Digestive protease,lipase and amylase activities in stage i larvae of the american lobster,Homarus americanus

• 1.1. Digestive protease, lipase, and amylase of Stage I larvae of the American lobster Homarus americanus are characterized.
• 2.2. A sensitive method for detection of crustacean lipase was developed using an latroscan which combines thin-layer chromatography and flame ionization detection to quantify free fatty acids generated by lipase digestion.
• 3.3. pH optima of the three enzymes occurred at or near the pH of gastric fluid.
• 4.4. A time course study demonstrated slight increases in protease and amylase activities during the first larval stage, regardless of whether the lobsters were fed or not, whereas lipase activity was constant.

     

Protease,amylase and lipase activities in the midgut and hindgut of the cricket,Gryllus rubens and mole cricket,Scapteriscus acletus

• 1.1. Midgut is the major source of protease, amylase and lipase in a cricket, Gryllus rubens and in a mole cricket, Scapteriscus actetus.
• 2.2. Hindgut makes a significant contribution, and possibly even the major contribution, to digestion in both crickets, with enzyme activities from 20% (amylase and lipase) to 30% (protease) of midgut level, and a pH favorable to action of all three.
• 3.3. Ingested food helps regulate digestive enzyme levels, and crickets starved for 5 days had only 50–60% of normal levels of enzyme activity.

     

Compensations for temperature in the activities of digestive enzymes of Periplan eta americana (L.)

• 1.1. Differential thermal acclimatory responses of maximal catalytic rates (V_max) of digestive enzymes have been measured in both sexes of Periplaneta americana adapted to 16 and 32°C.
• 2.2. Salivary amylase of females and gastric protease of males exhibit “translational” acclimation, the former showing a “complete” but the latter only a “partial” compensation. The value of Q₁₀ is not altered in the adaptive response.
• 3.3. An alteration of the thermal coefficient is evidenced by the “translational-cum-rotational” compensation of gastric amylolytic activity, with significant warm acclimation but no cold acclimation in both sexes.
• 4.4. Gastric protease of female cockroaches and gastric lipase of both sexes are characterized by the lack of an adaptive compensation to temperature, while salivary amylase of male appears to manifest an “inverse” acclimation.
• 5.5. Sexual dimorphism in the levels of the activities and in the patterns of thermal acclimation of the digestive enzymes is indicated.

     

Studies on intestinal protease: Isolation,purification and effect of dietary proteins on alkaline protease activity of the air-breathing fish,Clarias batrachus (Linn.)

1. The effect of dietary protein levels on the proteolytic activity in the intestines of the air-breathing fish, Clarias batrachus (Linn.) has been studied
2. Activity of proteolytic enzymes increased significantly in fishes maintained with a 50% protein diet from those maintained with a 25% protein diet; still higher dietary protein percentage showed no further stimulation of enzyme activity.
3. In a study on the determination of sub-cellular localisation, it has been found that protease activity is more prominent in lysosomes than in other organelles of the cell.
4. A sixty fold purification of alkaline protease from the intestine of Clarias batrachus has been achieved by ion exchange chromatography on DEAE cellulose which has been further checked by polyacrylamide gel electrophoresis.

     

Protease and Amylase in the digestive tract ofBarbus paludinosus

1. Protease and amylase activity in the digestive system ofBarbus paludinosus Peters (Pisces, Cyprinidae) has been investigated.
2. Chromatographic analysis showed seven amino acids to be present in both the anterior and posterior intestine. Only leucine, phenylalanine, valine, glycine and aspartic acid were positively identified.
3. In the anterior intestine chromatography revealed two sugars, but only one in the posterior intestine which was identified as glucose.
4. The pH of the intestinal fluid was found to be 5.8 and 7.8 for the fore and hind gut respectively, This correlates well with the enzyme pH optima found in in vitro experiments.
5. Protease and amylase activity was found throughout the digestive tract. Maximum proteolytic activity being present in the anterior intestine. Amylase activity is similar in both regions of the gut.
6. Correlation between the digestive enzymes and the fishes diet is briefly discussed.

     

Intracellular proteolytic activity in developing myxospores of Myxococcus xanthus

1. Cell-free extracts from vegetative cells and developing myxospores of Myxococcus xanthus were found to contain similar amounts of proteolytic activity, approximately 80% of which was due to one or more neutral metal proteases.
2. Sixty per cent of the proteolytic activity was particulate.
3. The specific activity of the proteases was high throughout all stages of myxospore formation and displayed small increases in activity at two stages of development: (1) during cell shortening and (2) immediately following the conversion to spheres. The first peak in activity was apparent in assays conducted at pH 8 or 10 whereas the second peak was obvious only at pH 6.
4. A mutant which develops into myxospores only after a lag of approximately 7–8 h possessed levels of proteases similar to the wild type and displayed a peak in proteolytic activity after a delay of 7–8 h.
5. Low levels of serine protease activity were occasionally detected in both vegetative cells and myxospores; no sulfhydryl proteases were detectable in either cell type.
6. Extracellular proteases accumulated in the medium throughout myxospore development but differed from the intracellular proteases in pH optima and sensitivity to inhibitors.

     

Digestive enzymes in the exocrine pancreas of the frog Rana esculenta

• 1.1. The activity of some digestive enzymes has been investigated in a crude pancreas homogenate of frog Rana esculenta. The levels of amylase, trypsin and chymotrypsin depend on nutritional status being lower in fasted animals; ribonuclease and lipase levels do not seem to be affected by fasting.
• 2.2. Frog pancreatic enzymes show pH optima and thermostability similar to those reported for higher vertebrates.
• 3.3. The effects of PMSF and EDTA on proteolytic enzymes suggest that in the frog pancreas besides serine-proteases which represent the major proteolytic activity, other enzymes, possibly metalloenzymes, are present.

     

The digestive enzymes of the pacific brown shrimp Penaeus californiensis.: I—Properties of amylase activity in the digestive tract

• 1.1. Crude extract of the whole digestive tract from the brown shrimp (P. californiensis) was investigated for digestive amylase activity.
• 2.2. Considerable amylase activity was found at pH 6.5–8.0, with optimum pH at around 7.5.
• 3.3. Optimum temperature was found between 30–40°C, similar to amylases from other crustaceans.
• 4.4. Amylase activity was highly halotolerant, having 50% maximum activity at 3 M NaCl.
• 5.5. Maximum amylase activity was found at 0.01 M NaCl.
• 6.6. Amylase activity was partially inhibited by the divalent ions Hg²⁺, Zn²⁺, Cu²⁺ and Cr²⁺.
• 7.7. Mg²⁺ and Ca²⁺ ions seemed to enhance amylase activity.

     

The reproductive cycle in a typical estuarine mollusc Nausitora hedleyi Schepman based on a study of the Gonad Index

1. Previous work on the methods employed for the determination of the breeding season of shipworms is briefly reviewed.
2. The method adopted for studying the reproductive cycle by using the “gonad index” is described.
3. The reproductive cycle of Nausitora hedleyi is described in detail based on a study of the gonad index of different sexes collected at monthly intervals from the estuarine environment of Cochin harbour.
4. The fact that breeding is restricted as marked by seasonal activity is shown from the size and activity of the gonad during the different months of the year.
5. The environment, and the hydrographic conditions prevailing in the habitat of N. hedleyi in the Cochin harbour are described.

     

The anaerobic metabolism of malate of Saccharomyces bailii and the partial purification and characterization of malic enzyme

1. The main pathway of the anaerobic metabolism of l-malate in Saccharomyces bailii is catalyzed by a l-malic enzyme.
2. The enzyme was purified more than 300-fold. During the purification procedure fumarase and pyruvate decarboxylase were removed completely, and malate dehydrogenase and oxalacetate decarboxylase were removed to a very large extent.
3. Manganese ions are not required for the reaction of malic enzyme of Saccharomyces bailii, but the activity of the enzyme is increased by manganese.
4. The reaction of l-malic enzyme proceeds with the coenzymes NAD and (to a lesser extent) NADP.
5. The K _m-values of the malic enzyme of Saccharomyces bailii were 10 mM for l-malate and 0.1 mM for NAD.
6. A model based on the activity and substrate affinity of malic enzyme, the intracellular concentration of malate and phosphate, and its action on fumarase, is proposed to explain the complete anaerobic degradation of malate in Saccharomyces bailii as compared with the partial decomposition of malate in Saccharomyces cerevisiae.

     

Seasonal variation in the biochemical composition of Mytilus viridis at Ratnagiri on the West Coast of India

1. The seasonal variation in the water, protein, fat and glycogen contents of the mussel, Mytilus viridis has been studied for the year March, 1974 to March, 1975.
2. The water level increased during the monsoon season and decreased in summer.
3. The level of protein, fat and glycogen showed correlation with the reproductive cycle of the mussel.
4. The protein level was high when the mussels were mature and dropped during the breeding period.
5. During sex change from male to female in May the protein level remained high whereas during sex change from female to male in October and November it was low.
6. The fat level was high in mature mussels and declined on spawning.
7. The glycogen level was at its peak in immature mussels and low in mature.

     

A study on the rate of water transport of the clam katelysia opima in relation to environmental conditions

1. The neutral red technique was employed to study the rate of filtration in Katelysia opima.
2. The weight specific water filtration was found to be greater for younger clams compared to the older ones.
3. The rate of water filtration increased with decreasing salinity.
4. Water filtration was found to increase as temperature increased, reaching a maximum at 35°C. but then sharply decreasing at 39°C.
5. Light had no significant effect on the rate of filtration.
6. Suspended matter was found to affect the rate of water filtration.
7. The rate of filtration was low at high pH and high in low pH.
8. The rate of water filtration was found to be faster during high tide than during low tide.
9. The presence of the parasitic crab, Pennotheris sp., in the mantle cavity of clams had a marked effect on the particle filtration.
10. Accidental cut of the siphon tips had no effect on the rate of filtration.

     

Function of laccase in the white-rot fungus Fomes annosus

1. Thioglycolic acid, a Cu-chelating agent, totally inhibited extracellular laccase activity without affecting growth and morphology of Fomes annosus.
2. In the presence of thioglycolic acid Fomes annosus cleaved high molecular weight lignosulfonate with a molecular weight range of 2×10⁶ to 1000. In the absence of thioglycolic acid the polymerizing activity of laccase prevented the detection of lignosulfonate breakdown products.
3. Oxidative polymerization of a lignin monomer, coniferyl alcohol, occurred in the presence but not in the absence of laccase activity.
4. Catechol and guaiacol added to the medium at a concentration of 2 mmol, are normally oxidized by fungal laccase and strongly inhibit growth. Presence of thioglycolic acid prevented the oxidation of these phenols and simultaneously permitted normal growth.

     

Survival,osmoregulatory ability,and respiration of idotea chelipes (Crustacea,Isopoda) from lake veere in different salinities and temperatures

1. An ecological and physiological study ofI. chelipes from Lake Veere, The Netherlands, was made.
2. Both osmoregulatory capacity and survival decrease with increasing temperature as well as with decreasing salinity.
3. Respiration experiments suggest that the need of energy by osmoregulatory activity may be supplied at the cost of other physiological processes, at any rate at temperatures of 10°C and higher.
4. It may be expected that, if temperatures higher than 15°C and salinities lower than 8‰ coincide, the population ofI. chelipes will be affected negatively.

     

An ecological study of the Ponggol Estuary in Singapore

1. An ecological study of the Ponggol Estuary was conducted from July 1965 to June 1966 and the seasonal data on physical, chemical and biological characteristics were presented.
2. The Ponggol River represents a short, narrow and shallow estuary in Singapore. The river mouth is open throughout the year and water from eastern Johore Straits drains in twice a day at high tide. The upper reach, however, is left exposed at low tide.
3. The Ponggol River was classified as a vertically and laterally homogeneous estuary and was found to exhibit a mesohaline to polyhaline environment.
4. Significant hydrological gradients from the river mouth 10 the upper reach were noted in the river system. Salinity, dissolved oxygen and pH increased towards the mouth of the river and other parameters such as nutrients, dissolved organic matter and turbidity increased towards the source.
5. Although the river received organic pollutants at the upper reach the estuary was able to discharge them fairly rapidly through regular flushing by the tides. The transient rise of organic matter did not appear to impart any serious affect on the biota in the estuary.
6. Over 98% of the phytoplankton consisted of diatoms, most of which were brought into the estuary from eastern Johore Straits. Freshwater forms were relatively few.
7. Phytoplankton biomass was considerably higher than the adjoining waters. and was reduced at the upper reach due to high turbidity of the water.
8. 80% of the zooplankton was composed of dinoflagellates,Difflugia, copepods and bivalve larvae dominating at all sections of the estuary.
9. Percentage composition of the zooplankton showed that dinoflagellates and copepod nauplii predominated at high tide whileDifflugia and bivalve larvae were abundant at low tide.
10. Zooplankton standing crop, in general, was higher towards the source at high tide but the reverse was found at low tide, i.e. standing crop increased towards the river mouth. This was attributed to the process of concentration.
11. Species composition of zooplankton was found to be more or less similar to that of the eastern Johore Straits.
12. The nekton consisted predominantly of small and juvenile fish. Close correlation of fish and copepods was found to be statistically valid and it was concluded that the fish entered the estuary to feed rather than to spawn.
13. The squids formed an important catch of the beach seine unit and were caught throughout the year.
14. The fish population could be grouped into four categories: estuarine components, euryhaline components, marine components and migratory components.
15. Benthic invertebrates were abundant. Commercially important species consisted of prawns,Metapenaeus andPenaeus, and crabs,Neptunus pelagicus andScylla serrata.
16. The river bed was inhabited predominantly by molluscs and the distribution resembled that of the sheltered shore of muddy-sand type.

     

Metabolism of the anaerobic formation of succinic acid bySaccharomyces cerevisiae

1. Succinic acid is formed in amounts of 0.2–1.7 g/l by fermenting yeasts of the genusSaccharomyces during the exponential growth phase. No differences were observed between the various species, respiratory deficient mutants and wild type strains.
2. At low glucose concentrations the formation of succinic acid depended on the amount of sugar fermented. However, the nitrogen source was found to be of greater importance than the carbon source.
3. Of all nitrogen sources, glutamate yielded the highest amounts of succinic acid. Glutamate led to an oxidative and aspartate to a reductive formation of succinic acid.
4. A reductive formation of succinic acid by the citric acid cycle enzymes was observed with malate. This was partially inhibited by malonate. No evidence was obtained that the glyoxylate cycle is involved in succinic acid formation by yeasts.
5. Anaerobically grown cells ofSaccharomyces cerevisiae contained α-ketoglutarate dehydrogenase. Its activity was found in the 175000 x g sediment after fractionated centrifugation. The specific activity increased 6-fold after growth on glutamate as compared with cells grown on ammonium sulfate.
6. The specific activities of malate dehydrogenase, fumarase, succinate dehydrogenase, succinylcoenzymeA synthetase, α-ketoglutarate dehydrogenase and glutamate dehydrogenase (nicotinamide adenine dinucleotide dependent) were determined in yeast cells grown on glutamate or ammonium sulfate. Similar results were obtained with a wild type strain and a respiratory deficient mutant. The latter did not contain succinate dehydrogenase.
7. In fermenting yeasts succinic acid is mainly formed from glutamate by oxidation.

     

Ontogenic change of digestive enzymes in Penaeus monodon

• 1.1. Ontogenic changes of both proteases and carbohydrases of Penaeus monodon from different larva stages to adult were investigated.
• 2.2. Total protease activity was low during nauplius and zoea but peaked up in mysis. This was due to the activity increase of both trypsin and chymotrypsin.
• 3.3. The change of isozyme pattern of these two enzymes from different life stages of the shrimp was further determined by functional staining on an electrophoregram.
• 4.4. Activity of α-amylase increased after the post-larva stage, while that of chitinase and maltase showed a peak in zoea then gradually decreased to adult.
• 5.5. The ratio of α-amylase activity to protease coincided with the dietary change of the shrimp in different life stage.

     

Enzyme activities and isozyme composition of triglyceride,diglyceride and monoglyceride lipases in Periplaneta americana,Locusta migratoria and Polia adjuncta

• 1.1. Measurements of maximal enzyme activities were combined with an electrophoretic study of isozyme make-up in an examination of triglyceride, diglyceride and monoglyceride lipases from the flight muscle, fat body and gut of the cockroach, Periplaneta americana and the locust, Locusta migratoria and from the flight muscle and fat body of the moth, Polia adjuncta.
• 2.2. Tri-, di- and mono-glyceride lipases were present in all tissues of the insects with diglyceride lipase ≥ triglyceride lipase activity in all cases and monoglyceride lipase ≥ diglyceride lipase activity in locust and moth.
• 3.3. In the flight muscle, a strong correlation was found between the activities of lipases and the known use of lipid as a fuel for flight in these insects. Lipase activities were lowest in the cockroach (a carbohydrate-based flight metabolism), intermediate in the locust (both carbohydrate and lipid-fueled flight), and highest in the moth (a non-feeding, lipid-catabolizing adult) flight muscle.
• 4.4. Polyacrylamide gel electrophoresis, using substrate-impregnated gels and stained for fatty acids released by lipase action, demonstrated the presence of tissue specific isozymes of tri-, di- and mono-glyceride lipases in the three insects. In addition, some, but not all, tissues showed multiple molecular forms of one or more of the lipases.
• 5.5. Diglyceride and monoglyceride lipase activities in both flight muscle and fat body of the insects coelectrophoresed suggesting the possibility that these two lipase activities might be catalyzed by a single enzyme protein.

     

In vivo buccal nerve activity that distinguishes ingestion from rejection can be used to predict behavioral transitions in Aplysia

1. We are studying the neural basis of consummatory feeding behavior in Aplysia using intact, freely moving animals.
2. Video records show that the timing of radula closure during the radula protraction-retraction cycle constitutes a major difference between ingestion (biting or swallowing) and rejection. During ingestion, the radula is closed as it retracts. During rejection, the radula is closed as it protracts.
3. We observed two patterns of activity in nerves which are likely to mediate these radula movements. Patterns I and II are associated with ingestion and rejection, respectively, and are distinguished by the timing of radula nerve activity with respect to the onset of buccal nerve 2 activity.
4. The association of ingestion with pattern I is maintained when the animal feeds on a polyethylene tube, the same food substrate used to elicit rejection responses. Under these conditions, pattern I is associated with either swallowing or no net tube movement.
5. Most transitions from swallowing to rejection were preceded by one or more occurrences of pattern I in which there was no net tube movement, suggesting that these transitions can be predicted.
6. Our data suggest that these two patterns can be used to distinguish ingestion from rejection.

     

Biogenesis of mitochondria 20 the effects of altered membrane lipid composition on mitochondrial oxidative phosphorylation inSaccharomyces cerevisiae

1. The lipid composition of mitochondria isolated from a fatty acid desaturase mutant ofSaccharomyces cerevisiae may be extensively manipulated by growing the organism on defined supplements of unsaturated fatty acid (UFA).
2. The fatty acid composition of the mitochondrial lipids closely follows that of the whole cells from which the mitochondria are isolated. UFA-depleted mitochondria contain normal levels of sterols, neutral lipids and total phospholipids, but have much lower levels of phosphatidyl inositides.
3. UFA-depleted mitochondria possess a full complement of cytochromes, oxidase both NAD-linked and flavoprotein-linked substrates at normal rates, and have levels of succinate and malate dehydrogenases similar to those of UFA-supplemented mitochondria. However, UFA-depletion has a marked effect on the ability of cytochromec to reactivate the NADH oxidase activity of cytochromec-depleted mitochondria.
4. The efficiency of oxidative phosphorylation decreases progressively with the UFA content of the mitochondria, and oxidative phosphorylation is completely lost in mitochondria containing approximately 20% UFA.
5. The incorporation of UFA into the lipids of UFA-depleted mitochondriain vivo results in a recoupling of oxidative phosphorylation. Recoupling is insensitive to both chloramphenicol and cycloheximide, indicating that all the proteins necessary for oxidative phosphorylation are present in UFA-depleted mitochondria, and that the less of oxidative phosphorylation is a purely lipid lesion.
6. ATPase activity is apparently unaffected by UFA-depletion, but³²P_i-ATP exchange activity is lost in mitochondria which have been extensively depleted in UFA.
7. Valinomycin stimulates the respiration of UFA-supplemented mitochondria in media containing potassium, but has no effect on the respiration of UFA-depleted mitochondria, suggesting that active transport of potassium is lost as a result of UFA-depletion.