinoids.     

Fertilization induced changes in sea urchin sperm: mitochondrial deformation and phosphatidylserine exposure

This study demonstrates that the single mitochondrion of the sea urchin sperm undergoes a shape change at fertilization that is linked to respiration. The mitochondrion swells and shifts to the lateral side of the sperm head on contact with the homologous egg jelly or egg surface; Mg(2+)- or Na(+)-free seawater or respiratory inhibitors also induce this change. During the mitochondrial deformation, the sperm decreases the rate of oxygen consumption and their redox-state of cytochromes is disrupted b-c(1)/c. Simultaneously, the adenine nucleotides content changes precipitously. This suggests that mitochondrial morphology is strongly associated with respiratory activities in the sea urchin sperm. These changes in mitochondrial morphology and function are similar to the mitochondrial changes in apoptotic cells such as swelling, decrease in its membrane potential, and release of cytochrome c. In apoptotic cells, the exposure of phosphatidylserine from the inner to outer leaflet of the plasma membrane is one of prominence phenomena. This change was visualized by staining the sea urchin sperm with Annexin V-Fluorescein. It is possible that mitochondrial deformation is an initial sign of sperm destruction, which like as apoptotic cells.     

The sperm of Hexagenia (Pseudeatonica) albivitta Walker (Ephemeroptera: Fossoriae: Ephemeridae)

This study describes the sperm morphology of the mayfly Hexagenia (Pseudeatonica) albivitta (Ephemeroptera). Its spermatozoon measures approximately 30 μm of which 9 μm corresponds to the head. The head is composed of an approximately round acrosomal vesicle and a cylindrical nucleus. The nucleus has two concavities, one in the anterior tip, where the acrosomal vesicle is inserted and a deeper one at its base, where the flagellum components are inserted. The flagellum is composed of an axoneme, a mitochondrion and a dense rod adjacent to the mitochondrion. A centriolar adjunct is also observed surrounding the axoneme in the initial portion of the flagellum and extends along the flagellum for at least 2 μm, surrounding the axoneme in a half‐moon shape. The axoneme is the longest component of the flagellum, and it follows the 9+9+0 pattern, with no central pair of microtubules. At the posterior region of the flagellum, the mitochondrion has a dumb‐bell shape in cross sections that, together with the rectangular mitochondrial‐associated rod, is responsible for the flattened shape of the flagellum. An internal membrane is observed surrounding both mitochondrion and its associated structure.     

Creatine ethyl ester: A new substrate for creatine kinase

The creatine kinase/phosphocreatine system plays a key role in cell energy buffering and transport, particularly in cells with high or fluctuating energy requirements, like neurons, i.e. it participates in the energetic metabolism of the brain. Creatine depletion causes several nervous system diseases, alleviated by phosphagen supplementation. Often, the supplementation contains both creatine and creatine ethyl ester, known to improve the effect of creatine through an unknown mechanism. In this work we showed that purified creatine kinase is able to phosphorilate the creatine ethyl ester. The K _m and V _max values, as well as temperature and pH optima were determined. Conversion of the creatine ethyl ester into its phosphorylated derivative, sheds light on the role of the creatine ethyl ester as an energy source in supplementation for selected individuals.     

Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Adenylyl cyclases (ACs) synthesize cAMP and are present in cells as transmembrane AC and soluble AC (sAC). In sperm, the cAMP produced regulates ion channels and it also activates protein kinase-A that in turn phosphorylates specific axonemal proteins to activate flagellar motility. In mammalian sperm, sAC localizes to the midpiece of flagella, whereas in sea urchin sperm sAC is along the entire flagellum. Here we show that in sea urchin sperm, sAC is complexed with proteins of the plasma membrane and axoneme. Immunoprecipitation shows that a minimum of 10 proteins is tightly associated with sAC. Mass spectrometry of peptides derived from these proteins shows them to be: axonemal dynein heavy chains 7 and 9, sperm specific Na+/H+ exchanger, cyclic nucleotide-gated ion channel, sperm specific creatine kinase, membrane bound guanylyl cyclase, cyclic GMP specific phosphodiesterase 5A, the receptor for the egg peptide speract, and alpha- and beta-tubulins. The sAC-associated proteins could be important in linking membrane signal transduction to energy utilisation in the regulation of flagellar motility.     

Properties of pyruvate kinase and flagellar ATPase in rabbit spermatozoa: relation to metabolic strategy of the sperm cell

Rabbit sperm pyruvate kinase remains bound to the cell structure of hypotonically treated mature rabbit epididymal spermatozoa (HTRES). It displays kinetic behavior very similar to that of rabbit muscle pyruvate kinase with regard to KM values for substrates, activation by monovalent and divalent cations, inhibition by phenylalanine which is reversed by alanine, and lack of activation by fructose-1,6-biphosphate. The flagellar ATPase also remains bound to the cell structure of HTRES, whose motility may be reactivated by a source of ATP. It requires Mg+2 for activity; the KM for both ATP and MG+2 is 0.2 mM, implying that MgATP is the substrate. The ATPase activity is not inhibited by ouabain, oligomycin, or vanadate, which also do not affect reconstituted motility, and is not affected by cyclic AMP in the presence of an inhibitor of phosphodiesterase. The activities of pyruvate kinase and the flagellar ATPase in a given preparation of HTRES are comparable. Rabbit spermatozoa have a metabolic strategy which is very similar to muscle cells. This suggests that the major use of the sperm cell's metabolic machinery is maintenance of energy for the contractile work of motility and that only minor amounts of metabolic energy appear to be consumed in other reactions, including those involved in fertilization.     

Myristoylation of flagellar creatine kinase in the sperm phosphocreatine shuttle is linked to its membrane association properties.

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.     

Variation of phosphagens in sea urchin eggs before and after fertilization]

Improved methods of separation, identification and determination of phosphagens have been applied to the study of monosubstituted guanidines and phosphagenes in sea urchin eggs, before and after fertilization. We have been able not only to identify in these materials phosphoarginine, but also phosphocreatine and to detect an unknown phosphagen. After fertilization, free arginine and creatine decrease, within respect to unfertilized eggs, whereas phosphocreatine undergoes a large increase; phosphoarginine remains almost constant, and an unknown phosphagen appears. These observations allow to interprete the results of some authors, who, applyng less refined methods, found only an increase in phosphoarginine in the same materials.     

Asparagine 285 plays a key role in transition state stabilization in rabbit muscle creatine kinase

To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site-directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with the major cause of activity loss being a reduction in k(cat) in comparison to the recombinant native CK. The data for N285Q still fit a random-order, rapid-equilibrium mechanism, with either MgATP or creatine binding first with affinities very nearly equal to those for native CK. However, the affinity for the binding of the second substrate is reduced approximately 10-fold, suggesting that addition of a single methylene group at position 285 disrupts the symphony of substrate binding. The data for the N285A mutant only fit an ordered binding mechanism, with MgATP binding first. Isosteric replacement to form the N285D mutant has almost no effect on the K(M) values for either creatine or MgATP, thus the decrease in activity is due almost entirely to a 5000-fold reduction in k(cat). Using the quenching of the intrinsic CK tryptophan fluorescence by added MgADP (Borders et al. 2002), it was found that, unlike native CK, none of the mutants have the ability to form a quaternary TSAC. We use these data to propose that asparagine 285 indeed plays a key role in transition state stabilization in the reaction catalyzed by creatine kinase and other phosphagen kinases.     

Inverse metabolic engineering with phosphagen kinase systems improves the cellular energy state

Inverse metabolic engineering attempts to identify or construct desired phenotypes of applied interest to endow them on appropriate host organisms. A particular desirable phenotype is the ATP homeostasis exhibited by animal cells with high and variable ATP turnover through temporal and spatial energy buffering. This buffering is achieved by phosphagen kinase systems that consist of a specific kinase and its cognate phosphagen, which functions as a large pool of 'high-energy phosphates' that are used to replenish ATP during periods of high energetic demand. This review discusses recent advances and potentials of inverse metabolic engineering of cell types that do not normally contain such systems--bacteria, yeast, plants, and liver--with creatine or arginine kinase systems. Examples are discussed that illustrate how microbial metabolism can be tailored for large-scale industrial processes with imperfect mixing and how the liver can be protected from metabolic insults or stimulated for better regeneration.     

Tandem mass spectrometry identifies proteins phosphorylated by cyclic AMP-dependent protein kinase when sea urchin sperm undergo the acrosome reaction

The exocytotic acrosome reaction (AR), which is required for fertilization, occurs when sea urchin sperm contact the egg jelly (EJ) layer. Among other physiological changes, increases in adenylyl cyclase activity, cAMP and cAMP-dependent protein kinase (PKA) activity occur coincident with the AR. By using inhibitors of PKA, a permeable analog of cAMP and the phosphodiesterase inhibitor IBMX, we show that PKA activity is required for AR induction by EJ. A minimum of six sperm proteins are phosphorylated by PKA upon exposure to EJ, as detected by a PKA substrate-specific antibody. The phosphorylation of these proteins and the percentage of acrosome reacted sperm can be regulated by PKA modulators. The fucose sulfate polymer (FSP), a major component of EJ, is the molecule that triggers sperm PKA activation. Extracellular Ca(2+) is required for PKA activation. Six sperm proteins phosphorylated by PKA were identified by tandem mass spectrometry (MS/MS) utilizing the emerging sea urchin genome. Based on their identities and localizations in sperm head and flagellum, the putative functions of these proteins in sperm physiology and AR induction are discussed.     

Phosphagen kinase evolution. Expression in echinoderms

Arginine kinase and creatine kinase that catalyze the transfer of a phosphate group between ATP and arginine and creatine, respectively, play an important role in cellular energetics. In contrast to most animals which exhibit a single phosphagen kinase activity (creatine kinase in chordates and arginine kinase in protostomians), echinoderms exhibit both arginine kinase and creatine kinase activities, sometimes in the same tissue. In contrast to chordates in which creatine kinases are dimers (consisting of two subunits of 40 kDa) and protostomians in which arginine kinases are usually monomers (40 kDa), echinoids contain specific phosphagen kinases: a dimeric arginine kinase (consisting of two subunits of 42 kDa) in eggs and a monomeric creatine kinase (145 kDa) in sperm. We have examined echinoderms from the five existing classes (echinoids, asteroids, ophiuroids, holothurians and crinoids) for the expression of these specific phosphagen kinases in different tissues. Gel filtration was used to determine the molecular masses of the native enzymes. Antibodies specific for arginine kinase or for creatine kinase were used to characterize the subunit composition of arginine kinase and creatine kinase after SDS/PAGE and transfer. In all echinoderms analyzed, arginine kinase always occurred as an enzyme of about 81 kDa consisting of two subunits of 42 kDa and creatine kinase as a monomeric enzyme of 140-155 kDa. The occurrence in echinoderms of both phosphagen kinases with distinct specificities and specific molecular structures is discussed from both a developmental and evolutionary point of view.