Long-term starvation in Xenopus laevis daudin—III. Effects on enzymes in several tissues

• 1.1. Adult, female Xenopus laevis were subjected to 12 months of starvation.
• 2.2. Starvation resulted in a continuous reduction in the activity of both hepatic and renal glucose-6-phosphate dehydroganse.
• 3.3. Fructose-1,6-diphosphatase was significantly reduced at months 10 and 12 in the liver, and at months 4, 10, and 12 in the kidney.
• 4.4. Pyruvate kinase activity of muscle and liver decreased during the experimental period whereas the renal enzyme remained essentially unchanged.
• 5.5. Both hepatic and renal glutamate-pyruvate transaminase (GPT) and hepatic glutamate-oxaloacetate transaminase (GOT) showed a reduction of activity after 2 and 4 months of starvation followed by an increase in GPT but not in GOT.

     

Purification and characterization of elastase from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

• 1.1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography.
• 2.2. The approximate molecular weight of the elastase was 27 kDa and the isoelectric point was remarkably basic.
• 3.3. The pH optimum of this enzyme was 8.0, when assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide.
• 4.4. When assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide, the enzyme activity had a temperature optimum of 45°C, and the enzyme was stable up to this temperature.
• 5.5. The trout elastase exhibited a higher specific activity than porcine elastase against Succinyl-Ala-Ala-Ala-p-Nitroanilide and elastin-orcein.
• 6.6. The trout elastase was inhibited by elastatinal, PMSF, TPCK, SBTI and Bowman-Birk inhibitor.

     

Effects of a purine inhibitor,allopurinol, on urate metabolism in the german cockroach,Blattella germanica L. (Dictyoptera: Blattellidae)

• 1.1. Adult male and female cockroaches (Blattella germanica) were maintained on a positive nitrogen balance diet (66% protein) containing various levels of allopurinol (0–3%) to determine the effects of allopurinol on urate synthesis and storage.
• 2.2. Each insect was injected with [¹⁴C]hypoxanthine and after 1 week was analyzed for whole-body hypoxanthine, xanthine and urate radiolabel.
• 3.3. There was a general trend of decreased whole-body radiolabel retention, radiolabeled body urates and total-body urate content in both sexes with increasing amounts of dietary allopurinol.
• 4.4. Virgin female adults were allowed to feed on diets containing 0, 25 and 66% protein plus 0.1% allopurinol and were injected with [¹⁴C]xanthine.
• 5.5. After 1 week radiolabel content in the whole-body xanthine and urate pools was determined.
• 6.6. Females on the 0% protein diets contained less radiolabel in the whole-body and body urates than those on either 25 or 66% protein diets.

     

Purification and partial characterization of an AMP deaminase from the marine invertebrate Palaemon serratus

• 1.1. AMP deaminase from Palaemon serratus tail muscle was partially purified by chromatography on cellulose phosphate.
• 2.2. Muscle homogenates expressed very low enzyme activities and the presence of ATP was necessary to detect AMP deaminase. The specific activity and substrate affinity of the purified enzyme were also very low.
• 3.3. The purified prawn muscle AMP deaminase was contaminated by contractile proteins, one of the major contaminants being actin.
• 4.4. The enzyme displayed a very high affinity for actomyosin which was only partially abolished by pyrophosphate.

     

The purification of kallikrein from human whole saliva

• 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
• 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
• 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
• 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
• 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
• 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
• 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
• 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

     

Main serum protein of rainbow trout (Salmo gairdnerii): its biological properties and significance

• 1.1. Main serum α₁-protein (α₁P) of rainbow trout was purified and its biochemical and physico-pathological properties were studied.
• 2.2. α₁P was suggested to be a primitive protein having both properties of albumin and AFP in serum proteins of mammals according to the following results.
• 3.3. Molecular weight (75,000), two kinds of molecules (pI 4.55 and 5.05) and amino acid composition.
• 4.4. Dye- or ConA binding activity.
• 5.5. Estrogen binding activity and inhibitory effect on lymphoblastoid-forming activity.
• 6.6. Possible osmotic regulator.
• 7.7. Significant elevation of blood α₁P level in the course of hepatoma induction.

     

Distribution of glutamate dehydrogenase in the intestine of the earthworm (Lumbricus terrestris), and some physiological implications

• 1.1. Intestines of fresh and dehydrated-starved L. terrestris were compared to tissue and anterior-posterior distribution of glutamate dehydrogenase (GDH) and other mitochondrial or cytosol dehydrogenases.
• 2.2. For any dehydrogenase, including GDH, practically all the activity was in the gut epithelium. This distribution of GDH supports Tillinghast (1967, 1968) as to the excretory route for ammonia.
• 3.3. While the distributions of the marker dehydrogenases were reasonably uniform along the intestine, the GDH activity was predominantly (80–90% of the total activity) in the last third of the mid-intestine, indicating a true physiological differentiation of the midgut tube. The GDH activity of the typhlosole was about two times the activity in the peripheral epithelium. The GDH distribution was independent of the physiological state of the worm.
• 4.4. From the distribution of GDH it follows that the mid-intestine, immediately before the hindgut, is the main region both for amino acid uptake and catabolism. As regards amino acids, it typifies the primitive digestive tube by having both the absorptive and the liver functions.

     

Potassium balance in freshwater-adapted xtrout Oncorhynchus mykiss

• 1.1. Potassium loss occurs through the gills of trout.
• 2.2. This loss is compensated by intestinal absorption.
• 3.3. During a fasting period, branchial regulation appears after 2–3 days.
• 4.4. The maintenance of potassium balance is discussed as a function of experimental conditions.

     

Specific dynamic action (SDA) in the supralittoral isopod,Ligia pallasii: Effect of ration and body size on SDA

• 1.1. Ration and body size effects on specific dynamic action (SDA) were investigated in the supralittoral isopod Ligia pallasii using seaweed and chemical diets.
• 2.2. SDA increased asymptotically with ingested meal size for all diets.
• 3.3. Body weight had a significant positive effect on SDA for only one of the six diets tested, but weak tendencies were present in the data for the other diets.
• 4.4. SDA appeared to increase geometrically with increasing concentration of amino acids at high ration levels.

     

Modification of glyceraldehyde-3-phosphate dehydrogenase,EC 1.2.1.12, isolated from rainbow trout during acclimation at 5 or 15°C—I. Changes in the activity of individual muscle isolates as evidence for variable adaptive responses

• 1.1. The mean K_m and V_max values for G3PDH isolated from the lateral muscle of cold-adapted (5°C) rainbow trout, Salmo gairdneri, were twice those of enzyme from warm-adapted (15°C) trout when assayed at 7°C but not at any other temperature.
• 2.2. The entropy of activation of warm enzyme was about 3 times that of cold enzyme. However, enthalpy or free energy of activation among acclimation groups differed less or not at all.
• 3.3. Individual G3PDH isolates within either adaptation group differed in kinetic characteristics.

     

Identification of cathepsin L-like proteinase and aminopeptidase in yolk granules of the sand worm,Nereis diversicolor

• 1.1. Two proteinases have been identified in yolk granules of Nereis diversicolor mature oocytes, an aminopeptidase and an acid cysteine proteinase.
• 2.2. The aminopeptidase was identified as a metallo-enzyme having a molecular weight of about 260 kDa.
• 3.3. Except that the acid cysteine proteinase is a high molecular weight protein (200 kDa) and has a very low pH optimum (3.0), the enzyme possesses properties resembling those of mammalian cathepsin L.
• 4.4. The cathepsin L-like proteinase was found to be liable to the in vitro proteolysis of the yolk granule proteins and is therefore suggested to be involved in yolk protein processing.

     

Growth and hemolymph trehalose levels of Heliothis zea larvae fed diets containing various sugars

• 1.1. The amount of sugar required for growth of Heliothis zea larvae on a chemically defined diet was determined.
• 2.2. Larvae grew well on fructose, galactose, sucrose, trehalose and raffinose diets but not on diets containing more than 0.5% glucose.
• 3.3. A starch diet did not promote rapid larval growth.
• 4.4. Hemolymph trehalose levels in 12-day-old larvae ranged from none to 45μmoles/ml.
• 5.5. A method for analysis of hemolymph trehalose by gas chromatography is described.

     

Purification and some particular kinetic properties of rat spleen cytosolic deoxynucleotidase

• 1.1. Rat spleen cytosolic deoxynucleotidase was purified 40,000-fold to almost homogeneity and had a specific activity of 3000 μmol/min per mg.
• 2.2. Molecular mass of the native enzyme was 45 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that the native enzyme comprises two identical 27-kDa subunits.
• 3.3. Specific enzyme activity increases with increasing concentration of enzyme protein and approaches a plateau at high enzyme concentrations.
• 4.4. Enzyme activity increases gradually and nonlinearly with increasing concentration of enzyme in the low concentration range. Above a certain concentration the increase attains a maximal and constant slope.
• 5.5. The kinetic properties can be explained by assuming dissociation of the enzyme into subunits with low or no activity.

     

Growth and haematological response of sturgeon hybrids Russian sturgeon (Acipenser guldenstadti brandt) × beluga (Huso huso L.) to protein and lipid contents in the diet

• 1.1. Fingerlings of intergenious hybrid Russian sturgeon (Acipenser guldenstadti) × beluga (Huso huso) weighing 22 g reared in water with salinity 18 ppt were fed nine diets differing in protein and fat content.
• 2.2. The increase of dietary protein content (from 45 to 52%) improved the fingerlings growth rate, food and protein conversion efficiencies. No effect of further protein content increase to 60% was observed.
• 3.3. The increase of dietary fat content from 10 to 20% positively influenced all growth results.
• 4.4. The muscular lipid content increased following the increase in dietary fat due to accumulation of triacylglycerols.
• 5.5. Distinctive leucopenia in neutrophils and leucophilia in lymphocytes following dietary protein and fat content increase were observed.
• 6.6. It was concluded that within the analysed range of values the increase of dietary protein and lipid content improved the physiological status of sturgeon hybrid fingerlings.

     

Regulatory properties of rabbit liver phosphorylase kinase

• 1.1. Purified native rabbit liver phosphorylase kinase becomes activated during the assay of its activity while low molecular weight forms of the same enzyme do not.
• 2.2. The activation requires ATP and maganesium ions, suggesting the phosphorylation of the enzyme by a protein kinase as the mechanism involved.
• 3.3. The activation of the enzyme can be reverted by the action of a type 1 protein phosphatase isolated from the same tissue.
• 4.4. The activation can also be catalyzed by the catalytic subunit of cAMP-dependent protein kinase in a process that requires a much lower ATP concentration to proceed.
• 5.5. The activation is believed to be due to an autocatalytic phosphorylation of phosphorylase kinase itself. In support of this hypothesis are the regulation of the process through calcium ions, the low levels of endogenous protein kinase detected in the purified preparation, the high ATP concentrations required in the absence of cAMP dependent protein kinase and the fact that the process cannot be blocked by an excess of the heat stable inhibitor specific for the later enzyme.
• 6.6. The low molecular weight forms of the enzyme on their side are not affected by the action of neither protein phosphatase 1 nor cyclic AMP dependent protein kinase.
• 7.7. Both activated and nonactivated phosphorylase kinase are partially dependent on calcium ions, the affinity of the former being higher than that of the latter. The low molecular forms do not require calcium ions to express their activity.

     

Effects of lysine administration on plasma arginine and on some nitrogenous catabolites in rainbow trout

• 1.1. Rainbow trout were fed either graded levels of lysine (0.8, 1.8 and 3%) at a constant level of arginine (1.4%) or excess arginine (2.4%) at a fixed level of lysine (1.8%).
• 2.2. Increasing the dietary lysine level affected plasma urea, plasma arginine and ammonia excretion.
• 3.3. Trout fed graded levels of lysine received an arginine challenge (U¹⁴C-l-arginine) and it was found that excess dietary lysine led to a decrease in arginine degradation.
• 4.4. Injection of l-lysine induced a decrease in urea excretion, while injection of l-arginine increased both urea and ammonia excretion in control well-fed trout.
• 5.5. These results are discussed in the light of current knowledge on the antagonism between lysine and arginine.

     

δ-Aminolevulinic acid dehydratase inactivation by uroporphyrin I in light and darkness

• 1.1. The action of uroporphyrin I on erythrocytic ALA-D activity under dark and light conditions was examined.
• 2.2. Photo and non-photoinactivation of ALA-D induced by uroporphyrin I were observed.
• 3.3. Both effects were dependent on uroporphyrin concentration, temperature and time of exposure of the protein to the porphyrin.
• 4.4. Light-dependent effect of uroporphyrin I is related with the phototoxicity of porphyrins and could be produced by primary amino acid photooxidation followed by secondary cross-linking of the protein.
• 5.5. Light-dependent effect of uroporphyrin I could be ascribed to a direct enzyme inhibition due to binding of the porphyrin to the protein inducing structural changes at or near its active site.

     

Proteolytic cleavage of band 3 protein in relation to anion transport in fish (Oncorhynchus mykiss) red blood cells

• 1.1. The effects of trypsin and chymotrypsin on HCO₃⁻/Cl⁻ exchange through red blood cell membranes of humans and trout were studied.
• 2.2. To measure the anion exchange we used a right-angle light-scattering technique by applying the Jacobs-Stewart cycle in ammonium solution and the osmotiration method at constant cell volume.
• 3.3. The Cl⁻ flux in human red blood cells remained unaltered after treatment with external trypsin and chymotrypsin while in trout red blood cells the flux decreased.
• 4.4. This partial inhibition of anion transport in fish, ranging from 30 to 40%,suggest that one or several of the cleavage sites in band 3 protein, essential for anion transport function, are exposed in fish red blood cells.
• 5.5. In human red blood cells the fragments of band 3 which are affected by proteolytic digestion, retain their tertiary structure because there is no influence on anion transport.

     

Essential tyrosyl residues of human placental alkaline phosphatase

• 1.1. Human placental alkaline phosphatase was inactivated with tetranitromethane in a biphasic process.
• 2.2. Spectral and amino acid analysis demonstrated that the inactivation was due to the conversion of tyrosine residues to 3-nitrotyrosine.
• 3.3. The inactivation process showed saturation kinetics.
• 4.4. Protection of the enzyme against tetranitromethane inactivation was afforded by inorganic phosphate.
• 5.5. The binding affinity between the modified enzyme and inorganic phosphate was decreased.
• 6.6. Our results suggest the involvement of tyrosyl residues in the locus of phosphoryl site of the phosphorylated enzyme forms.

     

The effect of temperature on oxygen equilibrium curves of trout blood (Salmo gairdnerii)

• 1.1. Oxygen equilibrium curves were measured on trout red blood cell suspensions at pH 7.8 and 8.4 at 15, 20 and 25 C. Normal red cells and red cells that had been depleted of their ATP content were used.
• 2.2. The equilibrium data were fitted to the Adair's model and the enthalpy (ΔH) and entropy (ΔS) changes for the first and fourth steps of oxygenation and for overall oxygenation were calculated from the temperature dependencies of the Adair constants.
• 3.3. For normal red blood cells, the apparent heat for the first oxygenation step, δh₁, is close to zero.
• 4.4. Temperature insensitivity of this step at physiological pH, combined with a large pH dependence, probably denotes a property of Hb4, the Root effect Hb of trout blood.
• 5.5. At pH 7.8, ΔH₄ is about —4kcal/mol, a small value which may be attributed to the large release of Bohr protons that occurs at the last oxygenation step and corresponds to an endothermic process which opposes to the exothermic oxygenation of the haem.
• 6.6. The ΔH₄ value appears to have a large influence on the enthalpy for overall oxygenation.
• 7.7. Results for ATP-free red cells are consistent with a mere increase in the intracellular pH and suggest that ATP has no specific effect at and above pH_i ~ 7.7.
• 8.8. Effects of temperature and pH on trout red blood cell isotherms emphasize the primary importance of the major component of trout blood, namely Hb4, in trout blood functional properties.