Characterization of NaK ATPase from the gills of the freshwater prawn Macrobrachium rosenbergii (de Man)

• 1.1. The specific activity of Na-K ATPase was determined from the microsomal preparation of gills dissected from adult Macrobrachium rosenbergii.
• 2.2. Maximal ATPase activity was achieved at a substrate concentration of 0.5 mM ATP.
• 3.3. Optimal enzyme activity was obtained at pH of 7.5.
• 4.4. The Arrhenius plot of Na-K ATPase activity revealed a marked discontinuity at 30°C. “Mg” ATPase activity did not exhibit a marked discontinuity.
• 5.5. The E_a for Na-K ATPase and “Mg” ATPase was 14.6 kCal/mole and 9.31 kCal/mole respectively. Q₁₀ values for Na-K ATPase was 2.34 and for “Mg” ATPase 1.65.
• 6.6. ATPase activity and gill homogenate protein concentration exhibited a linear relationship up to 130 μg protein/ml.
• 7.7. Na-K ATPase activity was inhibited by 10⁻³ M ouabain. It was equally inhibited by the removal of K⁺ from the reaction medium.

     

Chemical characterization of the brain of a coelacanth,Latimeria chalumnae

• 1.1. The chemical composition of coelacanth brain was studied and compared with some other species of bony fishes.
• 2.2. Almost all lipid classes generally seen in vertebrate brains were detected: 22:1 and 22h:1 acids were abundant in cerebroside and 24:1 acid in ganglioside. The hydroxy fatty acid content of cerebroside was high.
• 3.3. The myelin protein composition was unusual in that a 28,000-dalton protein was a major component.
• 4.4. 2′,3′-Cyclic nucleotide 3′-phosphodiesterase was 10 times more active than in the other bony fishes.
• 5.5. The present data suggest that molecular construction of coelacanth myelin is more advanced than that of the other bony fishes.

     

Renal response to hypoxia in carp,Cyprinus carpio: Changes in glomerular filtration rate,urine and blood properties and plasma catecholamines of carp exposed to hypoxic conditions

• 1.1. Changes in glomerular nitration rate (GFR), urine and blood properties and plasma catecholamines of carp were investigated during and following hypoxia.
• 2.2. GFR and urine flow decreased with increased urinary concentrations of bio-components, except protein, in the course of hypoxia.
• 3.3. Decreases in blood pH, and increases in haematocrit value and plasma K⁺, Ca²⁺, Mg²⁺, inorganic phosphate (P_i), ammonia, lactic acid and catecholamines (CAs) were observed as hypoxia progressed.
• 4.4. Increased GFR and urine flow, and higher values for urinary components, except protein, compared with those of the control were found in the initial post-stress stage.
• 5.5. The possible significance of increased plasma CAs in relation to changes in renal function in hypoxic carp is discussed.

     

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.

     

31P-NMR study on effects of electric shock on swimming leeches,Hirudo medicinalis

• 1.1. We evaluated the effect of electric shock on swimming leeches by measuring changes in high-energy phosphate metabolism using in vivo³¹P-NMR.
• 2.2. Leeches electrically stimulated during swimming showed anodal galvanotaxis, and stopped swimming with stimulation at strong current.
• 3.3. Comparison of the concentrations of high-energy phosphate metabolites before and after electric shock using ³¹P-NMR revealed a marked decrease in β-ATP and an increase in that of Pi.
• 4.4. Electric shock apparently induces excessive muscle fatigue in leeches, resulting in transient paralysis.

     

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.

     

Functional properties of the two major hemoglobin components from Leporinus friderici (Pisces)

• 1.1. The hemoglobins of Leporinus friderici were separated by liquid chromatography on DEAE-Sepharose in order to isolate the two major electrophoretic components.
• 2.2. The chromatographic fraction I (electrophoretically slow anodic) showed no Bohr effect and no nucleoside triphosphate modulation.
• 3.3. The chromatographic fraction III (electrophoretically fast anodic) showed a normal Bohr effect and addition of nucleoside triphosphate decreased oxygen affinity but did not alter the Bohr effect.
• 4.4. The whole hemolysate showed a normal Bohr effect and phosphate modulation altered both Bohr effect and oxygen affinity.
• 5.5. No or little difference between the effect of adenosine or guanosine triphosphates on hemoglobin function was observed.

     

A novel inhibitor of glycogen phosphorylase from crayfish hepatopancreas

• 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
• 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
• 3.3. The inhibitor is a small protein (M_r = 23,000) which did not show proteolytic activity.
• 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
• 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
• 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.

     

Changes in high-energy phosphate metabolism in the water scorpion,Ranatra chinensis,under cold water-warm water stress

• 1.1. To evaluate changes in high-energy phosphate metabolism in the water scorpion (Ranatra chinensis) under restraint and cold water-warm water stresses, in vivo [³¹P]NMR spectra were obtained.
• 2.2. Under restraint stress, arginine phosphate (Arg-P) decreased by 10% after 1 hr and remained at that level thereafter, while β-ATP showed negligible changes over 6 hr.
• 3.3. As the water temperature gradually increased or decreased, the relative concentration of Arg-P decreased due to enzyme regulation.
• 4.4. Repeated cold water-warm water stress, which consisted of repeated 15 min exposures to cold water (5°C) followed by 15 min exposures to warm water (30°C) caused distinct decreases in Arg-P and β-ATP concentration. These decreases were dependent on the frequency of exposure.
• 5.5. Phosphomonoesters (PME) increased not only with restraint stress but also with cold water-warm water stress.
• 6.6. The effect of cold water-warm water stress on high-energy phosphate metabolism was greater than that of restraint stress.

     

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Glucosylation of mannose containing dolichyl diphosphate oligosaccharide

• 1.1. A maximum rate of dolichyl phosphate [¹⁴C]glucose synthesis from 55-day embryos was achieved at 16nM concentration of exogenous dolichyl phosphate and exceeded about 3 times that without addition of dolichyl phosphate.
• 2.2. The highest values of [¹⁴C]glucose incorporation from UDP-[¹⁴C]glucose into dolichyl phosphate [¹⁴C]glucose, dolichyl diphosphate [¹⁴C]Glc-oligosaccharides and proteins were reached at 5 min time point of incubation of liver microsomes both from embryos and sows.
• 3.3. The radioactive incorporation into proteins was about 7-fold higher in liver microsomes from sows compared to that from embryos, probably due to the greater content of acceptor proteins in microsomes from sows.
• 4.4. The enzymatic transfer of Glc₃-oligosaccharide from a lipid carrier to endogenous protein acceptor in microsomes from pig embryonic and adult livers was considerably faster than the removal of glucose residues during the initial stages of processing of protein-bound oligosaccharides.
• 5.5. One labelled compound was discovered in the Chcl₃-Ch₃Oh-H₂O (1:1:0.3, by vol) extract after incubation of liver microsomes from embryos and sows with UDP-[¹⁴C]glucose. On the basis of its mobility on the chromatogram it appears to be GlcNAc₂Man₉Glc₃.

     

Biomines-stimulated phosphorylation and (Na+, K+-ATPase in the gills of the chinese crab,Eriocheir sinensis

• 1.1. Subcellular distribution of (NA⁺, K⁺-ATPase and ouabain-insensitive ATPase (Mg²⁺-ATPase) are compared in branchial tissues of the euryhaline crab, Eriocheir sinensis, acclimated to fresh water.
• 2.2. Both the anterior and posterior gills contain cAMP-dependent protein kinase and endogenous protein substrate for phosphorylation.
• 3.3. Phosphorylation occurs in both “particulate” and “soluble” subcellular fractions but its stimulation by cAMP is restricted to the “soluble” fraction.
• 4.4. serotonin (5-HT) and dopamine receptors are present only in the “light particulate” fraction isolated from the posterior gills.

• 1.(a) Serotonin and dopamine have no effect on the phosphorylation observed in a subcellular fraction alone.
• 2.(b) Activation of the phosphorylation by serotonin and dopamine is found when the soluble fraction (source of cAMP-dependent protein kinase) is added to the fraction P₃ from the posterior gills.
• 3.(c) No activation occurs with the fractions P₃ as well as P₁ or P₂ (not shown) from anterior gills of fresh water crab.
• 4.(d) Cyproheptadine, a serotonin receptor antagonist, inhibits the 5-HT dependent increase in phosphorylation.
• 5.(e) The dopamine receptor antagonist, chlorpromazine, inhibits dopamine-stimulated phosphorylation.
• 6.5. Ouabain mimics the effect of cyproheptadine on the serotonin-stimulated phosphorylation found in the posterior gills.

     

In situ phosphorylation of contractile proteins of a molluscan Mytilus edulis catch muscle in different functional states

• 1.1. The incorporation of ³²P into the contractile proteins of the anterior byssus retractor muscle of Mytilus edilus L. was analyzed during the different stages of a contraction-catch-relaxatin cycle.
• 2.2. The experiments were performed with saponin-skinned fibers preincubated with γ-³²P-ATP.
• 3.3. The total amount of ³²P incorporated into the fiber proteins was anlyzed by measuring the label of TCA-insoluble protein in a scintillation counter.
• 4.4. The dose incorporated was about twice as high during Ca²⁺ induced contraction and serotonin induced accelerated relaxation as during test and catch.
• 5.5. The molecular mass of the phosphorylated proteins was analyzed by autoradiography of the proteins separated by SDS-PAGE.
• 6.6. Up to 26 protein spots of different molecular masses were labelled, including such well characterized protein spe+cies as myosin heavy and light chains, paramyosin and tropomyosin.

     

Possible involvement of adenylylation in the modification of a 26 kDa protein in rat parotid acinar cells

• 1.1. Adenylylation, a posttranslational modification of proteins, was investigated in saponin-permeabilized acinar cells of the rat parotid gland.
• 2.2. When cells were incubated with [2,8-³H]ATP, several proteins, including a 26 kDa protein in the particulate fraction, were labeled.
• 3.3. Upon incubation of cells with [α-³²P]ATP in the presence of cAMP and 3-isobutyl-lmethylxanthine, ³²P-labeling of the 26 kDa protein was observed.
• 4.4. After treatment with snake venom phosphodiesterase, [³²P]AMP was released from the 26kDa protein. Such release was not observed when cells were labeled with [γ-³²P]ATP.
• 5.5. The ³²P-labeling pattern of proteins with [α-³²P]ATP was clearly different from that with [adenylate-³²P]NAD⁺.
• 6.6. The results suggest that the 26 kDa protein is one of the adenylylation substrates in rat parotid acinar cells.

     

Ion transport in crab gills: A new method using isolated half platelets of Eriocheir gills in an ussing-type chamber

• 1.1. A half platelet preparation from Chinese crab (Eriocheir sinensis) gill is described which allows electrophysiological investigations of ion transport by gill epithelial monolayer when mounted in a modified Ussing chamber.
• 2.2. The resistance of these preparations equals half that of complete gill platelets (containing the gill epithelium and cuticle twice) indicating that cell damage during preparation of half platelets is negligible.
• 3.3. The transepithelial resistance (resistance of cuticle subtracted previously) was determined to be about 140 Ω cm² when both sides are bathed with identical salines.
• 4.4. Similarities to the results obtained with perfused complete gills demonstrates the reliability of this preparation.
• 5.5. When identical salines are applied on both sides of the epithelium an outside positive transepithelial potential difference (PD_te) up to 40 mV was measured.
• 6.6. The occurrence of such a high PD_te under symmetric conditions and its sensitivity to CN⁻ suggests the PD_te to be generated by active transport processes.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

Contrast in adenine uptake by chicken and rabbit erythrocytes in vitro

• 1.1. Relative to rabbit erythrocytes, chicken red blood cells exhibit a much greater capacity to utilize [³H]adenine for nucleotide synthesis in vitro, even at 5°C and in the absence of added inorganic phosphate.
• 2.2. This difference is largely due to a higher concentration of phosphoribosylpyrophosphate and greater activity of adenine phosphoribosyltransferase in the avian cells. lli]3. The capacity of avian erythrocytes for utilization of guanine and hypoxanthine is several fold less than that of adenine.
• 3.4. The data are consistent with lower activity for hypoxanthine/guanine phosphoribosyltransferase than for adenine phosphoribosyltransferase in intact chicken erythrocytes.
• 4.5. The results indicate that reutilization of adenine by chicken erythrocytes may be physiologically significant.

     

Phosphorylation in crayfish (Procambarus clarkii) eggs during hatching

• 1.1.|The high-energy phosphorylation metabolism in crayfish, Procambarus clarkii eggs during brooding and juvenile crayfish after hatching was studied by in vivo³¹P nuclear magnetic resonance (³¹P NMR) spectroscopy.
• 2.2.|Inorganic phosphoric acid (Pi) and adenosine-5′-triphosphate ATP(γ-,α-,β-) were detected in the dark brownish red eggs after oviposition.
• 3.3.|In orange unhatched eggs, only sugar phosphate (SP), Pi and resolved phosphometabolite from ATP were observed.
• 4.4.|Peaks of SP, Pi, arginine phosphate (Arg-P), and ATP (γ,α,β) appeared in larvae of crayfish after hatching (nauplius, zoea and juvenile crayfish).
• 5.5.|The high-energy phosphorylation metabolism changed to an anaerobic condition along with a decrease in the concentration of dissolved oxygen in fresh water.

     

An alkaline phosphatase from Thermus sp strain Rt41A

• 1.1. A thermostable orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from Thermus sp strain Rt41A has been purified 400-fold to give a specific activity of 25 U/mg at 60°C in IM diethanolamine (pH 11.1).
• 2.2. The enzyme has a M_r of 160,000 and is trimeric.
• 3.3. The half-life of the enzyme is 5 min at 85°C.
• 4.4. The enzyme has a wide specificity for a number of phosphate monoesters.
• 5.5. The H_m of the enzyme is pH dependent, so the pH optimum of the enzyme is affected by the substrate concentration.
• 6.6. The enzyme is inhibited 50% by 20 mM Ca²⁺ or Mg²⁺.
• 7.7. The K_i for phosphate, EDTA-di sodium salt and arsenate (in 1 M diethanolamine, pH 11.1) is approx 1.2, 1.6 and 4mM respectively.
• 8.8. Urea (200 mM) is not inhibitory.

     

Effects of water stress on hemolymph volume,osmotic potential and chemical composition in Megetra cancellata

• 1.1. Rates of water loss in Megetra cancellata were very high compared to those reported for other xeric arthropods.
• 2.2. Hemolymph weight in hydrated animals was 43.0% of the total body weight while it was 24.7% in desiccated animals that had lost 16.1% of their body weight as water.
• 3.3. Hemolymph osmotic potential increased from 417 to 447 mOsm/kg in desiccated beetles, but osmotic regulation was evident.
• 4.4. Total hemolymph protein mass and concentration decreased in desiccated beetles while amino acid concentrations remained constant (at about 70 mM).
• 5.5. Na⁺ and −PO₄ concentrations increased in desiccated beetles.
• 6.6. Cl⁻ and K⁺ concentrations in desiccated beetles were equal to those in undesiccated beetles.

     

Effect of inorganic phosphate on synthesis of 5-phosphoribosyl-1-pyrophosphate in cultured plant cells

• 1.1. Inorganic phosphate (Pi) was absorbed rapidly by suspension-cultured cells of Catharanthus roseus which had previously been cultured in Pi-free Murashige Skoog medium.
• 2.2. The intracellular levels of ATP, ADP and 5-phosphoribosyl-l-pyrophosphate (PRPP) increased markedly during the 24 hr which followed the addition of Pi (1.25mM).
• 3.3. Availability of PRPP in vivo, estimated by the measurement of nucleotide synthesis from [8-¹⁴C]adenine, was also increased by addition of Pi.
• 4.4. Only a 20% increase in the maximum catalytic activity of PRPP synthetase was observed in extracts of cells, prepared 24 hr after addition of Pi.
• 5.5. In contrast to results for mammalian PRPP synthetase, the activity of PRPP synthetase, partially purified from Catharanthus roseus, was inhibited by concentration of Pi greater than 5mM.
• 6.6. The mechanisms involved in the increased availability of PRPP and the synthesis of adenine nucleotides in the plant cells cultured in Pi-containing medium are discussed.