Comparative study of proteolytic enzymes in the digestive tracts of the european sea bass and hybrid striped bass reared in freshwater

• 1.1. A comparative study of the proteolytic activity in four different sections of the digestive tracts of the European sea bass (Dicentrarchus labrax) and hybrid striped bass (Morone chrysops × M. saxatilis) reared in freshwater revealed minor differences between these fish.
• 2.2. Tryptic activity plays a major role in the proteolytic process in both fish.
• 3.3. The activity of seven intestinal proteolytic enzymes was detected utilizing a combination of specific substrates and inhibitors.
• 4.4. High levels of proteolytic activity were detected in both the proximal and distal sections of the fish intestine at a high pH range (9–10).
• 5.5. In situ monitoring of pH levels revealed a lower pH level in the intestinal proximal section of hybrid striped bass compared with the distal section.
• 6.6. In contrast, higher pH levels were detected at the proximal compared with the distal sections of D. labrax intestine.

     

Activities of the three ammonia-forming enzymes in the tissues of the brackish-water bivalve Corbicula japonica

• 1.1. Activities of the three ammonia-forming enzymes, glutamate dehydrogenase, AMP deaminase and serine dehydrase (SerDH), were measured in tissues of gill, digestive diverticula, mantle and foot muscle of the brackish-water bivalve Corbicula japonica.
• 2.2. High levels of SerDH activity were detected in gill and digestive diverticula, while the activity levels of the other two enzymes were low.
• 3.3. The result suggests the significance of SerDH in amino acid degradation of this species.

     

Trypanosoma cruzi and Trypanosoma rangeli: Glutamate dehydrogenases and proteolytic activities

• 1.1. Trypanosoma (Herpetosoma) rangeli contains proteolytic activity with azocasein, casein and BAPA as substrates, and aminopeptidase activity with Arg-BNA as substrate. The respective pH optima were 5.5, 7.0, 8.5 and 7.0.
• 2.2. The effect of the protease inhibitors PMSF, TLCK and trasylol was studied. 0.5 mM TLCK caused considerable inhibition of all these activities, whereas 1 mM PMSF was much less effective. Trasylol (0.14 mg/ml) inhibited the activities with azocasein and Arg-BNA as substrates.
• 3.3. Trypanosoma (Schizotrypanum) cruzi contains very similar proteolytic activities, with some slight differences in pH optima and in response to inhibitors. Thus Trasylol was not effective on any activity, and the activity on Arg-BNA was little sensitive to TLCK.
• 4.4. The levels of these enzymes, and also of the NAD- and NADP-linked glutamate dehydrogenases, were studied in six stocks of T. (Sch.) cruzi and three stocks of T. (H.) rangeli. There was no significant difference in the NAD-gluDH, nor in the proteolytic activity with BAPA. On the other hand, the other enzymes tested presented differences which ranged from about 3-fold for the aminopeptidase to nearly 100-fold for the NADP-gluDH. The electrophoretic behaviour of the latter was identical in all the stocks of both species, thus showing that the difference was only quantitative.
• 5.5. The three stocks of T. (H.) rangeli were more similar to some T. (Sch.) cruzi stocks (Tul 2) than the latter were to each other, thus emphasizing at a biochemical level the similarities between these two species.

     

Digestive proteases in the midgut gland of the atlantic blue crab,Callinectes sapidus

• 1.1. Digestive proteases from the midgut gland of male Atlantic blue crabs, Callinectes sapidus, were investigated. Tentative identities of proteolytic enzymes were determined with synthetic substrates and inhibitors.
• 2.2. Trypsin, chymotrypsin, carboxypeptidase A and B and leucine aminopeptidase activities were found and quantified.
• 3.3. Activity against Succinyl-(Ala)₃-nitroanalide was also found. This as yet unidentified enzyme has a mol. wt of about 26,000 and has elastolytic activity.

     

Alterations in proteases,protease inhibitors and ecdysone levels: a profile of stress in insects

• 1.1. A comparison of proteolytic and protease inhibitory activity, and ecdysteroid levels in body fluids was made between normal larvae of the flesh fly, Sarcophaga bullata, and those that had been water-stressed for two days.
• 2.2. The course of proteolytic activity in water stressed flies decreases 6 hr after beginning the experiment and remains low in comparison with control flies.
• 3.3. The course of protease inhibitors exhibits a mirror image pattern to proteases.
• 4.4. Ecdysteroid pattern shows two peaks in control animals: minor at 24 hr and major at pupariation, in experimental animals: at 1 hr, at 6 hr and at white pupal stage.

     

Changes in glycolytic and associated enzyme activities during rana ridibunda oocyte development

• 1.1. Most of glycolytic and associated enzymes in the oocytes of the frog Rana ridibunda exhibit a higher activity at the early growth stages; the activity declines by the time the oocyte reaches full growth. Citrate syntase follows a similar pattern.
• 2.2. Enzymes related to gluconeogenesis have non-detectable activity.
• 3.3. It is suggested that at the early stages of oocyte growth glycogen could contribute as a fuel mainly for the pentose phosphate pathway; in the full-grown oocyte glycogen could serve mainly as a fuel for the glycolytic pathway.

     

The fate of the bowman-birk trypsin inhibitor from soybeans in the digestive tract of chicks

• 1.1. Intestinal absorption of the Bowman-Birk trypsin inhibitor from soybeans was studied comparatively in chicks by direct follow-up of absorbed radioactive inhibitor and by radioimmunological estimation of the unlabelled inhibitor.
• 2.2. Initial studies, performed in vitro by the inverted sac technique, suggested that both the native inhibitor and its degradation products were absorbed.
• 3.3. However, the results obtained for in situ and in vivo experiments indicated that the absorption of the native inhibitor is negligible.
• 4.4. Most of the inhibitor is degraded during its passage through the intestine, and the majority of the degradation products is excreted in the feces.
• 5.5. The effects of ingested inhibitor on pancreas enlargement and on secretion of pancreatic enzymes stems from an indirect effect on the intestine.

     

Hypocalcemic factor in the ultimobranchial gland of the frog,Rana rugosa

• 1.1. The hypocalcemic activity of the ultimobranchial gland of the frog, Rana rugosa, was estimated using a rat bioassay method.
• 2.2. Extracts of the ultimobranchial gland showed a very high hypocalcmic activity. The value corresponded to 6,340 mU (MRC)/kg b.w.
• 3.3. Serum inorganic phosphorus values of rats received the extract decreased in proportion to the dose, although no changes were found in serum sodium concentration.

     

Effect of developmental derangements on the proteolytic and protease-inhibitory activities in Galleria mellonella (Insecta)

• 1.1. Protease inhibitory activity in the whole body homogenates of Galleria mellonella larvae exhibits maxima at the beginning of the last larval and pupal instars. Injury, chilling, immobilization, and ligations of larvae cause an increase of inhibition.
• 2.2. The inhibitory activity is high in the haemolymph but low in midgut and faty body. By contrast, the proteolytic activity is low in haemolymph and high in both midgut and fat body.
• 3.3. Starvation and ligations cause a dramatic fall of the proteolytic activity and increase of the inhibitory activity in examined organs.

     

Tyrosine aminotransferase activity of frog (Rana esculenta) liver—III. A circannual study

• 1.1. A circannual study of tyrosine aminotransferase and other metabolic enzymes in frog liver is reported. The subcellular distribution of all enzymatic activities under investigation was also studied.
• 2.2. Results show significant oscillations of all enzymatic activities throughout the year; in particular tyrosine aminotransferase has a marked summer maximum.
• 3.3. The subcellular distribution of tyrosine aminotransferase shows significant variations: the soluble activity of the enzyme presents a bimodal circannual distribution, which has its counterpart in an increased activity of heavier fractions.

     

Purification of two lipases with high phospholipase A1 activity from guinea-pig pancreas

• 1.1. Two cationic lipases (Ia and Ib) were purified from homogenates of fresh guinea-pig pancreas by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose (twice for the latter) followed by gel filtration on Sephadex G-100.
• 2.2. Both enzymes were homogeneous upon polyacrylamide gel electrophoresis. Their molecular weights are 37000 and 42000 for lipases Ia and Ib, respectively, as determined by gel filtration on Sephadex G-100. Very close values for isoelectric points were found in the pH range 9.3–9.4.
• 3.3. The cationic lipases are characterized by a high phospholipase A activity (500 IU/mg protein using a potentiometric assay with egg yolk lecithin as substrate), resulting in an unusual phospholipase/lipase activity ratio of 1.
• 4.4. Using doubly labelled phosphatidylcholine, a specificity, A₁, was described for the two enzymes, which are unaffected by N-ethylmaleimide, diisopropylfluorophosphate and p-bromophenacylbromide. The enzymes are insensitive to EDTA and slightly inhibited by CaCl₂and MgCl₂, whereas sodium deoxycholate is required for maximal activity.

     

Bioassay and proteolytic activity of digestive enzymes from octopus saliva

• 1.1. A bioassay for octopus saliva, based on detachment of crab dactylopodite flexor muscle under standard conditions, has been developed.
• 2.2. There is a direct relationship between increasing caseinolytic activity of saliva from Eledone cirrhosa and decreasing muscle detachment time.
• 3.3. Fractionation of saliva, using preparative isoelectric focusing, shows that muscle releasing activity is restricted to fractions containing proteins with high isoelectric points and maximum caseinase activity.
• 4.4. It is concluded that proteolytic enzyme(s) in octopus saliva selectively release crab muscle from attachment to the carapace.

     

Amino acid composition and the protein form of procarboxypeptidase a purified from the pancreas of the sei whale Balaenoptera bolealis

• 1.1. Procarboxypeptidase (W-PCPA) was purified from the pancreas of the sei whale Balaenoptera bolealis.
• 2.2. W-PCPA was obtained as a homogeneous protein in polyacylamide gel disc electrophoresis.
• 3.3. W-PCPA has a molecular weight of 75,000.
• 4.4. Amino acid composition of W-PCPA was compared with that of bovine procarboxypeptidase as A S5 (PCPA-S5).
• 5.5. W-PCPA may be two subunits, and the aggregate form may resemble PCPA-S5.

     

Proteolysis post mortem in North Atlantic krill

• 1.1. The autoproteolytic processes in selected species of North Atlantic krill, Meganyctiphanes norvegica (M. Sars), Thysanoessa inermis (Krøyer) and T. raschii (M. Sars) have been examined at 0°C by following the release of peptides and free amino acids.
• 2.2. The krill contains high levels of peptide hydrolases, and autoproteolysis seems to be due mainly to digestive enzymes localized in the hepatopancreas and the intestinal tract of the animals.
• 3.3. During autoproteolysis the individual amino acids were generally released at rates corresponding to their proportion in the bulk protein of the krill. The major exceptions were alanine which accumulated in amounts larger than was to be expected from the composition of the krill protein, and glutamic acid/glutamine, aspartic acid/asparagine, arginine, and to some extent glycine, proline and serine, which accumulated to a lesser extent than was to be expected.
• 4.4. Storage of krill for 1 week resulted in only minor changes in the total content of amino acids as determined after acid hydrolysis, with the exception of alanine which increased in concentration.
• 5.5. The results suggest that the formation of free alanine is partly due to reactions other than proteolysis.
• 6.6. The release of free amino acids was accompanied by a considerable increase in the amount of small peptides, and glutamic acid/glutamine, aspartic acid/asparagine, glycine and proline tended to accumulate in these peptides.
• 7.7. The autoproteolytic activity of the Thysanoessa species showed seasonal variations, probably in response to food availability. In the case M. norvegica, the results suggest that there are smaller fluctuations in the level of proteolytic enzymes, probably indicating less pronounced variations in the food intake over the year.

     

Mixed function oxidase activity in the harbour seal (Phoca vitulina) from sable is., N.S.

• 1.1. One adult male, eight pups (including two full term foetuses) and nine adult female harbour seals (Phoca vitulina) were analysed for indices of mixed function oxidase (MFO) activity.
• 2.2. MFO activity was present in liver samples, but was at or below detection limits in samples of kidney, lung and pancreas.
• 3.3. Hepatic ethoxyresorufin O-de-ethylase and benzo[a]pyrene hydroxylase activities were similar to those reported in other seals and in other mammals.
• 4.4. Cytochromes P-450 and b₅ concentrations were slightly lower than those observed in other mammals.
• 5.5. MFO activities in newborn pups and foetuses were significantly lower than those in adult females.
• 6.6. No qualitative differences in cytochrome P-450 isozyme distribution between foetal and adult samples could be discerned by electrophoresis.

     

Characterization of the Melanoplus sanguinipes hemolymph after infection with Beauveria bassiana or wounding

• 1.1. The phenoloxidase activity, protein and carbohydrate levels were studied for 24 hr in the hemolymph of the migratory grasshopper, Melanoplus sanguinipes after artificial wounding of the insect cuticle or the injection of Beauveria bassiana conidia.
• 2.2. Injection or wounding induced a primary response and phenoloxidase activity was found to increase within 10–60 min. The values for phenoloxidase activity in viable B. bassiana-injected insects exhibited a secondary response, i.e., an increase 24 hr after injection.
• 3.3. In wounded insects and those injected with inactivated conidia, the phenoloxidase activity receded after the initial increase and remained at low levels.
• 4.4. Protein concentrations in the hemolymph increased immediately after infection and wounding and returned to basal levels during the course of the experiment.
• 5.5. Injection of viable B. bassiana resulted in a gradual increase in the protein concentrations between 12 and 24 hr.
• 6.6. There was no apparent change in the carbohydrate levels in either B. bassiana-infected or wounded insects.
• 7.7. These results are discussed in relation to their possible role(s) and interrelationships in the immune response to infection or wounding. Furthermore, we suggest that a “factor” is released after mechanical injury of the integument.

     

Purification and characterization of the amylolytic enzymes of Saccharomycopsis fibuligera

• 1.1. A complex of extracellular amylolytic enzymes produced by Saccharomycopsis fibuligera KZ, grown on fine fibre (waste product from corn starch production) and corn-steep liquor, has been studied.
• 2.2. α-Amylases and glucoamylases, as the main representatives of this complex, were separated by hydrophobic chromatography on Spheron 300 LC.
• 3.3. Individual isoenzymes of one type were separated on FPLC-Mono Q.
• 4.4. The relative molecular weight of α-amylases is 54,000, glucoamylases 62,000, maximal activity is reached by both enzymes between pH 5.0 and 6.2 at a temperature of 40–50°C.
• 5.5. Glucoamylases have a higher stability of the native structure than α-amylases, they retain 55% of their original activity, even after 10 min of incubation at 100°C.

     

S-Adenosyl-l-methionine decarboxylase activities in Mytilus edulis

• 1.1. The activities of S-adenosylmethionine decarboxylase (EC 4.1.1.50) were measured in cell extracts of mantle, hepatopancreas and foot from Mytilus edulis.
• 2.2. The apparent molecular weights of the enzymes estimated by gel filtration chromatography were 65,000 ± 10,000.
• 3.3. The enzymes do not require bivalent cations for catalysis and show optimum pH between 7.0–8.0 in phosphate buffer.
• 4.4. The hepatopancreas enzyme shows different behavior to the other two enzymes against temperature and its activity is strongly inhibited by NH₄⁺.
• 5.5. The apparent K_ms for S-adenosylmethionine were found to be 300, 200 and 250 μM for the hepatopancreas, mantle and foot enzymes, respectively.

     

Effects of photoperiod on plasma corticoid levels in the goldfish,Carassius auratus—role of the pineal

• 1.1. The effects of photoperiod and pinealectomy on plasma corticoid levels in the goldfish (Carassius auratus) were examined.
• 2.2. Plasma corticoid levels differed in goldfish maintained under different photoperiod regimes, but this response varied seasonally.
• 3.3. Pinealectomy altered the effects of photoperiod on plasma corticoid levels but this effect varied with season.
• 4.4. Plasma corticoid levels were correlated with ovarian activity. The effects of photoperiod on plasma corticoid levels appear to be related to the influence of light on reproduction.
• 5.5. The alteration of plasma corticoid levels in pinealectomized fish may be due to the role this organ plays in mediating photoperiod effects on gonadal activity.

     

Role of antiproteolytic heparin-binding serum protein(s) in modulating the levels of sialyl- and galactosyltransferase activity released during the incubation of rat jejunal slices

• 1.1. Sialyltransferase released into the medium during the incubation of rat jejunal slices in serum-free buffer, was susceptible to proteolytic degradation. Heat inactivated horse serum or its antiproteolytic heparin-binding fraction was found to be necessary in determining the activity of sialyltransferase released (Nadkarni et al., 1991).
• 2.2. In the present study, we have shown that heat inactivated rat serum (HRS) or its antiproteolytic heparin-binding fraction (HBF) had a role in determining the sialyltransferase activity released during jejunal slice incubations.
• 3.3. Galactosyltransferase was also released during incubations, but was not proteolytically degraded and the presence of HRS or HBF in incubations did not alter the levels of galactosyltransferase activity released.
• 4.4. Trypsin activity in serum-free incubation medium was higher compared to medium containing HRS.
• 5.5. Addition of serum-free medium obtained from 4 hr incubations of the jejunal slices, to medium obtained from parallel incubations done in the presence of HRS, caused inhibition of sialyl- but not galactosyltransferase activity.
• 6.6. In jejunal homogenates stored at −20°C, sialyltransferase activity was decreased during 0–45 days of storage, whereas galactosyltransferase activity remained fairly stable for upto 56 days.
• 7.7. Inclusion of HRS or HBF in homogenates resulted in higher sialyl- but not galactosyltransferase activity compared to serum-free homogenate samples.
• 8.8. The results suggest that HRS or its antiproteolytic heparin-binding proteins have a role in determining the sialyltransferase activity released from the jejunal slices. In contrast galactosyltransferase released was not susceptible to proteolysis, and HRS or HBF was not required to express its activity.