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A F Quest

Publications and source records attributed to A F Quest.

24 records · Page 2Linked to original sources
PubMed1991

Membrane association of flagellar creatine kinase in the sperm phosphocreatine shuttle.

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.

Animals↗
PubMed1990

Phosphorylation of chicken brain-type creatine kinase affects a physiologically important kinetic parameter and gives rise to protein microheterogeneity in vivo.

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.

Animals↗
PubMed1990

Two different B-type creatine kinase subunits dimerize in a tissue-specific manner.

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.

Animals↗
PubMed1990

Molecular mechanisms of sea-urchin sperm activation before fertilization.

Several mechanisms are used to control the behaviour of sea urchin spermatozoa while fertilizing eggs. These include discrete regulatory steps that modulate the sperm activation sequence from spawning to gamete membrane fusion. After release from the testis, sperm motility is instantaneously activated, by using intracellular pH as a throttle mechanism to control the rate of the dynein motor that catalyses axonemal bending. To support motility, energy is transported from the mitochondrion to the tail, by using a shuttle mechanism involving phosphocreatine diffusion. This shuttle employs a novel, endotriplicated, creatine kinase of Mr 140,000 in the flagellar axoneme as its terminus. The steering mechanism that determines where the spermatozoon swims is unknown, but may involve an egg peptide-induced guanylate cyclase activation, mediated by a cGMP-dependent Ca2+ channel, and attenuated by a plasma membrane cGMP phosphodiesterase. Upon arriving at the egg, which is identified by virtue of its proteoglycan coat (egg jelly), the spermatozoon undergoes a univesicular secretion that prepares it to fuse with the egg. This acrosome reaction involves several altered ionic fluxes in its mechanism, terminating in a massive Ca2+ uptake. If the spermatozoon is fortunate enough to fuse with an egg, a new member of the species is generated; if the acrosome reaction occurs without gamete fusion, the spermatozoon rapidly dies. All of these activation processes involve changes in the intracellular ionic milieu that are co-ordinated with altered enzyme activities, often in a causal fashion. Even with our current imperfect understanding of the process, a few of the steps in sperm activation may be defined by biochemical pathways that include specific modulatory control points.

Acrosome↗
PubMed1989

Purification of brain-type creatine kinase (B-CK) from several tissues of the chicken: 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.

Animals↗