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J R Dedman

Publications and source records attributed to J R Dedman.

125 records · Page 7Linked to original sources
PubMed1978

Calcium-dependent regulator protein: localization in mitotic apparatus of eukaryotic cells.

Calcium-dependent regulator protein is a low molecular weight (17,000), thermostable, calcium binding protein which is structurally homologous to skeletal muscle troponin C. This protein is present in all nonmuscle cells and has been shown to decorate stress fibers in interphase cells by indirect immunofluorescence. Using this procedure we have investigated the distribution of the protein during mitosis of eukaryotic cells. As the cells enter prophase, the distinct cytoplasmic localization disappears commensurate with the dissolution of the cytoskeleton. The regulator protein seems to be randomly distributed throughout the prophase cell, including the region around the condensed chromosomes. However, at prometaphase, it is localized in association with the half-spindles of the mitotic apparatus. Through metaphase and most of anaphase, the protein remains localized between the chromosomes and the poles of the spindle. During late anaphase the protein is also found in the interzone region but rapidly condenses into two small regions, one on each side of the midbody that separates the daughter cells. The regulator protein is not localized in the cleavage furrow during telophase, whereas actin is demonstrable in this region. Indeed, placement of the protein during mitosis is distinct from both that of actin and that of tubulin. The localization of calcium-dependent regulator protein during mitosis suggests that it may mediate the calcium effects on the mitotic apparatus and thus play a role in chromosome movement.

Actins↗
PubMed1978

Control of microtubule assembly-disassembly by calcium-dependent regulator protein.

The Ca2+-dependent regulator (CDR) protein of cyclic nucleotide phosphodiesterase is a low molecular weight, acidic, Ca2+-binding protein which has been implicated in a number of Ca2+-dependent enzymatic functions. Indirect immunofluorescence has revealed that CDR is specifically associated with the chromosome-to-pole region of the mitotic apparatus during metaphase-anaphase in a pattern distinctly different from that of similar cultured cells stained with antitubulin. This characteristic localization in the mitotic half-spindle suggested a role for CDR in the control of microtubule assembly-disassembly during mitosis. Thus, CDR was examined for its effects on microtubule polymerization in vitro. It was determined that stoichiometric concentrations of CDR and a homologous Ca2+-binding protein, skeletal muscle troponin C, both inhibited and reversed microtubule assembly in a Ca2+-dependent manner. CDR-dependent inhibition of in vitro microtubule assembly occurred at physiological Ca2+ concentrations (approximately 10 micron) that, in the absence of CDR, caused only a slight reduction in polymerization. At Ca2+ concentrations in the low physiological range (less than 1 micron), no inhibition was observed. These biochemical results, together with the immunofluorescent localization of CDR in the mitotic half-spindle, provide evidence that Ca2+ is an endogenous regulator of microtubule disassembly through the activity of CDR.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1977

Ca2+-dependent regulation of cyclic-AMP phosphodiesterase by parvalbumin.

Carp parvalbumin has been shown to activate rat brain phosphodiesterase in a Ca2+-dependent manner. The concentration of Ca2+ required for half-maximal stimulation is 1.4 X 10(-7) M, whereas rat testis Ca2+-dependent regulator (CDR) of phosphodiesterase required 1.2 X 10(-6) M Ca2+. The difference in the slopes of the two curves demonstrated that the activation induced by parvalbumin was not the result of a small contamination by CDR. In addition, it has been shown that Ca2+ binding to parvalbumin parallels its activation of phosphodiesterase. These data suggest that Ca2+ must bind to a single specific metal binding site before phosphodiesterase can be fully activated.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1977

Biological cross-reactivity of rat testis phosphodiesterase activator protein and rabbit skeletal muscle troponin-C.

Phosphodiesterase activator protein and troponin-C have been purified from rat testis and rabbit skeletal muscle, respectively. The two proteins appear to be structurally distinct since the activator protein migrates faster than troponin-C on sodium dodecyl sulfate-polyacrylamide gels. Each of the calcium-binding proteins will, however, substitute for the other in their respective biological systems. Testis activator protein forms a complex with rabbit muscle troponin subunits TnI and TnT soluble in low salt. This hybrid complex (AIT) can regulate rabbit skeletal muscle actomyosin ATPase activity. AIT regulation, although influenced by free Aa2+ levels, is distinct from that of native troponin. Likewise, muscle troponin-C can substitute for activator protein in the stimulation of cyclic nucleotide phosphodiesterase. Troponin-C will fully stimulate phosphodiesterase although its affinity is 600-fold lower than that of activator protein. Ca2+ regulation studies demonstrate that both proteins require micormolar levels of free Ca2+ to induce phosphodiesterase activation. Activator protein requires 1.2 x 10(6) M and troponin-C, 1.9 X 10(6) M free Ca2+ for half-maximal stimulation of phosphodiesterase. The biological cross-reactivity of these proteins supports the sequence homology recently reported by Watterson et al. (Watterson, D.M., Harrelson, W.G., Keller, P.M., Sharief, F., and Vanaman, T.C. (1976) J.Biol. Chem. 251, 4501-4513). In addition, this preliminary study suggests that this nonmuscle troponin-C-like protein potentially may function in other Ca2+-regulated cellular events in addition to its moculation of cyclic nucleotide levels.

Adenosine Triphosphatases↗
PubMed1977

Characterization of a spectrophotometric assay for cAMP phosphodiesterase.

The hydrolysis of cAMP can be monitored spectrophotometrically through the conversion of 5' AMP to 5' IMP using a specific 5' AMP amino-hydrolase (EC3.5.4.6). The optical properties and extinction coefficient differences of these compounds have been quantitatively determined. Phosphodiesterase assayed in this manner is linear with respect to time and enzyme concentration, and is more reliable than conventional assay procedures. Due to the high specificity of the assay system phosphodiesterase can be selectively assayed in unfractionated cytosol. The assay, when conducted on a conventional spectrophotometer, can detect the hydrolysis of 0.5 nmoles cAMP per min.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1976

Biochemical actions of follice-stimulating hormone in the sertoli cell of the rat testis.

The sequenc of biochemical events associated with the action of follicle-stimulating hormone (FSH) in the testis has been investigated using a Sertoli cell-enriched testis model system. The Sertoli cell-encriched testis, created by irradiation of male rats in utero, is devoid of germinal elements but contains a normal complement of supportive Sertoli cells. Comparison of the Sertoli cell-enriched testis with normal testis, demonstrates that the two types of testes contain equal numbers of FSH specific receptors, judged by the binding of labeled hormone. In addition, FSH over a concentration range from 6 X 10(-11) to 6 X 10(-9)M will stimulate the production of adenosine 3',5' monophosphate (cAMP) in the Sertoli cell-enriched testis in a manner indistinguishable from that of the normal testis. Incubation of Sertoli cell enriched testis also results in the activation of soluble cAMP-dependent protein kinase. This response to FSH is dependent upon the age of the animal and disappears at about 32 days of age. While sensitivity to the hormone can still be detected in mature Sertoli cell-enriched animals by the addition of the phosphodiesterase inhibitor 1-methyl-3-isobutyl-xanthine, no detectable increase in phosphodiesterase activity is apparent after 30 days of age. Injection of FSH into Sertoli cell-enriched animals results in an increase in total testicular protein synthesis as well as in the production of the Sertoli cell-specific protein, androgen-binding protein within 30 minutes. Furthermore, while hypophysectomy of Sertoli cell-enriched animals result in a decline of the testicular concentration of androgen-binding protein, the injection of FSH will stimulate and maintain the levels of androgen-binding protein in such animals. These results demonstrate that the Sertoli cell-enriched testis is capable of carrying out the sequence of biochemical events previously described for FSH in the normal testis and therefore, suggest that the Sertoli cell is the primary target cell for FSH action.

Age Factors↗
PubMedDate not supplied

Mutation of highly conserved arginine residues disrupts the structure and function of annexin V.

BACKGROUND: Annexins are a family of structurally related proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Annexins are characterized by highly conserved canonical domains of approximately 70 amino acids. Annexin V contains four such domains. Each of these domains has a highly conserved arginine (R). METHODS: To evaluate the role of the conserved arginines in the molecular structure of annexin V, negatively charged amino acids were substituted for arginines at positions R43, R115, R199, and R274 using site-directed mutagenesis. RESULTS: Mutants R199D and R274E were rapidly degraded when expressed in bacteria, and were not further characterized. R43E exhibited an electrophoretic mobility similar to the wild-type protein, while R115E migrated significantly in a slower fashion, suggesting a less compact conformation. R43E and R115E exhibited much greater susceptibility to proteolytic digestion than the wild type. While Ca(2+)-dependence for phospholipid binding was similar in both mutants (half-maximal 50-80 microM Ca2+), R43E and R115E exhibited a 6- and 2-fold decrease in phospholipid affinity, respectively. Consistent with the different phospholipid affinities of the annexins, a phospholipid-dependent clotting reaction, the activated partial thromboplastin time (aPTT), was significantly prolonged by the wild-type protein and mutants R115E and R115A. The aPTT was unaffected by R43E. CONCLUSIONS: Our data suggest that mutation of these highly conserved arginine residues in each of the four canonical domains of annexin have differential effects on the phospholipid binding, tertiary structure, and proteolytic susceptibility of annexin V. The site I mutation, R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolytic susceptibility. The site II mutation, R115E, produced a small change in phospholipid binding but a significant modification of electrophoretic mobility. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of annexin V by establishing hydrogen bonds and ionic bridges.

Amino Acid Sequence↗