Search PubMed⌕ Search

Biomedical subjects

B M Shapiro

Publications and source records attributed to B M Shapiro.

At least 37 records · Page 2Linked to original sources
PubMed1988

Ovothiol replaces glutathione peroxidase as a hydrogen peroxide scavenger in sea urchin eggs.

Despite its potential toxicity, H2O2 is used as an extracellular oxidant by Stronglylocentrotus purpuratus eggs to cross-link their fertilization envelopes. These eggs contain 5 mM 1-methyl-N alpha,N alpha-dimethyl-4-mercaptohistidine (ovothiol C), which reacts with H2O2. In consuming H2O2 and being reduced by glutathione, ovothiol acts as a glutathione peroxidase and replaces the function of the enzyme in eggs. The ovothiol system is more effective than egg catalase in destroying H2O2 at concentrations produced during fertilization and constitutes a principal mechanism for preventing oxidative damage at fertilization.

Amino Acids, Sulfur↗
PubMed1988

Regional differentiation of the sea urchin sperm plasma membrane.

In order to study the molecular basis for the functional localization and behavioral control of sperm, we have partially characterized plasma membranes prepared from isolated head and tail fractions. These membranes have similar amounts of the Na+ pump (as reflected by (Na+,K+)-ATPase activity), whereas they differ in protein composition, binding sites for Ca2+ channel antagonists, and in the localization of enzymes of cyclic nucleotide metabolism. The Ca2+ channel antagonist D600 (and related phenylalkylamines) binds to plasma membrane preparations from sperm heads and tails with much higher affinity than do the dihydropyridine antagonists. This binding is inhibited greatly by certain monovalent (but not divalent) ions, especially Na+, Tris+, glycine ethyl ester+, and methylamine+.K+,Li+, and choline+ are less effective. In media of ionic composition resembling seawater, sperm tail membranes exhibit 6.5-fold more binding sites for D600 than do membranes from sperm head. cGMP phosphodiesterase and adenylate cyclase are also enriched in plasma membranes from the tail. Thus, the highly polarized sperm cell exhibits a regional differentiation of plasma membrane proteins implicated in behavioral control.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

Hierarchies of protein cross-linking in the extracellular matrix: involvement of an egg surface transglutaminase in early stages of fertilization envelope assembly.

The involvement of transglutaminase activity in fertilization envelope (FE) formation was investigated using eggs from the sea urchin, Strongylocentrotus purpuratus. Eggs fertilized in the presence of the transglutaminase inhibitors, putrescine and cadaverine, had disorganized and expanded FEs with inhibition of the characteristic I-T transition. The permeability of the FE was increased by these agents, as revealed by the loss of proteins from the perivitelline space and the appearance of ovoperoxidase activity in supernates from putrescine-treated eggs. [3H]putrescine was incorporated into the FE during fertilization in a reaction catalyzed by an egg surface transglutaminase that could also use dimethylcasein as a substrate in vitelline layer-denuded eggs. Egg secretory products alone had no transglutaminase activity. The cell surface transglutaminase activity was transient and maximal within 4 min of activation. The enzyme was Ca2+ dependent and was inhibited by Zn2+. We conclude that sea urchin egg surface transglutaminase catalyzes an early step in a hierarchy of cross-linking events during FE assembly, one that occurs before ovoperoxidase-mediated dityrosine formation (Foerder, C. A., and B. M. Shapiro. 1977. Proc. Natl. Acad. Sci. USA. 74:4214-4218). Thus it provides a graphic example of the physiological function of a cell surface transglutaminase.

Animals↗
PubMed1988

Oxidative stress and the role of novel thiol compounds at fertilization.

A new class of thiols, the 1-methyl-4-mercaptohistidines, has been found in high concentrations in invertebrate eggs. This family, called the ovothiols, has unusual redox properties, including the ability to confer a CN- -resistant NAD(P)H oxidase activity on ovoperoxidase, the enzyme that catalyzes the physiological crosslinking of the fertilization envelope with dityrosine residues. Ovothiol has a redox potential of 44 mV positive to glutathione and thus is maintained in the reduced state in eggs by reduced glutathione, without the need for an ovothiol reductase. We propose that high concentrations of reduced ovothiol are present in eggs to protect them from the oxidative stress caused by the respiratory burst of fertilization.

Amino Acids, Sulfur↗
PubMed1987

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).

Amino Acids↗
PubMed1987

Purification and characterization of proteoliaisin, a coordinating protein in fertilization envelope assembly.

We report the purification and characterization of proteoliaisin, a protein that participates in the assembly of the sea urchin fertilization envelope. Proteoliaisin was purified from egg cortical granule exudate to greater than 99% homogeneity using chromatography on DEAE-Sepharose and on phenyl-Sepharose. Native proteoliaisin is a highly asymmetric protein (f/fo = 2.0) composed of a single Mr approximately 230,000 peptide. Its asymmetry was demonstrated both by analytical ultracentrifugation and by nondenaturing polyacrylamide gel electrophoresis, a novel analysis that detects molecular asymmetry in heterogeneous protein mixtures. Proteoliaisin is enriched in six amino acids: aspartic acid/asparagine, glutamic acid/glutamine, glycine, and cysteine, which account for over 50% of its mass. Nearly all of the cysteine residues are disulfide bonded. The protein contains a small proportion of aromatic amino acids with phenylalanine greater than tyrosine greater than tryptophan. At neutral pH its absorbance maximum is at 274.5 nm, with an extinction coefficient of 0.43 ml mg-1 cm-1. Proteoliaisin forms a 1:1 Ca2+-stabilized complex with ovoperoxidase, another component of the fertilization envelope, with Kd = 1.1 X 10(-6) M. Proteoliaisin, a constituent of the specialized echinoderm extracellular matrix called the fertilization envelope, has certain structural similarities to mammalian extracellular matrix proteins.

Amino Acids↗
PubMed1987

Ovothiols, a family of redox-active mercaptohistidine compounds from marine invertebrate eggs.

We have previously reported a novel thiol compound, 1-methyl-N alpha,N alpha-dimethyl-4-mercaptohistidine, or ovothiol, present at high concentration in the eggs of the sea urchin Strongylocentrotus purpuratus [Turner, E., Klevit, R., Hopkins, P. B., & Shapiro, B. M. (1986) J. Biol. Chem. 261, 13056-13063]. Here we report two related compounds, 1-methyl-N alpha-methyl-4-mercaptohistidine, or ovothiol B, from the scallop Chlamys hastata, and 1-methyl-4-mercaptohistidine, or ovothiol A, from the starfish Evasterias troschelii. These two compounds, as well as the S. purpuratus compound now designated ovothiol C, were isolated from eggs or ovarian tissue by S-carboxymethylation with [3H]iodoacetic acid, ion-exchange chromatography and ion-pairing high-pressure liquid chromatography. The structures of S-(carboxymethyl)ovothiols A and B were determined by 1H NMR, and that of ovothiol A was confirmed by comparison with authentic methylhistidine samples after desulfuration with Raney nickel. In the ovary of each species, the predominant methylation form of ovothiol accounts for at least 80% of the total 4-mercaptohistidine. The ovothiol concentration of the ovary far exceeds that of the testis or somatic tissues. The ovothiol C content of unfertilized S. purpuratus eggs is 1.14 mumol/10(6) eggs, equivalent to approximately 4.3 mM average concentration; the glutathione (GSH + GSSG) content is 0.9 mumol/10(6) eggs. In this species, high ovothiol levels persisted for the first 2 weeks of embryonic development. Ovothiol and glutathione account for virtually all of the trichloroacetic acid soluble-SH groups in the egg; these results are compared to several previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids, Sulfur↗
PubMed1987

Ovoperoxidase assembly into the sea urchin fertilization envelope and dityrosine crosslinking.

Ovoperoxidase, the enzyme implicated in hardening the extracellular coat of the fertilized sea urchin egg, is inserted into the assembling uncrosslinked (soft) fertilization membrane via specific interactions with a protein, proteoliaisin (P. Weidman, E. Kay, and B. M. Shapiro (1985). J. Cell. Biol. 100, 938-946), and the vitelline scaffold. Dityrosine crosslinks introduced by ovoperoxidase have been postulated to harden the assembled structure from such indirect data as the discovery of dityrosine in hard fertilization membranes (Foerder and B. M. Shapiro (1977). Proc. Natl. Acad. Sci. USA 74, 4214-4128; H. G. Hall (1978). Cell 15, 343-355). In this report, we show directly that soft fertilization membranes (SFM) contain no dityrosine residues but acquire these crosslinks in vitro only during hardening. In vitro hardening alters the susceptibility of the fertilization membrane to disruption in cation-depleted solutions and in detergent; the kinetics of these phenomena are all similar to those of hardening in vivo. Ovoperoxidase substrates were identified as a class of high-molecular-weight proteins of SFM by polyacrylamide gel electrophoresis after in vitro hardening or after an ovoperoxidase-catalyzed radioiodination reaction. The specificity of ovoperoxidase for particular substrates decreased once it was no longer associated with these polypeptides within the SFM. Moreover, after disruption of the SFM, ovoperoxidase had an increased capacity to iodinate an exogenous protein, myoglobin. These data suggest that assembly of ovoperoxidase into a specific locus within the soft fertilization membrane provides a regulatory mechanism to guarantee the crosslinking of only certain appropriately juxtaposed tyrosyl residues in the assembled structure.

Animals↗
PubMed1987

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.

Adenosine Triphosphatases↗
PubMed1987

Regulation of extracellular matrix assembly: in vitro reconstitution of a partial fertilization envelope from isolated components.

At fertilization, the glycocalyx (vitelline layer) of the sea urchin egg is transformed into an elevated fertilization envelope by the association of secreted peptides and the formation of intermolecular dityrosine bonds. Dityrosine cross-links are formed by a secreted ovoperoxidase that exists in a Ca2+-stabilized complex with proteoliaisin in the fertilization envelope. By using purified proteins, we now show that proteoliaisin is necessary and sufficient to link ovoperoxidase to the egg glycocalyx. Specifically, we have found that ovoperoxidase can associate with the vitelline layer only when complexed with proteoliaisin; proteoliaisin binds to the vitelline layer independently of its association with ovoperoxidase; proteolytic modification of the vitelline layer is not required for this interaction to occur; the binding of proteoliaisin to the vitelline layer is mediated by the synergistic action of the two major seawater divalent cations, Ca2+ and Mg2+; the number of proteoliaisin-binding sites on the vitelline layer of unfertilized eggs is equivalent to the amount of proteoliaisin secreted at fertilization; and the binding of ovoperoxidase to the vitelline layer, via proteoliaisin, permits the in vitro cross-linking of these two in vivo substrates. The association of purified ovoperoxidase and proteoliaisin with the vitelline layer of unfertilized eggs reconstitutes part of the morphogenesis of the fertilization envelope.

Animals↗
PubMed1986

Ovothiol: a novel thiohistidine compound from sea urchin eggs that confers NAD(P)H-O2 oxidoreductase activity on ovoperoxidase.

Sea urchin eggs contain a small molecular weight heat-stable factor that confers cyanide-resistant NAD(P)H-O2 oxidoreductase activity on ovoperoxidase (Turner, E., Somers, C. E., and Shapiro, B. M. (1985) J. Biol. Chem. 260, 13163-13171), the enzyme responsible for cross-linking the extracellular protein coat (fertilization membrane) of the egg. Here we report the isolation of the active cofactor and its identification by ultraviolet, NMR, and mass spectroscopy as a new sulfur-containing amino acid derivative, 1-methyl-alpha N,alpha N-dimethyl-4-thiohistidine, or ovothiol. Ovothiol reacts slowly with atmospheric oxygen or rapidly with micromolar concentrations of H2O2 to form ovothiol disulfide, which is inactive as a cofactor for the ovoperoxidase NAD(P)H oxidase. Reduced active ovothiol is regenerated by treatment with disulfide reductants and shows significant differences in its ultraviolet and NMR spectra from oxidized ovothiol. The oxidoreductase activity of the ovoperoxidase/ovothiol system is similar to that previously characterized with crude cofactor preparations; it is greatly enhanced by Mn2+ and is relatively insensitive to CN-, compared to the peroxidase activity of ovoperoxidase. The ovothiol content of eggs is estimated as 1.8 pmol/egg or an intracellular concentration of 6.8 mM. This concentration exceeds the amount of reductant needed for the CN-(-)insensitive oxygen consumption following fertilization and used in the production of H2O2 for fertilization membrane cross-linking. Whether ovothiol is involved in the cross-linking reaction, protects the egg from damage from H2O2, or has another role in development remains unclear.

Amino Acids, Sulfur↗
PubMed1986

Conformational control of ovoperoxidase catalysis in the sea urchin fertilization membrane.

The ovoperoxidase-catalyzed oxidation of iodide has been investigated as a function of pH for the homogeneous enzyme and for ovoperoxidase incorporated into several forms of the egg fertilization membrane. The pH dependent hysteresis previously observed in purified ovoperoxidase (Deits, T. L., Shapiro, B. M. (1985) J. Biol. Chem. 260, 7882-7888) is entirely absent in ovoperoxidase incorporated into the mature fertilization membrane, where the enzyme is bound noncovalently in vivo. The pH activity profile of ovoperoxidase incorporated into the mature fertilization membrane closely resembles the profile observed only transiently in purified ovoperoxidase subjected to a rapid downward pH shift. These observations can be accounted for by our previously presented mechanism for ovoperoxidase hysteresis (ibid.). We hypothesize that ovoperoxidase, upon incorporation into the fertilization membrane, is restricted to a limited subset of the conformational states available to the purified enzyme. This matrix-dependent conformational restriction is a novel control mechanism that serves to enhance the catalytic activity of ovoperoxidase upon its assembly into the fertilization membrane and thereby modulates ovoperoxidase catalysis in the vicinity of the developing egg.

Animals↗
PubMed1986

Ionic regulation of sea urchin sperm motility, metabolism and fertilizing capacity.

In order to pursue the significance of the ionic regulation of sea urchin sperm behaviour, alterations in the cation composition of sea water were tested for their effects on sperm fertilizing capacity. Nearly all changes which resulted in lowered sperm intracellular pH, including lowered sea-water pH, inclusion of the divalent ion chelator EGTA, addition of dithiothreitol, or removal of sea-water Na+, enhanced sperm viability for periods of up to a week. These conditions caused decreased cell motility and elevated ATP concentrations, and prevented the acrosome reaction. Conversely, changes which increased the intracellular pH, decreased sperm ATP concentrations, or induced the acrosome reaction, reduced sperm viability. A single medium, high sea-water K+ concentrations (greater than 100 mM), provided an exception to these general trends. At elevated K+ concentrations sperm were quiescent but became completely infertile. These data show that sperm fertilizing capacity is generally extended by maintenance of the sperm in an inactive state, and the results suggest that decreased cellular energy levels contribute to decreased fertility.

Adenosine Triphosphate↗
PubMed1986

Primary reconstruction of palatal defects.

Removal of the soft palate can cause marked functional deficit in deglutition and phonation. Most commonly, treatment of this deformity with prosthetic obturation has been less than ideal. Numerous reconstructive techniques have met with only partial success, while deforming distant structures. We will present a technique of reconstruction of the soft palate by use of a superiorly based pharyngeal flap. It has been used successfully in five patients who underwent soft palatectomy for malignant disease. The flaps have been the full width of the pharynx and extended down to the esophageal inlet. Viability of the flap is excellent, and the donor site is allowed to heal by secondary intention. Excellent function has been achieved in all cases with no compromise of oncological principles.

Female↗