Egg sialoglycans increase intracellular pH and potentiate the acrosome reaction of sea urchin sperm.

Sea urchin egg jelly (EJ) triggers sperm acrosome reaction (AR), an exocytotic event required for membrane fusion of the gametes. Purified fucose sulfate polymer (FSP) in EJ is one inducer of the AR. Binding of FSP to its receptor regulates opening of two distinct calcium channels and also elevates intracellular pH (pH(i)). EJ also contains sialic acid-rich glycans (sialoglycans (SG)) that were isolated by beta-elimination followed by DEAE chromatography. In the presence of limiting amounts of FSP, the SG fraction markedly potentiates the AR; however, by itself SG has no activity. The SG fraction increases the pH(i) of sperm without increasing intracellular Ca(2+). The SG-induced increase in pH(i) is not blocked by nifedipine or high K(+), whereas the FSP-induced pH(i) increase is sensitive to both these agents. Treatment of the SG fraction with neuraminidase or mild metaperiodate that specifically cleaves the glycerol side chain of sialic acid abolishes the AR potentiation and ability of SG to elevate pH(i). These data are the first to show that there are at least two pathways to induce sperm pH(i) increase and that egg surface sialic acid plays a role in triggering the sperm AR.     

PKA activation in concert with ARIS and asterosap induces the acrosome reaction in starfish

The acrosome reaction (AR) is a fundamental event for fertilization, which is induced in concert with acrosome reaction-inducing substance (ARIS) and asterosap, both of which are components of starfish egg jelly (EJ). During the AR, a spermatozoon undergoes a series of physiological changes, such as in intracellular cGMP concentration ([cGMP]i), pHi and intracellular Ca2+ concentration ([Ca2+]i). Affinity purification of cGMP-binding protein resulted in the isolation of a regulatory subunit of the cAMP-dependent protein kinase A (PKA), suggesting the involvement of a cAMP-dependent pathway in the AR. By using a cAMP enzyme immunoassay, [cAMP]i was found to increase in starfish spermatozoa when stimulated with ARIS and asterosap. ARIS could also increase the [cAMP]i in the presence of high pH seawater. Pretreatment of spermatozoa with two specific and cell-permeable PKA inhibitors, H89 and KT5720, prevented the induction of the AR in a concentration-dependent manner. These results suggest that PKA activity participates in the induction of the AR with ARIS and asterosap. To investigate this, we have cloned a gene that encodes a regulatory subunit of PKA that had been identified in starfish spermatozoa.     

Alteration of intracellular [Ca2+] in sea urchin sperm by the egg peptide speract. Evidence that increased intracellular Ca2+ is coupled to Na+ entry and increased intracellular pH

The egg peptide speract increases intracellular pH (pHi) and cyclic nucleotides in sperm of the sea urchin Strongylocentrotus purpuratus by a mechanism dependent on seawater Na+ but not Ca2+ (Hansbrough, J. R., and Garbers, D. L. (1981) J. Biol. Chem. 256, 2235-2241; Repaske, D. R., and Garbers, D. L. (1983) J. Biol. Chem. 258, 6025-6029). Using the Ca2+ indicators quin2 and indo-1, we show that speract stimulates a transient rise in intracellular [Ca2+] ([a2+]i) when millimolar Ca2+ is present in seawater. The rise is increased and extended by the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), which also enhances 22Na+ uptake with or without Ca2+. Without MIX, speract initiates a rise in [Ca2+]i that peaks within approximately 5 s and decreases with a t1/2 of approximately 9 s. Activation of Na+:H+ exchange without speract by either Na+ addition to sperm in Na+-free seawater (NaFASW) or by monensin also increases [Ca2+]i, but neither change is transient. Inhibition of Na+:H+ exchange by increased seawater [K+] prevents the rise in [Ca2+]i initiated by either speract or Na+ addition to sperm in NaFASW. Increasing pHi by adding 10 mM NH4+ or by addition of Li+ to sperm in NaFASW does not increase [Ca2+]i. The data suggest that speract binding leads to rapid activation of Na+:H+ exchange; and, as a consequence, [Ca2+] entry increases transiently through either Na+:Ca2+ exchange or else through a verapamil-insensitive Ca2+ channel. MIX prevents the inactivation of this entry mechanism.     

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.     

Real-time measurements of the interactions between fluorescent speract and its sperm receptor

Lytechinus pictus sea urchin sperm express receptors for speract, a sperm-activating peptide derived from the homologous egg jelly coat. We found that the fluorescence of fluorophore-labeled, active, speract analogs is quenched upon receptor binding. This property allowed us to perform real-time measurements of speract-receptor interactions using intact sperm and to determine, for the first time, their association (k(on)) and dissociation (k(off)) rate constants. The high k(on) (2.4 x 10(7) M(-1 )s(-1)) and low k(off) (4.4 x 10(-6) s(-1) (95%) and 3.7 x 10(-4) s(-1) (5%)) can account for the sperm response to picomolar concentrations of speract. We also examined the influence of extracellular ions on speract-receptor interactions using the fluorescence quenching method described in this study. The association rate of speract to the receptor is dramatically reduced in Na(+)-free seawater (NaFSW), divalent cation-free seawater (DCFSW), and high-K(+) seawater (HKSW). In seawater speract induces an increase in intracellular pH (pHi), while it is unable to do so in either NaFSW or HKSW. To test if the lack of this pHi change causes the reduction in the speract association rate, pHi was increased with NH(4)Cl (10 mM) at the time labeled speract was added. Interestingly, this procedure completely (in HKSW) or partially (in NaFSW and DCFSW) restored the speract association rate to its receptor. These findings indicate that an increase in sperm pHi positively affects the receptor binding activity for this peptide and may partially explain the positive binding cooperativity displayed by the speract receptor.     

Store-operated calcium channels trigger exocytosis of the sea urchin sperm acrosomal vesicle

The acrosome reaction (AR) of sperm is a prerequisite for fusion with the egg. In sea urchins, the complete AR (CAR) consists of exocytosis of the acrosomal vesicle (AV) and polymerization of acrosomal actin to form the approximately 1 micro m long acrosomal process. The fucose sulfate polymer (FSP) of egg jelly stimulates Ca(2+) entry through two distinct Ca(2+) channels and induces the CAR. Here we report that the second channel is blocked by SKF96365 (SKF), an inhibitor of store-operated channels. SKF also blocks the thapsigargin (TG), trifluoperazine (TFP), and calmidizolium (CMZ) stimulated Ca(2+) entry into sperm. These data indicate that the second Ca(2+) channel is a store-operated channel (SOC) that may be regulated by calmodulin. The TG, TFP, and CMZ-induced intracellular Ca(2+) elevations are similar to those induced by FSP, but the sperm acrosomal process does not polymerize. An antibody to bindin, the major protein of the AV, showed that in a significant percentage of these drug-treated sperm, the AV had undergone exocytosis. When NH(4)Cl was added to increase intracellular pH, the TG-treated sperm polymerized actin to form the acrosomal process. We conclude that the second Ca(2+) channel of sea urchin sperm is a SOC that triggers AV exocytosis.     

High molecular mass egg fucose sulfate polymer is required for opening both Ca2+ channels involved in triggering the sea urchin sperm acrosome reaction.

A linear fucose sulfate polymer (FSP), >10(6) daltons, is a major component of sea urchin egg jelly. FSP induces the sperm acrosome reaction (AR), an exocytotic process required for animal fertilization. Two Ca(2+) channels activate during AR induction, the first opens 1 s after FSP addition, and the second opens 5 s after the first. Mild acid hydrolysis of FSP results in a linear decrease in polymer size. The ability of FSP to induce the AR and activate sperm Ca(2+) channels decreases with increasing time of hydrolysis. Hydrolyzed FSP of approximately 60 kDa blocks intact FSP from inducing the AR. At 44 microg/ml hydrolyzed FSP, Ca(2+) entry into sperm is almost equal to that occurring in 3.8 microg/ml intact FSP; however the AR is not induced. The shape of the [Ca(2+)](i) increase curve and use of the Ca(2+) channel blockers nifidipine and Ni(2+) indicate that hydrolyzed FSP opens the second Ca(2+) channel, but not the first, and thus does not induce the AR. The giant size of intact FSP is required to open both Ca(2+) channels involved in triggering the AR.     

Characterization of a monoclonal antibody that induces the acrosome reaction of sea urchin sperm

A monoclonal antibody, J18/29, induces the acrosome reaction (AR) in spermatozoa of the sea urchin Strongylocentrotus purpuratus. J18/29 induces increases in both intracellular Ca2+ and intracellular pH similar to those occurring upon induction of the AR by the natural inducer, the fucose sulfate-rich glycoconjugate of egg jelly. Lowering the Ca2+ concentration or the pH of the seawater inhibits the J18/29-induced AR, as does treatment with Co2+, an inhibitor of Ca2+ channels. The J18/29-induced AR is also inhibited by verapamil, tetraethylammonium chloride, and elevated K+. All these treatments cause similar inhibition of the egg jelly-induced AR. J18/29 reacts with a group of membrane proteins ranging in molecular mass from 340 to 25 kD, as shown by immunoprecipitation of lysates of 125I-labeled sperm and Western blots. The most prominent reacting proteins are of molecular masses of 320, 240, 170, and 58 kD. The basis of the multiple reactivity appears to reside in the polypeptide chains of these proteins, as J18/29 binding is sensitive to protease digestion but resistant to periodate oxidation. There are approximately 570,000 sites per cell for J18/29 binding. J18/29 is the only reagent of known binding specificity that induces the AR; it identifies a subset of sperm membrane proteins whose individual characterization may lead to the isolation of the receptors involved in the triggering of the AR at fertilization.     

Regulation of the starfish sperm acrosome reaction by cGMP, pH, cAMP and Ca2+

In the starfish, Asterias amurensis, three components in the jelly coat of eggs, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to induce the acrosome reaction (AR). Molecular recognition between the sperm surface molecules and the egg jelly molecules must underlie signal transduction events triggering the AR. Asterosap is a sperm-activating molecule, which stimulates rapid synthesis of intracellular cGMP, pH and Ca(2+). This transient elevation of Ca(2+) level is caused by a K(+)-dependent Na(+)/Ca(2+) exchanger, and the increase of intracellular pH is sufficient for ARIS to induce the AR. The concerted action of ARIS and asterosap could induce elevate intracellular cAMP levels in starfish sperm and the sustained increase in [Ca(2+)], which is essential for the AR. The signaling pathway induced by these factors seems to be synergistically regulated to trigger the AR in starfish sperm.     

Measurement of plasma membrane and mitochondrial potentials in sea urchin sperm. Changes upon activation and induction of the acrosome reaction

The relationship between the plasma membrane potential and activation of sperm motility and respiration, or induction of the acrosome reaction, was explored in sperm of the sea urchin Strongylocentrotus purpuratus. Plasma and mitochondrial membrane potentials were estimated by measuring the uptake of [14C]thiocyanate ( [14C]SCN-) and [3H]tetraphenylphosphonium ( [3H]TPP+) in intact sperm and sperm made permeant with digitonin. Mitochondrial potentials up to-185 mV were found, consistent with data for TPP+ uptake into mitochondria from other cell types. Values for TPP+ uptake corrected for mitochondrial accumulation and estimates of SCN- uptake both indicated that the plasma membrane potential was about -30 mV for actively respiring sperm in seawater and about -60 mV for quiescent sperm in Na+-free seawater. Activation of sperm motility and respiration induced by Na+ increased the intracellular pH and caused a depolarization of both the plasma membrane and mitochondrial potentials. However, membrane potential depolarization did not occur when the activation was induced by increased extracellular pH or by the peptide speract, although activation was always linked to increased intracellular pH. The acrosome reaction, on the other hand, was always associated with sperm plasma membrane potential depolarization, whether it was induced by the physiological effector from the egg surface or by several artificial triggering regimens. Thus, activation of respiration and motility is primarily controlled by increased intracellular pH (Christen, R., Schackmann, R. W., and Shapiro, B. M. (1982) J. Biol. Chem. 257, 14881-14890), whereas the acrosome reaction also requires depolarization of the plasma membrane potential.     

A depolarization can trigger Ca2+ uptake and the acrosome reaction when preceded by a hyperpolarization in L. pictus sea urchin sperm.

The acrosome reaction (AR) is an exocytotic event that allows sperm to recognize and fuse with the egg. In the sea urchin sperm this reaction is triggered by the outer investment of the egg, the jelly, which induces ionic movements leading to increases in intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi), a K(+)-dependent transient hyperpolarization which may involve K+ channels, and a depolarization which depends on external Ca2+. The present paper explores the role of the hyperpolarization in the triggering of the acrosome reaction. The artificial hyperpolarization of Lytechinus pictus sperm with valinomycin in K(+)-free seawater raised the pHi, caused a small increase in 45Ca2+ uptake, and triggered some AR. When the cells were depolarized with KCl (30 mM) 40-60 sec after the induced hyperpolarization, the pHi decreased and there was a significant increase in 45Ca2+ uptake, [Ca2+]i, and the AR. This waiting time was necessary in order to allow the pHi change required for the AR to occur. Thus, the jelly-induced hyperpolarization may lead to the intracellular alkalinization required to trigger the AR, and, on its own or via pHi, may regulate Ca2+ transport systems involved in this process. Because of the key role played by K+ in the triggering of the AR, the presence and characteristics of ion channels in L. pictus isolated sperm plasma membranes are being explored. Planar lipid bilayers into which these membranes were incorporated by fusion displayed 85 pS single channel transitions which were cation selective.     

Preloading of micromolar intracellular Ca2+ during capacitation of Sicyonia ingentis sperm, and the role of the pHi decrease during the acrosome reaction.

In studying the mechanism controlling the sperm acrosome reaction (AR) in the marine shrimp Sicyonia ingentis, intracellular Ca2+ and pH were measured using the fluorescent indicators Fura-2 and Fluo-3 for Ca2+, and SNARF-1 for pH. Capacitated sperm possessed an apparent resting Ca2+ concentration of 1-2 microM which remained constant upon induction of the AR with egg water. Uncapacitated sperm had extremely low Ca2+ levels and did not respond to egg water. These results suggest that, while in other species the Ca2+ is elevated to micromolar levels during initiation of the AR, S. ingentis sperm are preloaded with Ca2+ during capacitation and the trigger for the AR is downstream of the Ca2+ increase. The notion that Ca2+ influx is not involved at the actual time of the AR in capacitated S. ingentis sperm is supported by the inability of Ca2+ ionophore A23187 to induce the AR and the ineffectiveness of Ca2+ channel antagonists to block egg water-induced AR. Measurements of capacitated sperm pH showed a significant decrease during the first 10-15 min of the AR, which did not correlate temporally to either acrosomal exocytosis (at 5 min post-induction) or filament formation (after 45 min). Inhibition of egg protease activity required for induction of filament formation did not inhibit the pH drop, indicating that intracellular acidification is not the final trigger for filament formation, although it may be required prior to action of the protease.     

Sperm-activating peptide induces asymmetric flagellar bending in sea urchin sperm

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, induces various sperm responses including a transient increase in the intracellular Ca2+ concentration. However, it has not been clarified how speract modulates sperm motility and whether it functions as a chemoattractant. To confirm the effect of speract on sperm motility, we observed the flagellar bending response to speract in sperm of Hemicentrotus pulcherrimus, in experiments using caged speract and a lighting system for a microscope newly developed with a power LED. We found that speract induces increases in curvature of swimming paths and changes flagellar bending shape to asymmetric. These facts show that speract directly regulates flagellar motility, and suggest that speract-induced increases in intracellular Ca2+ concentration play an actual role in regulation of the flagellar movement.     

Receptor-mediated responses of plasma membranes isolated from Lytechinus pictus spermatozoa

Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), a peptide obtained from eggs, has been shown to bind to a plasma membrane receptor of Lytechinus pictus spermatozoa. Here, we show that the addition of speract to intact cells caused the appearance of a new protein-staining band (Mr = 140,000) on sodium dodecyl sulfate (SDS) polyacrylamide gels; concomitantly, a protein of apparent molecular weight (Mr) 150,000 disappeared. Guanylate cyclase activity also decreased approximately 50% after the addition of speract to intact cells. Plasma membranes were subsequently prepared from spermatozoa in the presence of fluoride at pH 6.0, conditions that resulted in retention of the speract receptor and the Mr 150,000 protein. Addition of speract to the membranes resulted in a disappearance of the Mr 150,000 protein and the appearance of a Mr 140,000 protein. Coincident with the apparent change in molecular weight, guanylate cyclase activity decreased 30% at maximal speract concentrations. A physiological event that occurs in the intact cell in response to speract can now be reproduced in isolated plasma membranes; it should, therefore, now be possible to define the molecular events that occur as a result of speract: receptor interaction.     

Rho-kinase (ROCK) in sea urchin sperm: its role in regulating the intracellular pH during the acrosome reaction

Sperm must undergo the acrosome reaction (AR) in order to fertilize the egg. In sea urchins, this reaction is triggered by the egg jelly (EJ) which, upon binding to its sperm receptor, induces increases in the ion permeability of the plasma membrane and changes in protein phosphorylation. Here, we demonstrated that the sperm expresses ROCK (∼135 kDa), which is a serine/threonine protein kinase. ROCK localized, as RhoGTPase (Rho), in the acrosomal region, midpiece and flagellum. H-1152, a ROCK antagonist, inhibited the two cellular processes defining the AR: the acrosomal exocytosis and the actin polymerization. The ionophores nigericin and A23187 reversed the AR inhibition induced by H-1152, suggesting that ROCK functions at the level of the EJ-induced ion fluxes. Accordingly, H-1152 blocked 70% the intracellular alkalinization induced by EJ. These results indicate that EJ activates a Na⁺-H⁺ exchanger (NHE) in the sperm through a Rho/ROCK-dependent signaling pathway that culminates in the AR.     

Inhibition of apoptosis in pulmonary endothelial cells by altered pH,mitochondrial function,and ATP supply

We investigated the effect of altered extracellular pH, mitochondrial function, and ATP content on development of apoptosis in human pulmonary artery endothelial cells after treatment with staurosporine (STS). STS produced a concentration- and time-dependent increase in caspase-3 activity in pH 7.4 medium that reached a peak at 6 h. The increase in caspase activity was associated with significant DNA fragmentation. Fluorescent imaging of treated monolayers in pH 7.4 medium with Hoechst-33342-propidium iodide demonstrated a large percentage of apoptotic cells ( approximately 40%) with no evidence of necrosis. Caspase activity, DNA fragmentation, and percentage of apoptotic cells were reduced after STS treatment in acidic media (pH 7.0 and 6.6). The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM inhibited STS-induced apoptosis, whereas the rise in intracellular Ca2+concentration in STS-treated cells in pH 7.4 medium was reduced in pH 7.0 medium. These results suggest that one mechanism for inhibitory effects of acidosis may be a pH-induced alteration in Ca2+ signaling. Treatment with STS in the presence of oligomycin (10 microM), an inhibitor of the mitochondrial F(0)F(1)-ATPase, in glucose-free media abolished caspase activation and DNA fragmentation in association with severe ATP depletion ( approximately 2% of control cells). Imaging demonstrated a change in the mode of cell death from apoptosis to necrosis under these conditions. This change was linked to the level of ATP depletion, because STS treatment in the absence of glucose or the presence of oligomycin in media with glucose still leads to apoptosis in the presence of only moderate ATP depletion. These results demonstrate that pH, mitochondrial function, and ATP supply are important variables that regulate STS-induced apoptosis in human pulmonary artery endothelial cells.     

Internal GTP stimulates the speract receptor mediated voltage changes in sea urchin spermatozoa membrane vesicles

A voltage-sensitive Na+/H+ exchanger in the flagellar membrane is responsible for regulating the intracellular pH of the sea urchin spermatozoa. A previous study has shown that the egg peptide speract can modulate this Na+/H+ exchanger through its hyperpolarizing effect on the membrane potential. The effect of GTP on this speract receptor mediated process is investigated in this study. Plasma membrane vesicles with an outwardly directed K+ gradient were prepared from the isolated flagella by osmotic lysis. Vesicular membrane potential was monitored by a cationic probe, diS-C3-(5), and an anionic probe, diS-BA-C2-(3). Results show that the presence of internal GTP greatly stimulated the speract induced membrane hyperpolarization in this vesicle system. The analog GTP gamma S was not only active but could, by itself, induce partial hyperpolarization which was further enhanced by speract addition. Internal GDP was partially active in supporting the speract effect, whereas GDP beta S, cGMP, GMP, and ATP were all inactive. The ionic selectivity of the speract effect was investigated by increasing the external concentration of various cations. K+ and Rb+ abolished the hyperpolarization while Cs+ had no effect. These results indicate that internal GTP is involved in the coupling between the speract receptor and the membrane hyperpolarization, which is most likely due to the activation of K+ selective channels.     

Co-localization of receptor and transducer proteins in the glycosphingolipid-enriched, low density, detergent-insoluble membrane fraction of sea urchin sperm

The low density, detergent-insoluble membrane fraction (LD-DIM), where gangliosides are likely to be highly enriched, was prepared from sperm of two sea urchin species, Hemicentrotus pulcherrimus and Strongylocentrotus purpuratus. Immunoblotting showed the presence in the LD-DIM of two receptors for egg ligands, a glycosylphosphatidylinositol (GPI)-anchored protein, and four proteins which may be involved in signal transduction. Co-immunoprecipitation revealed that at least three proteins, the speract receptor, the 63[emsp4 ]kDa GPI-anchored protein and the subunit of a heterotrimeric Gs protein, are localized in the LD-DIM. This suggests that the LD-DIM fraction may be a membrane microdomain for speract–speract receptor interaction, as well as the subsequent signal transduction pathway involved in induction of sperm respiration, motility and possibly the acrosome reaction.     

Modulation of the voltage-sensitive Na+/H+ exchange in sea urchin spermatozoa through membrane potential changes induced by the egg peptide speract

Sea urchin sperm motility can be activated by alkalinization of the internal pH, and previous studies have shown that the internal pH can be regulated by a voltage-sensitive Na+/H+ exchanger present in the flagellar plasma membrane. In this study, the effects of speract, a peptide purified from egg conditioned media, on the Na+/H+ exchange were investigated. Evidence presented indicates that speract activates K+ channels in the flagellar membrane and modulates the Na+/H+ exchange activity through resultant changes in membrane potential. In the presence of tetraphenylphosphonium, a lipophilic ion, or high external Na+, the isolated flagella were depolarized, and Na+/H+ exchanger was inhibited. Speract and valinomycin, a K+ ionophore, were able to reactivate 22Na+ uptake, H+ efflux, and alkalinization of intraflagellar pH under either of the depolarizing conditions. Membrane potential measurements using 3,3'-dipropylthiodicarbocyanide iodide indicated repolarization by either speract or valinomycin. The speract-induced voltage changes did not require Na+ but were sensitive to [K+]. Thus, speract induced a slight depolarization in Na+-free seawater with 10 mM K+ but a hyperpolarization with 2 mM K+. Further support for the activation of K+ channels in the flagella was the 2-5-fold stimulation of K+ efflux induced by speract as measured with a K+ electrode. The ionic selectivity of the speract-activated channel assessed by voltage measurements was K+ greater than Rb+ greater than Cs+. The half-maximally effective concentration of speract was about 0.2 nM. That the H+ and K+ efflux in response to peptide was receptor-mediated was confirmed by the use of speract or resact on intact sea urchin spermatozoa, where the peptides were found to stimulate K+ efflux and to reverse the tetraphenylphosphonium inhibition on H+ efflux only in the homologous spermatozoa. Modulation of the voltage-sensitive Na+/H+ exchange by egg peptides, therefore, appears to be indirect and is coupled through its action on membrane potential.     

Phanerochaete chrysosporium beta-Glucosidases: Induction, Cellular Localization, and Physical Characterization

Phanerochaete chrysosporium produces intracellular soluble and particulate beta-glucosidases and an extracellular beta-glucosidase. The extracellular enzyme is induced by cellulose but repressed in the presence of glucose. The molecular weight of this enzyme is 90,000. The K(m) for p-nitrophenyl-beta-glucoside is 1.6 x 10 M; the K(i) for glucose, a competitive inhibitor, is 5.0 x 10 M. The K(m) for cellobiose is 5.3 x 10 M. The intracellular soluble enzyme is induced by cellobiose; this induction is prevented by cycloheximide. The presence of 300 mM glucose in the medium, however, had no effect on induction. The K(m) for p-nitrophenyl-beta-glucoside is 1.1 x 10 M. The molecular weight of this enzyme is approximately 410,000. Both enzymes have an optimal temperature of 45 degrees C and an E(act) of 9.15 kcal (ca. 3.83 x 10 J). The pH optima, however, were approximately 7.0 and 5.5 for the intracellular and extracellular enzymes, respectively.