Shiro Horiuchi

Characterization of gill Na,K-ATPase in the freshwater crayfish, Procambarus clarki (Girard).

1. 1. Na,K-ATPase activity was demonstrated in heavy microsomal fractions of gill preparations of the freshwater crayfish, Procambarus clarki. 2. 2. The highest activity was found in 50 mM of sodium, and 20 mM of potassium in the presence of 2 mM of magnesium. 3. 3. The Km of gill Na,K-ATPase for ATP was 7.1 × 10−4M, and the Km of Mg-ATPase was 8.7 × 10−4 M. 4. 4. The Arrhenius plot for gill Na,K-ATPase showed a discontinuity at around 27°C. Such a distinct feature was not found in Mg-ATPase. 5. 5. A complete inhibitory effect on gill Na,K-ATPase by ouabain was found with 10−2 M ouabain. The pI 50 was 3.6. Gill Mg-ATPase was not inhibited by ouabain.

Effect of environmental temperature upon muscle lactate dehydrogenase in the crayfish, Procambarus clarki girard

Abstract 1. 1. Lactate dehydrogenase (LDH) in abdominal flexor muscle of crayfish, Procambarus clarki, showed a different type of pyruvate inhibition in the animals kept in cold, warm and hot environments, respectively. 2. 2. Pyruvate inhibition was studied in relation to the formation of the abortive ternary complex, LDH-NAD+-pyruvate. 3. 3. Arrhenius plots of log V max vs T −1 for crayfish LDH changed linearly in the range above and below the environmental temperature. 4. 4. LDH affinity for both pyruvate and lactate varies with temperature and the minimum apparent K m value was attained at the environmental temperature of the animal. 5. 5. These temperature-dependent kinetic properties of crayfish LDH were compared to those of LDH from leg gastrocnemius muscle of rat.

Characterization of cathepsin D in the regressing tadpole tail of bullfrog, Rana catesbeiana

Abstract 1. 1. Catheptic activity, measured by the hydrolysis of hemoglobin, increased remarkably during metamorphosis in the tadpole tail of Rana catesbeiana . 2. 2. Pepstatin inhibited about 75% of the catheptic activity, and the combination of pepstatin and monoiodoacetate inhibited it completely. Leupeptin slightly inhibited catheptic activity. 3. 3. By gel chromatography on Sephadex G-100, three molecular forms of catheptic activity were obtained. Their molecular weights were: Fraction I—larger than 100,000, Fraction II—50,000 and Fraction III—32,000. 4. 4. Fraction II, showing the highest activity was strongly inhibited by pepstatin and assumed to be cathepsin D. Fraction III was strongly inhibited by monoiodoacetate, indicating the existence of SH-proteinase.

ISOLATION OF PEPSINOGEN A FROM GASTRIC MUCOSA OF BULLFROG, RANA CATESBEIANA

Two pepsinogens, named pepsinogens II-2 and III, were purified from the gastric mucosa of the bullfrog, Rana catesbeiana. The two pepsinogens were distinct from each other with respect to activation rate, sensitivity to pepstatin, amino acid composition, immunogenicity and NH2-terminal sequence. The analysis of NH2-terminal sequence showed that pepsinogen II-2 is identical to progastricsin from the bullfrog esophagus. Pepsinogen III was thought to be pepsinogen A, which has so far not been found in the bullfrog.

Purification and characterization of cathepsin D-like proteinase from the tadpole tail of bullfrog, Rana catesbeiana

1. An acid aspartic proteinase in the regressing tadpole tail was purified about 800-fold with a 36% recovery. 2. The mol. wt of the enzyme was found to be 42,000 on gel filtration and 38,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively. 3. The purified enzyme had a maximum activity at pH 3.5 and an apparent Km of 0.084% with acid-denatured hemoglobin as substrate. 4. The enzyme activity was strongly inhibited by pepstatin. In addition, diazoacetylnorleucine methyl ester inactivated the enzyme in the presence of cupric ions. 5. The enzyme was identified as a cathepsin D (EC. 3.4.23.5)-like proteinase.

Acid proteinases of the fore-gut in metamorphosing tadpoles of Rana catesbeiana

1. Two types of acid proteinases were found in the adult stomach of the bullfrog, Rana catesbeiana. 2. The first type of enzyme appeared in the developing stomach and esophagus and contained more than two kinds of acid proteinases. 3. These enzymes were identified as pepsin-type enzymes. 4. The second type of enzyme existed from the larva to adult stage and was also present in the adult duodenum. 5. This enzyme was different from pepsin and thought to be cathepsin E.

Distribution of Acid Proteinase Activity in Molluscs

Abstract Acid proteinases with an optimum around pH 3 were demontrated in various tissues of 12 molluscan species. Enzymes strongly inhibited by pepstatin were predominant and the molecular weights of those from two species were in the region of 38,000–68,000, suggesting that they were cathepsin D-type proteinases.

Distribution of cathepsin E in the larval and adult organs of the bullfrog with special reference to the mature form in the larval fore-gut

The distibution of cathepsin E in several organs of the bullfrog, Rana catesbeiana, was analyzed at pre- and post-metamorphic stages by the acid proteinase assay, by visualization of enzyme activity on polyacrlamide fore-gut gels after electrophoresis and by immunoblotting with anti-cathepsin E serum. Cathepsin E was mainly distributed in the foregut at the larval stage and in the stomach, duodenum, large intestine and gall bladder at the post-metamorphic stage. In the larval fore-gut, a higher amount of the mature form of cathepsin E was observed in addition to the proform, but in other organs, including the stomach at the post-metamorphic stage, the mature form was barely detected. Developmental changes in the amount of cathepsin E were found in the digestive tract and the gall bladder by quantitative immunoblotting analysis. Finally, the larval fore-gut was stained immunohistochemically with anti-cathepsin E serum and the surface epithelium gave a strong immunoreactive signal.

Characterization of antennary gland Na,K-ATPase in the freshwater crayfish, Procambarus clarki girard

Abstract 1. 1. Na, K-ATPase activity was demonstrated in a heavy microsomal fraction of antennary gland preparations of the freshwater crayfish, Procambarus clarki . 2. 2. The highest activity was found in 100 mM of sodium and 4mM of potassium in the presence of 1 mM of magnesium at pH 8.5. 3. 3. K m of Na,K-ATPase for ATP was 9.0 × 10 −4 M . 4. 4. A complete inhibitory effect on Na,K-ATPase by ouabain was found with 10 −2 M ouabain. The pl 50 was 4.1. 5. 5. Both renal Na,K-ATPase and Mg-ATPase were inhibited by POMB. The pI 50 was 6.3 and 5.0, respectively. Inhibitory effect of POMB was restored by cysteine. 6. 6. The activity of gill Na,K-ATPase was increased only when the animals were maintained in 50% seawater.

Autolysis in the tail muscles of metamorphosing tadpoles

Degradation of muscle homogenate from the metamorphosing tadpole tail of bullfrog, Rana catesbeiana, was examined at acid and neutral pHs. More rapid and complete degradation was observed at acid pH. Proteinases working at acid pH were not inhibited by pepstatin but were inhibited by leupeptin. However, the inhibition by leupeptin was enhanced by pepstatin. These results show that lysosomal proteinases, a thiol proteinase(s) rather than cathepsin D, are involved in the degradation of tail muscle proteins.