An on-demand metalloprotease from psychro-tolerant Exiguobacterium undae Su-1, the activity and stability of which are controlled by the Ca2+ concentration

We reported an on-demand type of metalloprotease from Exiguobacterium undae Su-1. Although this species of bacterium is known to inhabit the permafrost, there are no reports on either strong proteases or peptidases. We found that Su-1 protease is superior to commercially available proteases in proteolytic activity in a lower to normal range of temperature (10-50 °C) as well as in rapid inactivation heat-dependently on the Ca2+ concentration. These characteristics meet well with the demands from food processing and manufacturing. Biochemical investigations of the purified enzyme and protein structural analysis after gene cloning confirmed that Su-1 protease conserved high identity in its primary sequence with thermophilic proteases of the M4 family. On the other hand, its flexibility was enhanced when one Ca2+ binding site was lost and by replacement for proline and isoleucine residues.     

Genetic and biochemical properties of an extracellular neutral metalloprotease from Staphylococcus hyicus subsp. hyicus

The gene encoding the extracellular neutral metalloprotease ShpI from Staphylococcus hyicus subsp. hyicus was cloned. DNA sequencing revealed an ORF of 1317 nucleotides encoding a 438 amino acid protein with Mr of 49698. When the cloned gene was expressed in Staphylococcus carnosus, a 42 kDa protease was found in the culture medium. The protease was purified from both S. carnosus (pCAshp1) and S. hyicus subsp. hyicus. The N-terminal amino acid sequences of the two proteases revealed that ShpI is organized as a pre-pro-enzyme with a proposed 26 amino acid signal peptide, a 75 amino acid hydrophilic pro-region, and a 337 amino acid extracellular mature form with a calculated Mr of 38394. The N-termini showed microheterogeneity in both host strains. ShpI had a maximum proteolytic activity at 55°C and pH 7.4–8.5. The protease, which had a low substrate specificity, could be inhibited by metal- and zinc-specific inhibitors, such as EDTA and 1,10-phenanthroline. Insensitivity to phosphoramidon separates ShpI from the thermolysin-like family. The conserved Zn²⁺ binding motif, the only homology to other proteases, and the reactivation of the apoenzyme by Zn²⁺, indicated that Zn²⁺ is the catalytic ion. Ca²⁺ very probably acts as a stabilizer. We also demonstrated the presence of a second extracellular protease in S. hyicus subsp. hyicus.     

Properties of a calcium-activated protease in squid axoplasm which selectively degrades neurofilament proteins

Axoplasm extruded from the giant axon of the squid contains Ca²⁺-activated proteases. The protease in the 100,000X g of supernatant of axoplasm is very specific and degrades only the 200,000 MW, neurofilament protein (NF₂₀₀), whereas the protease(s) in the pellet has a much wider range of substrate specificity. The activation of the supernatant protease is restricted to the Ca²⁺ ion, and no other divalent cation will substitute. The protease requires Ca²⁺ at a higher concentration than 0.5 mM for activation, and has a pH optimum of about 7.5. Degradation of the NF₂₀₀ appears to proceed through a 100,000 MW and possibly a 47,000–50,000-MW intermediate form before degradation to TCA-soluble peptides. Activity of the protease is inhibited by divalent cation chelators, Cu²⁺ and Fe²⁺, sulphydryl inhibitors, and leupeptin. This specific Ca²⁺-activated protease in squid axoplasm has identical properties to Ca²⁺-activated proteases found in various non-neural tissues. Despite its narrow protein substrate specificity, Ca²⁺-activated protease purified from human platelets effectively degrades squid NF₂₀₀, suggesting a possible structural relationship between platelet and muscle actin-binding proteins and neurofilament proteins.     

Purification and characterization of the cuticle-degrading protease produced by the entomopathogenic fungus,Beauveria bassiana in the presence of Sunn pest,Eurygaster integriceps (Hemiptera: Scutelleridae) cuticle

The extracellular protease from the entomopathogenic fungus, Beauveria bassiana in the presence of Eurygaster integriceps cuticle was isolated, purified and characterized. Isolate B1 of B. bassiana that shows high virulence against E. integriceps was examined for the production of the cuticle-degrading proteases. Results showed that both subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases were produced and the enzyme kinetic properties showed better activity of Pr1 in comparison with Pr2. The proteases were purified using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 5.09-fold increase in specific activity and 21.86% recovery. The enzyme molecular weight was estimated to be 47 kDa and the optimal pH and temperature were 8 and 45°C, respectively. The purified protease was activated by divalent cations, Ca^{2 +} and Mg^{2 +}, and inhibited by NaCl, KCl and determined as a serine protease by inhibition of its activity due to using PMSF, EDTA, mercaptoethanol and SDS. Studies on the timing of the protease secretion in the presence of cuticular substrates could provide information about the role of the accumulated hydrolytic enzymes during pathogenesis to better understand these processes.     

Extraction,purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ～?28?kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34?U/mL and 349.07?µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.     

Ca2+‐mediated exocytosis of subtilisin‐like protease 1: a key step in egress of Plasmodium falciparum merozoites

Egress of Plasmodium falciparum merozoites from host erythrocytes is a critical step in multiplication of blood‐stage parasites. A cascade of proteolytic events plays a major role in degradation of membranes leading to egress of merozoites. However, the signals that regulate the temporal activation and/or secretion of proteases upon maturation of merozoites in intra‐erythrocytic schizonts remain unclear. Here, we have tested the role of intracellular Ca²⁺ in regulation of egress of P. falciparum merozoites from schizonts. A sharp rise in intracellular Ca²⁺ just before egress, observed by time‐lapse video microscopy, suggested a role for intracellular Ca²⁺ in this process. Chelation of intracellular Ca²⁺ with chelators such as BAPTA‐AM or inhibition of Ca²⁺ release from intracellular stores with a phospholipase C (PLC) inhibitor blocks merozoite egress. Interestingly, chelation of intracellular Ca²⁺ in schizonts was also found to block the discharge of a key protease PfSUB1 (subtilisin‐like protease 1) from exonemes of P. falciparum merozoites to parasitophorous vacuole (PV). This leads to inhibition of processing of PfSERA5 (serine repeat antigen 5) and a block in parasitophorous vacuolar membrane (PVM) rupture and merozoite egress. A complete understanding of the steps regulating egress of P. falciparum merozoites may provide novel targets for development of drugs that block egress and limit parasite growth.     

Control of the onset of migration of neural crest cells in avian embryos

Summary To investigate the control of the timing in the epithelio-mesenchymal transformation of the neural crest into a migrating population, neural anlagen (neural tube plus crest) were isolated from 2-day quail embryos by proteases in the presence of Ca^{+ +} and explanted onto substrates favourable for neural crest cell migration. Explants isolated before normal migration had commenced required 3–8 h in vitro before neural crest cells started migration, but explants obtained at migratory stages showed an immediate onset of migration. The schedule was similar to that expected in vivo. When pre-migratory neural anlagen were isolated by protease in Ca^{+ +}- and Mg^{+ +}-free (CMF) medium, or when the protease was followed by a brief (5 min) exposure to CMF medium, neural crest cell migration commenced without delay, and the cohesion of the anlagen was impaired. Ca^{+ +}-free medium duplicated the effects of CMF, but neither Mg^{+ +}-free medium nor CMF treatment before treatment with protease stimulated migration and reduced cohesion. Precocious neural crest cell migration and reduced cohesion also followed when neural anlagen of pre-migratory stages were cultured with membrane. Ca^{+ +}-channel antagonists D600 and Nifedipine, without any exernal Ca^{+ +}-depletion.The decrease of cohesion of these tissues is consistent with results in other systems where protease/Ca^{+ +}-depletion inactivates Ca^{+ +}-dependent cell-cell adhesive mechanisms. Therefore, we suggest that Ca^{+ +}-dependent cell-cell adhesions play a part in preventing neural crest cells from migrating precociously and that the timed inactivation of this adhesion system normally helps trigger the onset of migration. The results with blockers of Ca^{+ +}-channels suggest that Ca^{+ +} levels may be involved in regulating this system.     

Role of electrostatics at the catalytic metal binding site in xylose isomerase action: Ca2+-inhibition and metal competence in the double mutant D254E/D256E

The catalytic metal binding site of xylose isomerase from Arthrobacter B3728 was modified by protein engineering to diminish the inhibitory effect of Ca²⁺ and to study the competence of metals on catalysis. To exclude Ca²⁺ from Site 2 a double mutant D254E/D256E was designed with reduced space available for binding. In order to elucidate structural consequences of the mutation the binary complex of the mutant with Mg²⁺ as well as ternary complexes with bivalent metal ions and the open-chain inhibitor xylitol were crystallized for x-ray studies. We determined the crystal structures of the ternary complexes containing Mg²⁺, Mn²⁺, and Ca²⁺ at 2.2 to 2.5 Å resolutions, and refined them to R factors of 16.3, 16.6, and 19.1, respectively. We found that all metals are liganded by both engineered glutamates as well as by atoms O1 and O2 of the inhibitor. The similarity of the coordination of Ca²⁺ to that of the cofactors as well as results with Be²⁺ weaken the assumption that geometry differences should account for the catalytic noncompetence of this ion. Kinetic results of the D254E/D256E mutant enzyme showed that the significant decrease in Ca²⁺ inhibition was accompanied by a similar reduction in the enzymatic activity. Qualitative argumentation, based on the protein electrostatic potential, indicates that the proximity of the negative side chains to the substrate significantly reduces the electrostatic stabilization of the transition state. Furthermore, due to the smaller size of the catalytic metal site, no water molecule, coordinating the metal, could be observed in ternary complexes of the double mutant. Consequently, the proton shuttle step in the overall mechanism should differ from that in the wild type. These effects can account for the observed decrease in catalytic efficiency of the D254E/D256E mutant enzyme. Proteins 28:183–193, 1997. © 1997 Wiley-Liss Inc.     

Calcium-activated proteolytic activity in rat liver mitochondria

Soluble extracts from sonicated rat liver mitochondria and rat liver cytosol were each chromatographed on DEAE-cellulose columns, and the fractions assayed for Ca²⁺-activated proteolytic activity using ¹⁴C-casein as a substrate. The mitochondrial preparations were shown to be free of cytosolic and microsomal contamination by the lack of alcohol dehydrogenase activity, a cytosolic marker enzyme, and by a lack of cytochrome P-450 activity, a microsomal marker enzyme. Two peaks of Ca²⁺-activated neutral endoprotease activity were resolved from the mitochondrial fractions. One protease was half-maximally activated with 25 μM Ca²⁺, and the other by 750 μM Ca²⁺. Rat liver cytosol contained only a high Ca²⁺-requiring protease peak. This is the first demonstration of Ca²⁺-activated proteases in mitochondria.     

Production of different proteases from fish gut microflora utilizing tannery fleshing

Three alkaline protease‐producing strains designated as ANFLR1, NPLR1, and PROLR15 were isolated from Labeo rohita fish gut. These strains are able to produce alkaline protease using tannery fleshing (TF) as the sole carbon and nitrogen source and were identified as Bacillus megaterium, Serratia marcescens, and novel Pontibacter sps. Proteases from these organisms were purified to electrophoretic homogeneity following ammonium sulphate precipitation, ion exchange, and column chromatography. SDS‐PAGE revealed molecular weights of the proteases to be 46 kDa (ANFLR1), 52 kDa (NPLR1), and 58 kDa (PROLR15). The optimum pH and temperature for the protease activity of ANFLR1, NPLR1, and PROLR15 were found to be 10.5, 11.5, 9, and 70^°C, 60^°C, and 50^°C, respectively. The maximum protease activities at the optimum conditions were 420 U/mL (ANFLR1), 550 U/mL (NPLR1), and 530 U/mL (PROLR15). Inhibition of the NPLR1 protease by pepstatin confirmed aspartate‐type enzymatic activity. Fe³⁺ enhanced the activity of PROLR15 protease. Unlike all other microbial proteases known so far, the PROLR15 enzyme did not require Ca²⁺ for activity and thermal stability. SDS‐PAGE and scanning electron microscopy analyses confirmed the conversion of high molecular weight substrate (TF) to low molecular weight peptides by these proteases. The alkaline metalloprotease production by novel Pontibacter sps. and aspartate protease production by S. marcescens remain unexplored. Hence, TF with its relatively abundant availability can be beneficially utilized for alkaline protease production through the fish gut microbial fermentation processes.     

EFFICIENT PROTEOLYSIS AND APPLICATION OF AN ALKALINE PROTEASE FROM HALOPHILIC Bacillus sp. EMB9

A salt-stable alkaline protease from moderately halophilic Bacillus sp. EMB9, isolated from the western coast of India, is described. This protease was capable of efficiently removing silver from used/waste X-Ray films, as well as hydrolyzing defatted soy flour with 31% degree of hydrolysis (DH). Production of the protease was optimized by using response surface methodology. Ca²⁺ and NaCl were the most critical factors in enhancing the yield. Under optimized culture conditions, a maximum of 369 U protease/mL was obtained, which is quite comparable to the yields of commercial proteases. The elevated production level coupled with ability to efficiently hydrolyze protein-laden soy flour and complete recovery of silver from used X-Ray films makes it a prospective industrial enzyme.     

Purification and some properties of the extracellular acid proteases from Mucor renninus

A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ～35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca²⁺, Mg²⁺, Cu²⁺, Co²⁺) on proteolytic activities of these enzymes studied.     

Calmodulin-like protein AtCML3 mediates dimerization of peroxisomal processing protease AtDEG15 and contributes to normal peroxisome metabolism

Matrix enzymes are imported into peroxisomes and glyoxysomes, a subclass of peroxisomes involved in lipid mobilization. Two peroxisomal targeting signals (PTS), the C-terminal PTS1 and the N-terminal PTS2, mediate the translocation of proteins into the organelle. PTS2 processing upon import is conserved in higher eukaryotes, and in watermelon the glyoxysomal processing protease (GPP) was shown to catalyse PTS2 processing. GPP and its ortholog, the peroxisomal DEG protease from Arabidopsis thaliana (AtDEG15), belong to the Deg/HtrA family of ATP-independent serine proteases with Escherichia coli DegP as their prototype. GPP existes in monomeric and dimeric forms. Their equilibrium is shifted towards the monomer upon Ca²⁺-removal and towards the dimer upon Ca²⁺-addition, which is accompanied by a change in substrate specificity from a general protease (monomer) to the specific cleavage of the PTS2 (dimer). We describe the Ca²⁺/calmodulin (CaM) mediated dimerization of AtDEG15. Dimerization is mediated by the CaM-like protein AtCML3 as shown by yeast two and three hybrid analyses. The binding of AtCML3 occurs within the first 25 N-terminal amino acids of AtDEG15, a domain containing a predicted CaM-binding motif. Biochemical analysis of AtDEG15 deletion constructs in planta support the requirement of the CaM-binding domain for PTS2 processing. Phylogenetic analyses indicate that the CaM-binding site is conserved in peroxisomal processing proteases of higher plants (dicots, monocots) but not present in orthologs of animals or cellular slime molds. Despite normal PTS2 processing activity, an atcml3 mutant exhibited reduced 2,4-DB sensitivity, a phenotype previously reported for the atdeg15 mutant, indicating similarly impaired peroxisome metabolism.     

Cytoplasmic proteases of rat liver parenchymal cells

Soluble extracts of isolated rat liver parenchymal cells contained three proteases with alkaline pH optima. One protease was a high molecular weight (Mr = 500,000) enzyme which was stimulated by ATP. The other two proteases were totally dependent on calcium for activity and displayed different calcium concentration requirements. One was half-maximally activated by 150 μM Ca²⁺ while the other required only 10 μM Ca²⁺ for half-maximal activation.     

Calcium and Phosphate Regulation of Nitrogen Metabolism in the Cyanobacterium Spirulina platensis Under the High Light Stress

High light stress (40 W/m²)-induced alterations in the nitrogen assimilatory enzymes in Spirulina platensis were studied under the Ca²⁺ and phosphate (Pi)-supplemented as well as starved conditions. Results revealed that activities of nitrate reductase (NR), amino acid transferases (AST/GOT and ALT/GPT), and protease enzymes in the high-light-incubated cells were relatively higher under the Ca²⁺- and Pi-starved conditions. On the contrary, relative rates of glutamine synthetase (GS) and ATPase activities were lower in the Ca²⁺- and Pi-starved cells. But the Spirulina cells under the Ca²⁺- and Pi-added conditions showed enhanced activity of both GS and ATPase enzymes. During the high-light stress, a decline in the GS activity, particularly under the Ca²⁺- and Pi-starved conditions, was indicative of a nitrogen starvation-like condition. This could be one of the reasons for induction of the NR and protease enzymes. A higher rate of GS activity was recorded under both the Ca²⁺- and Pi-supplemented conditions, perhaps owing to the enhanced rate of ATPase activity in such conditions. But a declining pattern of both NR and protease activities in the presence of Ca²⁺ and Pi, despite the higher rate of ATPase activity, might involve some other mechanism like the protein-kinase system. Received: 11 May 2000 / Accepted: 13 June 2000     

The cone-specific calcium sensor guanylate cyclase activating protein 4 from the zebrafish retina

Guanylate cyclase activating proteins (GCAPs) serve as neuronal Ca²⁺-sensor proteins in vertebrate rod and cone photoreceptor cells. Zebrafish express in their retina a variety of six different GCAPs, of which four are specific for cone cells. One isoform, zGCAP4, is mainly expressed in double cones and long single cones. We cloned the zGCAP4 gene, purified non-myristoylated and myristoylated forms of the protein after heterologous expression in Escherichia coli and studied its properties: zGCAP4 was a strong activator of membrane-bound guanylate cyclases from bovine and zebrafish retina, showing half-maximal activation at 520–570 nM free Ca²⁺ concentration. Furthermore, the Ca²⁺-sensitive activation properties of non-myristoylated and myristoylated zGCAP4 were similar, indicating no influence of the myristoyl moiety on Ca²⁺-sensor function. Myristoylated zGCAP4 showed low affinity for membranes and did not exhibit a Ca²⁺–myristoyl switch, a feature typical of some but not all neuronal Ca²⁺-sensor proteins. However, tryptophan fluorescence studies and Ca²⁺-dependent differences in protease accessibility revealed Ca²⁺-induced conformational changes in myristoylated and non-myristoylated zGCAP4, indicating the operation as a Ca²⁺ sensor. Thus, expression and biochemical properties of zGCAP4 are in agreement with its function as an efficient Ca²⁺-sensitive regulator of guanylate cyclase activity in cone vision.     

Degradation of purified neurofilament subunits by calcium-activated neutral protease: characterization of the cleavage products

Neurofilament proteins (NFP) purified from rat spinal cord were labeled with ¹²⁵-I and incubated with a crude extract from rat spinal cord containing Ca²⁺-activated protease(s). The protease(s) activated by mM Ca²⁺ cleaved the NFP and produced a series of breakdown products which were different for each NFP. The amount of cleavage was dependent upon the incubation time with proteases but the pattern remained constant. Some of the cleavage products were relatively stable. These observations suggest that the cleavage products produced by treating NFP subunits with the endogenous protease can be used as a finger print to further study NFP metabolism and to better understand their role in physiological and pathological conditions of the nervous system.     

An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations

An investigation on the properties of an alkaline protease secreted by Bacillus circulans BM15 strain isolated from a mangrove sediment sample was carried out in order to characterize the enzyme and to test its potency as a detergent additive. The protease was purified to apparent homogeneity by ammonium sulphate precipitation and was a 30-kDa protease as shown by SDS-PAGE and its proteolytic activity was detected by casein zymography. It had optimum activity at pH 7, was stable at alkaline pH range (7 to 11), had optimum temperature of activity 40°C and was stable up to a temperature of 55°C after incubation for one hour. Hg²⁺, Zn²⁺, Co²⁺, and Cu²⁺completely inhibited the enzyme activity, while Ca²⁺, Mg²⁺, K⁺ and Fe³⁺ were enhancing the same. The serine protease inhibitor PMSF and metal chelator EDTA inhibited the activity of this protease while the classic metalloprotease inhibitor 1, 10 phenanthroline did not show inhibition. The enzyme was stable in SDS, Triton-X-100 and H₂ O₂ as well as in various commercial detergents after incubation for one hour. The extracellular production of the enzyme, the pH and temperature stability and stability in presence of oxidants, surfactants and commercial detergents suggest its possible use as a detergent additive.     

Purification and characterization of two novel extra cellular proteases from Serratia rubidaea

A protease, producing bacterial culture (isolate ‘C’) was obtained from slaughterhouse waste samples, Hyderabad, India. It was related to Serratia rubidaea on the basis of 16S r RNA gene sequencing and biochemical properties. Cultural characters of S. rubidaea identified it as a psychrophile secreting protease at 10–30 °C. Single step purification of culture supernatant on sephacryl S-100 column revealed two proteases CP-1 and CP-2. The molecular masses of the enzymes determined by SDS-PAGE and zymography were approximately 97 and 45 kDa, respectively. N-terminal sequencing of CP-1 revealed a novel surface protein of S. rubidaea and CP-2 protease has shown 100% homology with protease of Serratia sp. A fold purification of 1.5 with 54% recovery was achieved in CP1 and purification of CP-2 resulted in 88% yield with a fold purification of 2. The optimum pH values of CP-1 and CP-2 were shown to be 10 and 8, respectively. The maximum activities for the enzymes were at 40 °C and 30 °C. Both the proteases are inhibited by EDTA indicating that they are metallo proteases. The activity of CP-1 was enhanced with Cu²⁺ that of CP-2 was enhanced with Zn²⁺ and Ca²⁺. These proteases have stability in presence of detergents, surfactants and solvents. These properties make these proteases an ideal choice for application in detergent formulations, food, leather industries, vaccine and enzyme peptide synthesis.     

The Ca increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca²⁺ increase occurs as a Ca²⁺ wave at each sperm entry site in the polyspermic egg. Some Ca²⁺ waves are preceded by a transient spike-like Ca²⁺ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca²⁺ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca²⁺ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca²⁺ store for the Ca²⁺ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.