Bioelectrical potentials across the mantle epithelium of Achatina fulica

• 1.1. The shell side of the mantle of Achatina fulica is several millivolts positive to the blood side in vitro.
• 2.2. The electrical potential does not depend on Na⁺, Ca²⁺, Mg²⁺, K⁺ or HCO₃⁻ but requires the presence of chloride on the shell side.
• 3.3. The potential difference and short-circuit current ranged from 3.0 to 30.0 mV and 15.0 to 75 μA/cm² with averages at 10m V and 50 μA/cm² respectively.
• 4.4. The electrical gradient is reduced by 2,4-dinitrophenol, thiocyanate and furosemide but not by ouabain, CO₂ or acetozolamide.
• 5.5. It is suggested that the nature and mechanism of electrogenesis in Achatina parallels that of the Helix mantle.

     

An in vitro study of short-chain fatty acid concentrations,production and absorption in pig (Sus scrofa) colon

• 1.1. Short-chain fatty acid concentration was 180mmol/l in the proximal colon and decreased to 108 mmol/l in the rectum.
• 2.2. Fermentation in chymus from different regions of the colon, showed the pattern of end products to reflect the substrate and not the site of the colon.
• 3.3. Isolated mucosa from proximal and distal colon had electroneutral sodium absorption of 4.8 ± 0.2 and 2.9 ± 0.8 μeq/cm² hr in bicarbonate free media, which was abolished in the absence of chloride.
• 4.4. Electroneutral sodium absorption was enhanced by short-chain fatty acids in the proximal colon and could be described by Michaelis-Menten kinetics with K_m 2.0–11 mmol/l and J_m 1.6–3.6μeq/cm² hr. In the distal colon the stimulation was smaller and propionate even inhibited sodium absorption.
• 5.5. Butyrate was absorbed in the proximal colon, whereas acetate and propionate, and butyrate in the distal colon had a flux ratio of one.
• 6.6. Amiloride (5 mmol/l) inhibited sodium absorption and net butyrate absorption.

     

Calcium-dependent potassium conductance in the photoresponse of a nudibranch mollusk

• 1.1. The response to light of Hermissenda photoreceptors when recorded intracellularly without interference from synaptic and action potentials consisted of three phases: an early depolarization (ED) followed by hyperpolarization (dip) and subsequent depolarization (tail).
• 2.2. The ED and the dip were associated with increased membrane conductance while decreased membrane conductance was involved with the tail.
• 3.3. The dip reversal potential was − 82.1 ± 5.3 mV and its amplitude varied inversely with the log of [K⁺].
• 4.4. Perfusing with agents which block K⁺ current like 4AP, Quinine, Quinidine or injection of TEA eliminated the dip and its associated increased membrane conductance, thus further supporting the role of K⁺ conductance in producing the dip.
• 5.5. The dip was enhanced by increased [Ca²⁺]_o, reduced by decreased [Ca²⁺]_o and abolished together with its associated increased membrane conductance when perfused with either D600, Cd²⁺, Mg²⁺, Mn²⁺, or Co²⁺, which block transmembrane Ca²⁺ current.
• 6.6. The dip and its associated increased membrane conductance were abolished by intracellular injection of EGTA and enhanced by perfusion with Ruthenium red.
• 7.7. Intracellular injection of Ca²⁺ mimicked the dip: membrane conductance was increased and the cell hyperpolarized.
• 8.8. These results indicate that the increase in intracellular [Ca²⁺] is primarily responsible for the light-induced increase of K⁺ conductance during the dip. The possible source of the Ca²⁺ is, at least in part, extracellular due to activation of an inward Ca²⁺ current.

     

Kinin-inactivating endopeptidase from rat liver

• 1.1. A kinin-inactivating serine-endopeptidase from rat liver was purified to an activity of 912 mU/mg of protein, when measured on bradykinin.
• 2.2. The endopeptidase molecular weight, estimated by gel filtration, was 68,000. Its isoelectric point was close to pH 4.9.
• 3.3. Vm for the hydrolysis of bradykinin, was 1.25 μmol/min/mg protein; K_m was 28μM. The two hydrolysis products from bradykinin were the pentapeptide Arg¹-Phe⁵ and the tetrapeptide Ser⁶-Arg⁹.

     

PLA2 activity in rat liver nuclei

• 1.1. Subcellular fractions of rat liver were assayed for PLA₂ activity.
• 2.2. The PLA₂ assay measures the release of [³ H]oleic acid from phospholipids, using labeled E. coli as substrate.
• 3.3. Nuclear fractions contained PLA₂ activity, which was Ca²⁺ dependent and could not be explained from mitochondrial, microsomal or plasma membrane contamination.
• 4.4. The V_max value of nuclear PLA₂ is 0.30 ± 0.04 pmol oleic acid/min/mg protein; its K_m value is 0.86±0.12μM, similar to that of mitochondrial PLA₂.
• 5.5. We conclude that rat liver nuclei contain PLA₂ activity.

     

Kinetic analysis of rat liver sorbitol dehydrogenase

• 1.1. A steady state kinetic investigation was performed on an improved preparation of rat-liver sorbitol dehydrogenase (l-iditol: NAD-oxidoreductase, EC 1.1.1.14).
• 2.2. Data analyses indicate the enzyme follows a rapid equilibrium random mechanism in the direction of sorbitol oxidation and a random mechanism in the direction of fructose reduction.
• 3.3. Kinetic constants were: K_m^NAD 0.082 mM; K_m^sorbitol 0.38 mM; K_m^NADH 67 μm; K_m^fructose 136 μM.
• 4.4. Evidence is adduced to indicate the more rapid reverse (fructose reduction) reaction is susceptible to metabolic control by formation of abortive enzyme-fructose-NAD and enzyme-NADH-sorbitol complexes.

     

Alkaline phosphatase from Basidiobolus haptosporosus: Comparison with alkaline phosphatases from rainbow lizard and man

• 1.1. DEAE-cellulose chromatography of mycelial alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) from Basidiobolus haptosporosus, produced three iso-enzymes “A”, “B” and “C”.
• 2.2. Fraction “A” was further characterized and showed maximum activity at pH 10 in 0.1 M sodium carbonate-bicarbonate buffer.
• 3.3. The enzyme was stimulated by Mg²⁺, Co²⁺ and Mn²⁺ and inactivated by Zn²⁺, Cu²⁺, EDTA, citrate and tartrate.
• 4.4. Phosphate ions inhibited it competitively, phenylalanine uncompetitively and urea noncompetitively.
• 5.5. It was heat stable for 60 min at 37°C but labile above 55°C.
• 6.6. Its K_m with p-nitrophenylphosphate was 0.5 mM; its estimated molecular weight was 160,000.
• 7.7. The results are compared with the properties of alkaline phosphatases from the rainbow lizard and man and discussed in terms of a triadic association between the fungus, the lizard and man.

     

l-lysinamidase from Cryptococcus laurenti 112. Purification and properties

• 1.1. The purified enzyme hydrolyzes the linear l-lysinamide and the cycle amide of l-lysine—l-α-amino-ϵ-caprolactam.
• 2.2. The apparent relative molecular mass is 180,000. The enzyme consists of four subunits and the molecular mass of a single subunit was found to be 47,000.
• 3.3. The coefficient of molecular sedimentation equals 8.3 S, the isoelectric point was determined to be pH 4.3
• 4.4. The enzyme is not a glycoprotein. p-Mercuribenzoate binds 10 SH-groups of the native enzyme molecule and 20 SH-groups in the presence of 0.7% SDS.
• 5.5. pH- optimum for the hydrolysis of l-lysine amides was observed to be 7.5–7.7. The enzyme is strictly dependent on Mn²⁺ and Mg²⁺.
• 6.6. The kinetic parameters for the hydrolysis of l-lysinamide where K_m = 3.8 mM and k_cat = 3000 sec⁻¹ For the hydrolysis of cyclic L-lysinamide K_m = 4.8 mM and k_cat = 2600 sec⁻.

     

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.

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

Decarboxylation of uroporphyrinogen I and III in 2,3,7,8-tetrachlorodibenzo-p-dioxin induced porphyria in mice

• 1.1. The decarboxylation of uroporphyrinogens I and III by porphyrinogen carboxy-lyase (EC 4.1.1.37) in mouse liver supernatant was compared in relation to substrate concentrations.
• 2.2. In this species uroporphyrinogen III was the best substrate judging by the criteria of K_m/V_max (estimated for total porphyrinogens) and was converted into coproporphyrinogen faster than its series I isomer.
• 3.3. The difference between the two isomers was mainly due to the first decarboxylation.
• 4.4. This difference was confirmed by calculation of the Hill coefficient and of Lineweaver-Burk plot which suggested that isomer I induced negative cooperativity in the active centre of the enzyme.
• 5.5. After treatment with a porphyrogenic dose of TCDD (25 μg/kg/week for 9 weeks) differences between uroporphyrinogen I and III as substrate were maintained.
• 6.6. In addition treatment reduced V_max and K_m (estimated for total porphyrinogens) of liver porphyrinogen carboxy-lyase to about half control values for both isomers.
• 7.7. V_max was reduced mainly because of the formation of smaller amounts of all products of decarboxylation, and K_m because more heptaporphyrinogen was formed than coproporphyrinogen.
• 8.8. Values of the Hill coefficient and Lineweaver-Burk plots suggested TCDD induced altered substrate affinity for isomer III too.
• 9.9. Treatment with TCDD did not affect the decarboxylation of uroporphyrinogen III by RBC porphyrinogen carboxy-lyase, estimated from K_m and V_max for total porphyrinogens formed.

     

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.

     

Preliminary studies on the characteristics of phenylalanine and β-methyl-glucoside transport in the tench intestine in vitro

• 1.1. The unidirectional transepithelial fluxes of L-phenylalanine, β-methyl-D-glucoside and sodium ions across emusculated sheets of tench mid-intestine were determined in flux chambers.
• 2.2. No net sodium flux was detectable, but phenylalanine was preferentially transferred from the mucosal to the serosal fluid.
• 3.3. There was also a net movement of β-methyl-glucoside towards the serosal medium, but it was much smaller than that of phenylalanine.
• 4.4. This transport was accompanied by an accumulation of each substrate from the mucosal medium into the tissue to a similar level and against a concentration gradient.
• 5.5. The poor transfer of the monosaccharide into the serosal medium could therefore be attributed to a low permeability of the baso-lateral membrane of the enterocyte for this substance.
• 6.6. The influx of L-phenylalanine and of β-methyl-d-glucoside into the epithelial cells of tench midintestine was examined by incubating slices of emusculated intestine in radioactively-labelled solutions of the substrate for 2 min.
• 7.7. The steady-state uptake was assessed after similar incubations lasting 45 min.
• 8.8. Phenylalanine influx obeys the Michaelis-Menten equation with a K_m of 2.9 mM and is dependent on the presence of sodium ions in the incubation medium.
• 9.9. β-Methyl-glucoside influx reveals the same characteristics with a K_m of 2.0 mM but a considerably lower V_max; in addition, it is inhibited by galactose.
• 10.10. The influx of both substrates is reduced by harmaline, which also inhibits the uptake of radioactive sodium by this preparation.
• 11.11. The steady-state uptake of β-methyl-glucoside is also inhibited by ouabain and by 2.4-dinitrophenol.
• 12.12. These results suggest that the mechanisms for sodium-dependent influx of monosaccharides and neutral amino-acids in the tench intestine are similar to those found in mammalian tissues.
• 13.13. The principal difference appears to involve the release of monosaccharides across the baso-lateral membrane of the enterocyte.

     

Acid phosphatase in the contents of the oesophagus and stomach of the snail,Helix pomatia L.

• 1.1. In the contents of the oesophagus and stomach, one form of acid phosphatase is found. Its electrophoretic mobility is identical to that of the multiple form 3 of acid phosphatase from the hepatopancreas.
• 2.2. The enzyme is not stimulated by divalent cations. It is inhibited by molybdate, Cu²⁺, Hg²⁺. F⁻ and tartrate L+.
• 3.3. The optimum pH of the enzyme is 4.5. The K_m for paranitrophenylphosphate as substrate amounts to 0.25 mM. The enzyme is stable at a temperature of up to 55°C.

     

Purification and properties of tetralysine endopeptidase from Escherichia coli AJ005

• 1.1. A new tetralysine endopeptidase from Escherichia coli AJ005 has been purified about 135-fold.
• 2.2. The peptidase seems to be specific to tetralysine among lysine homopolymers.
• 3.3. The optimal pH was about 7.5
• 4.4. The activity was inhibited by KCN but not inhibited by soybean trypsin inhibitor.
• 5.5. The apparent K_m value was 2.5 × 1O⁻³ M for tetralysine.

     

The exchange of radioactive cations by somatic and cardiac muscles of the crayfish

• 1.1. Intracellular concentrations of Na⁺, K⁺, Ca²⁺ and Mg²⁺ were measured in a somatic muscle and in the heart of the crayfish. The uptake and the efflux of Na²⁴, K⁴², Ca⁴⁵ and of Sr⁸⁹ were also measured.
• 2.2. The initial influx rates of the ions from van Harreveld's solution into resting somatic muscle (in μEq/g cell water/hr) are: K⁺ = 25; Na⁺ = 56; Ca²⁺ = 38. Similar figures were obtained for the heart muscle.
• 3.3. The calculated permeability constants (× 10⁸ cm/sec) are: P_K = 64; P_Na = 30 P_Ca = 10; P_Sr = 1·5.
• 4.4. The stimulation of the muscle fiber leads to an additional Ca²⁺ influx of about 2·8 pEq/cm² fiber surface. The additional Ca²⁺ uptake is sufficient to account for the change in potential on the membrane.
• 5.5. When muscles were immersed in Sr²⁺ solutions, no additional Sr⁸⁹ uptake was found with stimulation. However, there is a high resting Sr⁸⁹ uptake and the muscle in Sr²⁺ has a long refractory period, so a reasonable increase in Sr⁸⁹ uptake would not be detectable.
• 6.6. The results are discussed in relation to the divalent cation mechanism for generating action potentials and to the part played by Ca²⁺ in triggering contraction.

     

Reabsorption and accumulation of α-amino-iso-butyric acid in the nephridia of Sabella pavonina Sa vigny (Annelida polychaeta)

• 1.l. High amino acid concentrations were found in the anterior coelomic fluid of a Polychaeta (Sabella pavonina Savigny).
• 2.2. The concentrations being much higher in the fluid which penetrates the nephrostomia into the nephridia lumen than in the final urine indicates that the nephridia reabsorbs large amounts of amino acids.
• 3.3. Nephridial perfusion experiments showed that an amino acid analogue (α-amino-iso-butyric acid, AIB) is transported by the nephidia.
• 4.4. The transport took place across the nephridial wall owing to the presence of a carrier-mediated transport system and a diffusion system.
• 5.5. For the carrier-mediated transport, the V_max was 0.234 ± 0.025 nmol·min⁻ and the K_m 3.715 ± 0.315mmol·l⁻.
• 6.6. AIB accumulated in the nephridial cells up to a maximum rate of 01.17 nmol·min⁻.
• 7.7. Intracellular accumulation stopped increasing when the V_max for reabsorption was reached.
• 8.8. These results indicate that the carrier-mediated transport of AIB is located at the apical membrane of the nephridial cell, and that AIB transport by simple diffusion takes place through the paracellular pathway.

     

Sodium and potassium balance of captive meadow voles (Microtus pennsylvanicus) fed laboratory chow and vegetation diets

• 1.1. Mineral balance was studied in meadow voles (Microtus pennsylvanicus) maintained in the laboratory.
• 2.2. Urine and fecal Na⁺ contents of voles on low-Na⁺ diets were comparable to those reported for other herbivore species, but urine and fecal K levels were higher.
• 3.3. Voles approached Na⁺ balance (input = output) on diets with Na⁺ content as low as 56 ppm.
• 4.4. There was not a clearcut hypertrophy of the adrenal-gland zona glomerulosa in voles maintained on low-Na⁺ diets.
• 5.5. Plasma K content and bone water content were higher in voles maintained on high-Na ⁺ vegetation diets, suggesting expansion of extracellular fluid volume.

     

Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

Potassium channels in growth cones of leech retzius cells

• 1.1. Potassium-selective channels were analysed in growth cones of cultured leech Retzius cells.
• 2.2. In the cell-attached mode and at physiological bath and pipette solution little channel activity was observed at resting membrane potential. The channel open probability (p_o) increased with cell depolarization, and the slope conductance of the single K⁺ channel current was about 60 pS.
• 3.3. With symmetrical high KCl solution on both sides of the excised membrane patch three K⁺ -selective channels could be discriminated. Two channels exhibited a linear current-voltage relation of about 18 pS and 106 pS, respectively.
• 4.4. The most frequently observed K⁺ channel showed a non-linear current-voltage relation and p_o increased with increasing free cytoplasmic Ca²⁺ and during cell hyperpolarization.