Salinity-mediated carbonic anhydrase induction in the gills of the euryhaline green crab, Carcinus maenas

The euryhaline green crab, Carcinus maenas, is a relatively strong osmotic and ionic regulator, being able to maintain its hemolymph osmolality as much as 300 mOsm higher than that in the medium when the crab is acclimated to low salinity. It makes the transition from osmoconformity to osmoregulation at a critical salinity of 26 ppt, and new acclimated concentrations of hemolymph osmotic and ionic constituents are reached within 12 h after transfer to low salinity. One of the central features of this transition is an 8-fold induction of the enzyme carbonic anhydrase (CA) in the gills. This induction occurs primarily in the cytoplasmic pool of CA in the posterior, ion-transporting gills, although the membrane-associated fraction of CA also shows some induction in response to low salinity. Inhibition of branchial CA activity with acetazolamide (Az) has no effect in crabs acclimated to 32 ppt but causes a depression in hemolymph osmotic and ionic concentrations in crabs acclimated to 10 ppt. The salinity-sensitive nature of the cytoplasmic CA pool and the sensitivity of hemolymph osmotic/ionic regulation to Az confirm the enzyme's role in ion transport and regulation in this species. CA induction is a result of gene activation, as evidenced by an increase in CA mRNA at 24 h after transfer to low salinity and an increase in protein-specific CA activity immediately following at 48 h post-transfer. CA gene expression appears to be under inhibitory control by an as-yet unidentified repressor substance found in the major endocrine complex of the crab, the eyestalk.     

Differential induction of branchial carbonic anhydrase and NA(+)/K(+) ATPase activity in the euryhaline crab,Carcinus maenas,in response to low salinity exposure

The time course of induction of activity of carbonic anhydrase (CA) and Na/K ATPase, two enzymes that are central to osmotic and ionic regulation in the eyryhaline green crab, Carcinus maenas, was measured in response to a transfer from 32 to 10 ppt salinity. CA activity was low in all gills in crabs acclimated to high salinity. Activity was induced in the posterior three gills (G6-G9) starting at 96 hr following transfer to low salinity, with activity peaking at seven post-transfer. Na/K ATPase activity in posterior gills was already high in crabs acclimated to 32 ppt salinity, and it did not increase as a result of transfer to 10 ppt. Acclimation of crabs to hypersaline (40 ppt) conditions resulted in uniformly low levels of Na/K ATPase activity, and transfer from 40 ppt to 10 ppt stimulated a four-fold induction of activity in the posterior gills that was evident by seven days of low salinity exposure. Low salinity stimulates the activity of both enzymes, but a different degree of salinity change appears to be necessary to cause the induction of each enzyme. The Na/K ATPase activity is already high at a salinity (32 ppt) at which the crab is still an osmotic and ionic conformer. CA activity, however, even when expressed in low levels, is still present in excess of what is needed to supply counterions at a rate adequate to match the rate of active ion transport. It is possible that two strategies exist for the regulation of these two enzymes that coincide with the crab's intertidal and estuarine lifestyle: short-term modulation of activity of highly expressed enzyme (Na/K ATPase) and long-term modulation of enzyme concentration by changes in gene expression (CA). For all ranges of low salinity exposure, crabs undergo hemodilution, cell swelling, and subsequent cell volume readjustment as evidenced by the increase in concentration of TNPS in the hemolymph. This response takes place before the induction of enzyme activity, and it could serve as the initial signal in the induction pathway.     

Substrate–Inhibitor Analysis of Cholinesterase from Hemolymph of the Pacific Gastropod Mollusc Neptunea eulimata

Based on our own and literature data, a detailed substrate–inhibitor analysis of catalytic properties of cholinesterase of hemolymph of the Pacific gastropod mollusc Neptunea eulimata has been carried out. Using specific substrates and organophosphorus inhibitors, homogeneity of the enzyme preparation has been shown. The study of the substrate specificity, using 8 substrates—choline, fluorogenic and chromogenic esters—has revealed some features of similarity with mammalian erythrocyte cholinesterase. At the same time, testing of a large group (33 compounds) of organophosphorus inhibitors with various structure, including hydrophobic inhibitors studied for the first time, has established both quantitative and qualitative differences from the mammalian blood enzymes. It is concluded that cholinesterase from the neptunea hemolymph cannot be ascribed unanimously to the type of acetylhydrolases of acetylcholine (EC 3.1.1.7).     

Purification and characterization of a hemocyte proteinase of Sarcophaga, possibly participating in elimination of foreign substances.

We have reported that foreign protein injected into the abdominal cavity of Sarcophaga peregrina (flesh fly) larvae is degraded in the hemolymph by a proteinase secreted by hemocytes [Suzuki, T. and Natori, S. (1985) Comp. Biochem. Physiol. 81A, 191-193]. Here we report the purification and characterization of a proteinase from larval hemocytes. This enzyme is a cysteine proteinase consisting of 26-kDa and 29-kDa subunits with similar substrate specificity to mammalian cathepsin B. This enzyme was shown to be released from hemocytes into the hemolymph of larvae following injection of sheep red blood cells into the larvae, suggesting that it participates, at least in part, in elimination of foreign substances introduced into the body cavity.     

Enhanced enzymatic hydrolysis of langostino shell chitin with mixtures of enzymes from bacterial and fungal sources

A combination of enzyme preparations from Trichoderma atroviride and Serratia marcescens was able to completely degrade high concentrations (100 g/L) of chitin from langostino crab shells to N-acetylglucosamine (78%), glucosamine (2%), and chitobiose (10%). The result was achieved at 32 degrees C in 12 days with no pre-treatment (size reduction or swelling) of the substrate and without removal of the inhibitory end-products from the mixture. Enzymatic degradation of three forms of chitin by Serratia/Trichoderma and Streptomyces/Trichoderma blends was carried out according to a simplex-lattice mixture design. Fitted polynomial models indicated that there was synergy between prokaryotic and fungal enzymes for both hydrolysis of crab chitin and reduction of turbidity of colloidal chitin (primarily endo-type activity). Prokaryotic/fungal enzymes were not synergistic in degrading chitosan. Enzymes from prokaryotic sources had much lower activity against chitosan than enzymes from T. atroviride.     

Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln(181) as important for enzymatic activity and substrate specificity. Replacement of Gln(181) by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln(181) is critical for the enzymatic activity and substrate specificity of ERAP-1.     

Brachyurins--serine collagenolytic enzymes from crabs

The properties of brachyurins, proteolytic enzymes belonging to a new subfamily of chymotrypsin-like proteases, are considered. These enzymes, found in various species of crustacean, exhibit mixed substrate specificity and a marked collagenolytic activity. The enzymatic and physicochemical properties of brachyurins I and their primary and spatial structures are discussed in detail. A separate chapter is devoted to the preparations of collagenases from the hepatopancreas of king crab: their action on the damaged skin and use in medicine.     

Degradation of adipokinetic hormone family peptides by a circulating endopeptidase in the insect Manduca sexta.

The hemolymph (blood) of the Lepidopteran insect Manduca sexta contains an endopeptidase that metabolizes the nonapeptide Manduca adipokinetic hormone. In contrast to the situation in other insects, where the major site of inactivation is the Malpighian tubules (excretory organs), in Manduca the capacity of the hemolymph to metabolize adipokinetic hormone is comparable to that of the Malpighian tubules. The hemolymph enzyme cleaves Manduca adipokinetic hormone (pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-Gly-NH2) to give the fragment pGlu-Leu-Thr-Phe-Thr. Other fragments were not positively identified. The enzyme is present in the plasma and not in hemocytes, and occurs at similar levels in the hemolymph of larvae, pupae and adults. The enzyme is inactivated by boiling, has a neutral pH optimum (7.0-7.5), and an estimated molecular weight of 66 kDa. The enzyme was strongly inhibited by inhibitors of metalloprotease activity (EGTA and 1,10-phenanthroline), but not by serine protease inhibitors. The enzyme was capable of metabolizing a number of AKH family peptides with varying sequences around the presumed site of cleavage. An accurate assessment of enzyme kinetics was not possible with the assay method used, but the enzyme was not saturated at a substrate concentration of 10 microM, and the value of Km must be at least 1 microM. It is possible that the enzyme may represent a low affinity system of peptide removal rather than the principal means of inactivation.     

Brachyurins, Serine Collagenolytic Enzymes from Crabs

The properties of brachyurins, proteolytic enzymes belonging to a new subfamily of chymotrypsin-like proteases, are considered. These enzymes, found in various species of crustacean, exhibit mixed substrate specificity and a marked collagenolytic activity. The enzymatic and physicochemical properties of brachyurins I and their primary and spatial structures are discussed in detail. A separate chapter is devoted to the preparations of collagenases from the hepatopancreas of king crab: their action on the damaged skin and use in medicine.     

Enzymatic activities in European flat oyster, Ostrea edulis, and pacific oyster, Crassostrea gigas, hemolymph

Enzymatic activities in the hemolymph of healthy and Bonamia-infected Ostrea edulis and Crassostrea gigas were studied with a commercial kit for the detection of 19 enzymes: 15 and 16 enzymes, respectively, were detected in the hemolymph of O. edulis and C. gigas and 10 of them showed relatively high activity levels. Most of them existed in both the cell-free fraction of the hemolymph and in the hemocytes. The cell-free hemolymph fraction of Bonamia ostreae-infected European flat oysters showed an elevated enzymatic activity level compared with that of healthy individuals. C. gigas hemocytes possessed higher enzymatic activity levels than O. edulis hemocytes. Differences in enzymatic activities existed in granulocytes and hyalinocytes in both oyster species. The enzyme release from oyster hemocytes seemed to be selective. The infection by B. ostreae induced enzymatic activity variations in European flat oysters. Higher enzyme levels within hemocytes may contribute partly to the natural resistance of C. gigas to the infection by B. ostreae.     

Invertebrate red blood cell carbonic anhydrase

This is the first report documenting the presence of carbonic anhydrase (CA) for any invertebrate red cells. CA activity was measured in plasma, hemolysates of blood cells, and in hemolymph of selected species of invertebrates. Annelid red blood cells (RBC) and sipunculid pink blood cells both possessed significant levels of CA activity. Molluscan RBC, on the other hand, lacked CA activity. The distribution appears to have fallen along phylogenetic lines, with CA being present only in blood cells of the two more closely related groups. However, the presence of extracellular CA was confirmed in oyster hemolymph. Oyster hemolymph CA showed a similar affinity (Ki) for the sulfonamide inhibitors acetazolamide and ethoxzolamide, as did the vertebrate RBC CA II isozyme, supporting the idea that this isozyme could be the ancestral form of the enzyme.     

Hemolymph cholinesterase activity in the Mussel <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (Dunker, 1853) that was exposed to adverse natural and anthropogenic conditions

The influence of habitat conditions on the activity, the structure of the substrate specificity (the ratio of the substrate hydrolysis rates), and the kinetic parameters of substrate hydrolysis due to the effect of hemolymph cholinesterase of the mussel Crenomytilus grayanus was studied. Mussels were collected from areas that are influenced by seasonal and stationary upwelling, as well as from a polluted area. Upwelling and anthropogenic pressure were shown to alter the structure of hemolymph cholinesterase substrate specificity in mussels, up to complete loss of the ability to catalyze the hydrolysis of propionyland butyrylthiocholine. It was established that during the seasonal upwelling the efficiency of the cholinergic process in mussels is provided by a wide range of effective concentrations of the substrates and by decreasing their affinity to the enzyme. Under the conditions of chronic anthropogenic pollution, the cholinesterase of the mussel hemolymph loses its ability to hydrolyze substrates other than acetylthiocholine.     

Crab digestive lipase acting at high temperature: purification and biochemical characterization

In recent years, recovery and characterization of enzymes from fish and aquatic invertebrates have taken place and this had led to the emergence of some interesting new applications of these enzymes. However, much less is known about lipases from crustaceans. A lipolytic activity was located in the crab digestive glands (hepatopancreas), from which a crab digestive lipase (CDL) was purified. Pure CDL has a molecular mass of 65kDa as determined by SDS/PAGE analysis. Unlike known digestive lipases, CDL displayed its maximal activity on long and short-chain triacylglycerols at a temperature of 60 degrees C. A specific activity of 500U/mg or 130U/mg was obtained with TC(4) or olive oil as substrate, respectively. Only 10% of the maximal activity was detected at 37 degrees C. The enzyme retained 80% of its maximal activity when incubated during 10 min at 60 degrees C, and was completely inactivated at a temperature higher than 65 degrees C. Interestingly, neither colipase, nor bile salts were detected in the crab hepatopancreas. Which suggests that colipase evolved in invertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produce bile salts. No similarity between the 13 N-terminal amino acid residues of CDL was found with those of known other digestive lipases.     

Estimation of total hemolymph volume in the horseshoe crab Limulus polyphemus

Biomedical companies extract blood from the horseshoe crab, Limulus polyphemus, for the production of Limulus Amebocyte Lysate, used worldwide for detecting endotoxins in injectable solutions and medical devices. Despite the extensive use of horseshoe crabs by the biomedical industry, total hemolymph volume for this species is not known. The hemolymph volume of 60 adult horseshoe crabs was estimated using an inulin dilution technique. Blood volume of the horseshoe crab represented as a percentage of wet body weight was 25?±?2.2% for males and 25?±?5.1% (mean?±?SD) for females. Relationships between hemolymph volume and weight (p?=?0.0026, r ²?=?0.8762), hemolymph volume and prosomal width (p?<?0.0001), and hemolymph volume and inter-ocular width (p?<?0.0001) were observed. No significant differences were observed between males and females. The relationship of animal size and hemolymph volume can be used to predict how much blood can be drawn from horseshoe crabs used by the biomedical industry, and can be of further use in future bleeding mortality studies.     

Degradation of collagen substrates by a trypsin-like serine protease from the fiddler crab Uca pugilator

The collagenolytic properties of a trypsin-like protease from the hepatopancreas of the fiddler crab Uca pugilator have been examined. All collagen types, I-V, were attacked by this enzyme. Types III and IV were degraded much more rapidly than types I, II, and V. Crab protease produced multiple cleavages in the triple helix of each collagen at 25 degrees C; only in the case of type III collagen, however, was a major cleavage observed at a 3/4:1/4 locus that corresponded to the region of collagen susceptibility to mammalian collagenase action. Additionally, both the affinity and the specific activity of the crab protease for native collagen were lower than those which characterize mammalian collagenase. The results of this study, in conjunction with a previous report on the collagenolytic activity of another serine protease from the fiddler crab [Welgus, H. G., Grant, G. A., Jeffrey, J. J., & Eisen, A. Z. (1982) Biochemistry 21, 5183], suggest that the following properties distinguish the action of these invertebrate collagenolytic enzymes from the metalloenzyme collagenases of mammals: (1) broad substrate specificity, including both noncollagenous proteins and collagen types I-V; (2) ability to cleave the native triple helix of collagen at multiple loci; (3) reduced affinity or higher Km for collagen; and (4) lower specific activity on collagen fibrils.     

Cold-adapted and mesophilic brachyurins

Two different types of brachyurins, termed I and II, have been described in the literature. Within type I there are two subtypes, Ia and Ib. The prototype for the type I brachyurins is Fiddler crab collagenase I. Its cold-adapted analogue from Antarctic krill, termed euphaulysin, shares many of its characteristics. Both enzymes are distinguished by their broad substrate specificity as well as the ability to cleave collagen. The precursor form of euphaulysin has been expressed in Pichia pastoris and processed to its fully active form using cod trypsin. A molecular model of euphaulysin, based on the known crystal structure of crab collagenase I, indicates that the core structure of these enzymes is almost identical. As a cold-adapted enzyme, euphaulysin has a higher catalytic efficiency than crab collagenase I. It is also more sensitive to thermal inactivation and autolysis. Furthermore, euphaulysin has an increased length of several surface loops compared to crab collagenase I. Extended surface loops have been suggested to play a role in the cold activity of some bacterial enzymes. Sensitivity to autolysis is an important factor which contributes to the thermal instability of euphaulysin. Substitution of a highly exposed residue in the 'autolysis loop' of euphaulysin resulted in an increased stability of the enzyme towards thermal inactivation without altering its catalytic efficiency.     

Studies into factors contributing to substrate specificity of membrane-bound 3-ketoacyl-CoA synthases.

We are interested in constructing a model for the substrate-binding site of fatty acid elongase-1 3-ketoacyl CoA synthase (FAE1 KCS), the enzyme responsible for production of very long chain fatty acids of plant seed oils. Arabidopsis thaliana and Brassica napus FAE1 KCS enzymes are highly homologous but the seed oil content of these plants suggests that their substrate specificities differ with respect to acyl chain length. We used in vivo and in vitro assays of Saccharomyces cerevisiae-expressed FAE1 KCSs to demonstrate that the B. napus FAE1 KCS enzyme favors longer chain acyl substrates than the A. thaliana enzyme. Domains/residues responsible for substrate specificity were investigated by determining catalytic activity and substrate specificity of chimeric enzymes of A. thaliana and B. napus FAE1 KCS. The N-terminal region, excluding the transmembrane domain, was shown to be involved in substrate specificity. One chimeric enzyme that included A. thaliana sequence from the N terminus to residue 114 and B. napus sequence from residue 115 to the C terminus had substrate specificity similar to that of A. thaliana FAE1 KCS. However, a K92R substitution in this chimeric enzyme changed the specificity to that of the B. napus enzyme without loss of catalytic activity. Thus, this study was successful in identifying a domain involved in determining substrate specificity in FAE1 KCS and in engineering an enzyme with novel activity.     

Substrate specificity of beta-primeverosidase, a key enzyme in aroma formation during oolong tea and black tea manufacturing

We synthesized nine kinds of diglycosides and a monoglycoside of 2-phenylethanol to investigate the substrate specificity of the purified beta-primeverosidase from fresh leaves of a tea cultivar (Camellia sinensis var. sinensis cv. Yabukita) in comparison with the apparent substrate specificity of the crude enzyme extract from tea leaves. The crude enzyme extract mainly showed beta-primeverosidase activity, although monoglycosidases activity was present to some extent. The purified beta-primeverosidase showed very narrow substrate specificity with respect to the glycon moiety, and especially prominent specificity for the beta-primeverosyl (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosyl) moiety. The enzymes hydrolyzed naturally occurring diglycosides such as beta-primeveroside, beta-vicianoside, beta-acuminoside, beta-gentiobioside and 6-O-alpha-L-arabinofuranosyl-beta-D-glucopyranoside, but were unable to hydrolyze synthetic unnatural diglycosides. The purified enzyme was inactive toward 2-phenylethyl beta-D-glucopyranoside. The enzyme hydrolyzed each of the diglycosides into the corresponding disaccharide and 2-phenylethanol. These results indicate the beta-primeverosidase, a diglycosidase, to be a key enzyme involved in aroma formation during the tea manufacturing process.     

Thiol-activated serine proteinases from nymphal hemolymph of the African migratory locust,Locusta migratoria migratorioides

Two unique serine proteinase isoenzymes (LmHP-1 and LmHP-2) were isolated from the hemolymph of African migratory locust (Locusta migratoria migratorioides) nymphs. Both have a molecular mass of about 23 kDa and are activated by thiol-reducing agents. PMSF abolishes enzymes activity only after thiol activation, while the cysteine proteinase inhibitors E-64, iodoacetamide, and heavy metals fail to inhibit the thiol-activated enzymes. The N-terminal sequence was determined for the more-abundant LmHP-2 isoenzyme. It exhibits partial homology to that of other insect serine proteinases and similar substrate specificity and inhibition by the synthetic and protein trypsin inhibitors pABA, TLCK, BBI, and STI. The locust trypsins LmHP-1 and LmHP-2 constitute a new category of serine proteases wherein the active site of the enzyme is exposed by thiol activation without cleavage of peptide bonds.     

Glyceraldehyde-3-phosphate dehydrogenase of horseshoe crab (Tachypleus tridentatus).

Glyceraldehyde-3-phosphate dehydrogenase [ED 1.2.1.12] was purified from the horseshoe crab, a living fossil, and its properties were examined. 1 The purified enzyme was homogeneous as judged by various tests. The enzyme, like enzymes from other sources, was a tetramer with a subunit molecular weight of 36,000. The kinetic parameters and pH optimum were also similar to those of other enzymes, though the enzyme was more stable against heat and pH denaturations. 2 Analysis of SH groups showed that there were 4 SH groups per subunit, one of which was essential for the enzyme activity and was highly reactive. 3. CD spectra of the enzyme suggested that the enzyme had a very high content of beta-structure (ca. 45 per cent). 4. The horseshoe crab enzyme could form a hybrid in vitro with the rabbit muscle enzymes in concentrated salt solution at acidic pH. 5. There results indicate that the enzyme has overall structural similarity to other enzymes and that the enzyme is highly conserved during a long period of evolution. Some discussions on the structure and activity of the horseshoe crab enzyme are made in comparison with the enzymes from other sources.