Chymotrypsin-like and trypsin-like protease activities in the sea urchin (Hemicentrotus pulcherrimus) egg

Proteolytic activities in extracts of sea urchin eggs were examined using SDS (sodium dodecyl sulphate)-polyacrylamide gels. In the unfertilized eggs, proteases were detected as bands corresponding to the molecular weights of 40 kD and 26 kD on the gelatin gel, and 35 kD and 30 kD on the casein gel. Using various protease inhibitors, it was found that 40 kD, 30 kD, and 26 kD are chymotrypsin-like proteases and that 35 kD is a trypsin-like protease. The activity of the 40 kD chymotrypsin-like protease was found to be almost completely lost after insemination.     

Intracellular proteases from the extremely thermophilic archaebacteriumSulfolobus solfataricus

Proteolytic activities from the extremely thermoacidophilic archaebacteriumSulfolobus solfataricus were detected with the aid of synthetic substrates in a cell extract fractionated by gel filtration. Two aminopeptidases (aminopeptidase I and II), three endopeptidases (proteinase I, II and III) and one carboxypeptidase could be identified. Experiments carried out with protease inhibitors led to the identification of the exopeptidases as metalloproteases. Proteinases I and II behaved as chymotrypsin-like serine proteases, and proteinase III as a cysteine protease with a trypsin-like specificity. Molecular weight values assessed with the aid of marker proteins were as follows: aminopeptidase I, >450 kDa; aminopeptidase II, 170 kDa; carboxypeptidase, 160 kDa; proteinase I, 115 kDa; proteinase II, 32 kDa; proteinase III, 27 kDa. On incubation for 15 min they retained most of their activity up to a temperature of 90°C, with the sole exception of proteinase II, which was rapidly inactivated at 60°C. Protease content was also determined in crude extracts from cells grown in a mineral medium both to the stationary and to the exponential phase, with glucose or with yeast extract as carbon sources. No dramatic change was detected depending on the growth phase; however, carboxypeptidase level was three- to four-fold higher when yeast extract was present in the medium instead of glucose; this might suggest an involvement of this enzyme in the digestion of extracellularly available peptides.     

Timing of action of sperm proteases in ascidian fertilization

Summary The timing of action of three sperm proteases, acrosin, spermosin, and a chymotrypsin-like enzyme, in the fertilization of the ascidian,Halocynthia roretzi, was examined by adding specific protease inhibitors at various times after insemination. The results indicate that the last two enzymes both function at the early stage of the process of sperm penetration through the egg investment, while acrosin functions at the late stage.Acknowledgments. We are indebted to Dr M. Hoshi of Tokyo Institute of Technology for his helpful discussion, and to Dr T. Someno of Nippon Kayaku Kogyo Co. for his generous gifts of Z-Val-Pro-Arg-H and leupeptin. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, and from Naito Research Foundation.     

Zinc increases the longevity of unfertilized sea urchin eggs

Summary We have found that Zn²⁺ prevented lysis of unfertilized sea urchin eggs, and the eggs retained the ability to form fertilization membranes and to divide. SDS polyacrylamide gel electrophoresis showed that proteolysis of several proteins accompanied egg lysis, but Zn²⁺ inhibited this proteolysis. Therefore, Zn²⁺ blocks protease activity directly or indirectly and thereby prolongs the longevity of unfertilized sea urchin eggs.     

Proteases in apoptosis

The interleukin-1 β-converting enzyme (ICE)-like family proteases have recently been identified as key enzymes in apoptotic cell death. Among these proteases one can identify specific activities which may be involved in cytokine production or in resident protein cleavage. Several factors influence the constitutive apoptotic mechanism and may provide insight into the role of protease(s) in apoptosis. Although it appears that ICE family members play a most important role in promoting apoptotic cell death, evidence has been advanced that other proteases are also involved in sequential or parallel steps of apoptosis. Activation of a particular protease can lead to processing molecules either of the same or different proteases, leading to an activation of a protease cascade. Here we attempt to summarize the current thinking concerning these proteases and their involvement in apoptosis.     

ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae

Regulated protein degradation by ATP-dependent proteases plays a fundamental role in the biogenesis of mitochondria. Membrane-bound and soluble ATP-dependent proteases have been identified in various subcompartments of this organelle. Subunits composing these proteases are evolutionarily conserved from yeast to humans and, in support of an endosymbiotic origin of mitochondria, evolved from prokaryotic ancestors: the PIM1/Lon protease is active in the matrix of mitochondria, while the i-AAA protease and the m-AAA protease mediate the turnover of inner membrane proteins. Most of the knowledge concerning the biogenesis and the physiological role of ATP-dependent proteases comes from studies in the yeast Saccharomyces cerevisiae. Proteases were found to be required for mitochondrial stasis, for the maintenance of the morphology of the organelle and for mitochondrial genome integrity. ATP-dependent proteolysis is crucial for the expression of mitochondrially encoded subunits of respiratory chain complexes and for the assembly of these complexes. Hence, mitochondrial ATP-dependent proteases exert multiple roles which are essential for the maintenance of cellular respiratory competence.     

Snake venom thrombin-like enzymes: from reptilase to now

The snake venom thrombin-like enzymes (SVTLEs) comprise a number of serine proteases functionally and structurally related to thrombin. Until recently, only nine complete sequences of this subgroup of the serine protease family were known. Over the past 5 years, the primary structure of several SVTLEs has been characterized, and now this family includes several members. Of particular interest is their possible use in pathologies such as thrombosis. The aim of the present review is to summarize the state of the art concerning the evolutionary, structural and biological features of the SVTLEs.Received 16 August 2003; received after revision 26 September 2003; accepted 1 October 2003     

Protein digestion in the sea gooseberryPleurobranchia bachei A. Agassiz (Ctenophora: Tentaculata)

Summary Peaks of proteolytic activity of pharyngeal juice occur at pH 5.75 and pH 7.5. The proteases responsible for digestion include a tryptic alkaline protease and a thiol-activated acid protease which is probably cathepsin B. Levels of proteolytic activity parallel those of other carnivorous invertebrates which feed on zooplankton.This study was supported by operating grants from the National Research Council of Canada.We are grateful to Betsy Sweeney for her technical assistance.     

Dynorphin-degrading cysteine protease is highly specific for paired arginine residues.

The cleavage of dynorphin and three analogs containing paired basic residues by several proteases was investigated. The cysteine protease of neuroblastoma cells cleaved only the bond between Arg-Arg residues. Submandibular arginyl-endopeptidase, however, cleaved bonds between both Arg-Arg and Arg-Lys residues, and pancreatic trypsin at the carboxyl sides of both arginine and lysine residues. This shows that the cysteine protease is highly specific for paired arginine residues.     

Dynorphin-degrading cysteine protease is highly specific for paired arginine residues

The cleavage of dynorphin and three analogs containing paired basic residues by several proteases was investigated. The cysteine protease of neuroblastoma cells cleaved only the bond between Arg-Arg residues. Submandibular arginylendopeptidase, however, cleaved bonds between both Arg-Arg and Arg-Lys residues, and pancreatic trypsin at the carboxyl sides of both arginine and lysine residues. This shows that the cysteine protease is highly specific for paired arginine residues.     

Sperm chymotrypsin-like enzymes of different inhibitor-susceptibility as lysins in ascidians

Inhibitory effects of three peptidyl phenylalaninals on fertilization and on chymotrypsin-like enzyme activity of sperm in three species of ascidians were examined. The results suggest that a sperm chymotrypsin-like enzyme is indispensable for the fertilization in each of the ascidians, and that these enzymes have different susceptibilities to inhibitors.     

Aplysianin-A, an antibacterial and antineoplastic glycoprotein in the albumen gland of a sea hare, Aplysia kurodai

Aplysianin-A, an antibacterial and antineoplastic factor in the albumen gland of the sea hare Aplysia kurodai, was isolated. It had a molecular weight of approximately 320 kD and consisted of subunits with a molecular weight of 85 kD. It contained 9.8% neutral sugar. Aplysianin A showed 50% inhibition of Bacillus subtilis growth at a concentration of 4 microgram protein/ml and 50% lysis of murine MM46 tumor cells at 14 ng protein/ml. A partial identity of antigenic specificity of the purified specimen with an antineoplastic factor from Aplysia eggs was observed in immunodiffusion tests.     

Deciphering cryptic proteases

Proteases are deeply involved in physiology and pathology. For most, the mechanism is well defined but several fail to display typical protease features (as is the case of the four proteases contained in fibronectin, the inhibitor-resistant mesotrypsin and the proteosomal deubiquitinating enzyme) or have unclear physiological function (such as calpain-like proteins, transthyretin and factor seven activating protease). In other cases, such as in peroxisomal processing proteases, although substrates are defined, the enzyme remains undiscovered. Furthermore, several proteases were identified in pathological conditions, namely secretases in Alzheimers disease and gross cystic disease fluid protein 15 kDa in breast cancer, when most likely their physiological substrate is still hidden. Lastly, the evolutionary conservation of proteolytic enzymes raises questions related to the origin of biological events, such as the origin of cystein proteases and cell death responses. In this review we will discuss the above cryptic enzymes, as they will probably be relevant in the future.Received 7 December 2004; received after revision 5 January 2005; accepted 10 January 2005 Available online 09 March 2005     

Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins

Three antibacterial proteins were isolated from acid extracts of channel catfish (Ictalurus punctatus) skin by cation exchange chromatography and reverse-phase high-pressure liquid chromatography. The molecular masses of the proteins were 15.5, 15.5 and 30 kD as determined by SDS-polyacrylamide gel electrophoresis. Mass spectrometry, amino acid composition and amino acid sequence data suggest that the most abundant protein is closely related to histone H2B. The H2B-like protein was inhibitory to Aeromonas hydrophila and Saprolegnia spp., which are important bacterial and fungal pathogens of fish. These findings suggest that histones may be important defensive molecules in fish. Received 22 December 1997; received after revision 5 March 1998; accepted 5 March 1998     

Sperm chymotrypsin-like enzymes of different inhibitor-susceptibility as lysins in ascidians

Summary Inhibitory effects of three peptidyl phenylalaninals on fertilization and on chymotrypsin-like enzyme activity of sperm in three species of ascidians were examined. The results suggest that a sperm chymotrypsin-like enzyme is indispensable for the fertilization in each of the ascidians, and that these enzymes have different susceptibilities to inhibitors.12 November 1986Acknowledgments. We are indebted to Dr M. Hoshi of Tokyo Institute of Technology for his helpful discussion. This work was supportd by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

Effect of relative humidity on the free-living stages of Strongyloides papillosus (Rhabdiasoidea: Nematoda)

The eggs and larvae are affected by the changes in the humidity level around them. The eggs do not develop below 87% relative humidity at 30 degrees C and 25 degrees C. At sub-developmental rel. hum. they remain viable for 30 h at 81%, but at 73% rel. hum. level they do not survive beyond 18 h. Survival of larvae in 100% rel. hum. is longer at 30 degrees C than at 25 degrees C and 35 degrees C, and also they exhibit a poor resistance to desiccation.     

Regulated protein degradation in mitochondria

Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis. The matrix-localized PIM1 protease, a homologue of theEscherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity. Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria. The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, them- and thei-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins. In addition to its proteolytic activity, them-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes. It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes.     

Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63Ag8 cells

Using the suspension cell line P3X63Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (Mr 40 kD) and Semliki Forest virus core protein (Mr 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0 degrees C. Subsequent resealing of the pores was completed after a 5-min incubation at 37 degrees C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37 degrees C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K+, Mg2+, and Ca2+) and resealing (in the presence of K+, Mg2+, and Ca2+), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure.     

Recent progress in understanding the diversity of the human ov-serpin/clade B serpin family

The inhibitory mechanism against proteases is important in the maintenance of homeostasis or health in the body. The human ovalbumin serpin (ovserpin)/ clade B serpin family is one group of the human serpins, a family of serine protease inhibitors. They have acquired diversity in the profiles of target proteases, inhibitory mechanisms, and localization patterns during their evolution. Most serpins target serine proteases, however, some ov-serpins target only cysteine proteases or both serine and cysteine proteases and furthermore, several ov-serpins do not possess inhibitory activities. Although the ov-serpins act primarily as intracellular serpins, some show extracellular and nuclear localizations. Such diversity enables the ov-serpins to play multiple physiological roles in the body. Recent analyses have revealed that the functions of human ov-serpins are more diversified than we previously knew. In this article, we describe recent progress in our understanding of how the human ov-serpin/clade B serpin family demonstrates diversity.     

Effect of relative humidity on the free-living stages ofStrongyloides papillosus (Rhabdiasoidea: Nematoda)

Summary The eggs and larvae are affected by the changes in the humidity level around them. The eggs do not develop below 87% relative humidity at 30 °C and 25 °C. At sub-developmental rel. hum. they remain viable for 30 h at 81%, but at 73% rel. hum. level they do not survive beyond 18 h. Survival of larvae in 100% rel. hum. is longer at 30 °C than at 25 °C and 35 °C, and also they exhibit a poor resistance to desiccation.Acknowledgment. The author is grateful to Dr. Devendra Prasad of Patna University for his supervision and constant help in the work. The assistance of State University Grants Commission through a research scholarship is gratefully acknowledged.