Reduced external calcium or sodium stimulates calcium influx in Pelvetia eggs

The effect of external calcium and sodium ion concentrations on the calcium fluxes on the Pelvetia fastigiata De Toni egg was measured. Decreasing external [Ca²⁺] greatly increased the permeability of the eggs to Ca²⁺; at 1 mM external Ca²⁺ this permeability was 60 times as great as it was at the normal [Ca²⁺] of 10 mM. Lowering the external [Na⁺] also increased Ca²⁺ influx; at 2 mM Na⁺, the Ca²⁺ influx was 2–3 times as great as it was at the normal [Na⁺] if choline was used as a Na⁺ substitute. Lithium was less effective as a Na⁺ substitute in increasing Ca²⁺ influx. The extra Ca²⁺ influx in low [Na⁺] seemed to be dependent on internal [Na⁺]. The Ca²⁺ efflux increased transiently and then declined in low Na⁺ media.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Macrophyte-epiphyte biomass and productivity in an eelgrass (Zostera marina L.) community

The biomass, productivity (¹⁴C), and photosynthetic response to light and temperature of eelgrass, Zostera marina L. and its epiphytes was measured in a shallow estuarine system near Beaufort, North Carolina, during 1974. The maximum of the biomass (above-ground) was measured in March; this was followed by a general decline throughout the rest of the year. The average biomass was 105.0 g dry wt m^?2; 80.3 g dry wt m^?2 was eelgrass and 24.7 g dry wt m^?2 was epiphytes. The productivity of eelgrass averaged 0.88 mg C g^?1 h^?1 which was similar to that of the epiphytes, 0.65 mg C g^?1 h^?1. Eelgrass and epiphyte productivity was low during the spring and early summer, gave a maximum during late summer and fall, and declined during the winter; this progression was probably due to environmental factors associated with tidal heights. On an areal basis, the average annual productivity was 0.9 g C m^?2 day^?1 for eelgrass and 0.2 g C m^?2 day^?1 for the epiphytes. Rates of photosynthesis of both eelgrass and epiphytes increased with increasing temperature to an asymptotic value at which the system was light saturated. Both eelgrass and epiphytes had a temperature optimum of < 29 °C. A negative response to higher temperatures was also reflected in biomass measurements which showed the destruction of eelgrass with increasing summer temperatures. The data suggest that the primary productivity cycles of macrophytes and epiphytes are closely interrelated.     

Developmental changes in the45Ca2+ uptake byTrichoderma viride mycelium

The rate of the⁴⁵Ca²⁺ uptake by the submergedTrichoderma viride mycelium increased with the age of the culture from 6 h until a maximum which was reached at about 30 h, and then decreased until the uptake was virtually zero. The decrease in the rate of the⁴⁵Ca²⁺ uptake was accompanied by an increase of mycelial mass. The uptake rate could not be reactivated upon substituting the medium for a fresh one, without or with dilution of the mycelium. The results suggest that the rate of⁴⁵Ca²⁺ uptake reflects the biological age of the submerged culture. The surface-cultivated mycelium took up⁴⁵Ca²⁺ proportionally with time. The autoradiography of colonies showed that⁴⁵Ca²⁺ was distributed homogeneously throughout the mycelium during vegetative growth while conidiation was accompanied by a massive accumulation of⁴⁵Ca²⁺ in conidia. This work was supported by theSwiss National Science Foundation (joint Swiss-Slovak project 75LPJ041485) andSlovak Grant Agency (grant no. 1/1158/93).     

Purification and properties of a proteinase from Vipera lebetina (snake) venom

A proteinase from the venom of Vipera lebetima was purified by chromatography on Sephadex G-100 and CM-cellulose. The purified proteinase was homogeneous on SDS-polyacrylamide gel electrophoresis and consisted of a single chain with molecular weight of 37 000±1500. The isoelectric point of the proteinase was over 10. The enzyme was active on casein but not on esters and amides of arginine. It split the oxidized insulin B-chain at the peptide bonds of Tyr¹⁶-Leu¹⁷, Phe²⁴-Phe²⁵ and Phe²⁵-Tyr²⁶, and glucagon at the bonds Tyr¹⁰-Ser¹¹, Leu¹⁴-Asp¹⁵ and Leu²⁶-Met²⁷. The enzyme was inhibited by DFP and PMSF, and partially by soybean trypsin inhibitor, but not with EDTA.     

Volume changes of mammalian cells subjected to hypotonic solutions in vitro: evidence for the requirement of a sodium pump for the shrinking phase

A Coulter-orifice pulse-height analyzer system was used to measure volume spectra of mammalian cells in suspension at different times after the addition of an equal volume of water. In appropriate hypotonic medium, cultured mammalian cells rapidly increase in volume and then shrink, more slowly, approaching their initial volumes within 20 to 30 minutes at 37.5°C. The shrinking phase was found to be reversibly inhibited by ouabain and inhibited in both K⁺-free and Na⁺-free solutions; neither choline⁺ nor Li⁺ could substitute for extracellular Na⁺ in supporting the shrinking phenomenon but Rb⁺ and Cs⁺ were fairly good substitutes for K⁺. Under conditions similar to those with which the shrinking phenomenon was observed with cultured cells, it was not found with either human or mouse red blood cells. Two methods were used to determine intracellular Na⁺ and K⁺ content in osmotically shocked cells and in unshocked controls. An isotope equilibration method was employed with L5178-Y mouse lymphoblasts and a chemical determination by flame photometry was used with Ehrlich ascites tumor cells. The K⁺ content was significantly reduced and the Na⁺ content was unchanged or somewhat increased in cells which had returned to their original volumes in hypotonic medium. The K⁺ content was even more reduced but the Na⁺ content was greatly increased in cells which were osmotically shocked in the presence of ouabain.     

Further Studies on the Roles of Sodium and Potassium in the Generation of the Electro-Olfactogram : Effects of mono-, di-, and trivalent cations

In the negative EOG-generating process a cation which can substitute for Na⁺ was sought among the monovalent ions, Li⁺, Rb⁺, Cs⁺, NH₄⁺, and TEA⁺, the divalent ions, Mg⁺⁺, Ca⁺⁺, Sr⁺⁺, Ba⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, Co⁺⁺, and Ni⁺⁺, and the trivalent ions, Al⁺⁺⁺ and Fe⁺⁺⁺. In Ringer solutions in which Na⁺ was replaced by one of these cations the negative EOG's decreased in amplitude and could not maintain the original amplitudes. In K⁺-Ringer solution in which Na⁺ was replaced by K⁺, the negative EOG's reversed their polarity. Recovery of these reversed potentials was examined in modified Ringer solutions in which Na⁺ was replaced by one of the above cations. Complete recovery was found only in the normal Ringer solution. Thus, it was clarified that Na⁺ plays an irreplaceable role in the generation of the negative EOG's. The sieve hypothesis which was valid for the positive EOG-generating membrane or IPSP was not found applicable in any form to the negative EOG-generating membrane. The reversal of the negative EOG's found in K⁺- , Rb⁺- , and Ba⁺⁺-Ringer solutions was attributed to the exit of the internal K⁺. It is, however, not known whether or not Cl^- permeability increases in these Na⁺-free solutions and contributes to the generation of the reversed EOG's.     

Dual effects of K ions upon the inactivation of the anomalous rectifier of the tunicate egg cell membrane

Summary Inactivation of the K inward current through the anomalous rectifier channel of the egg cell membrane of a tunicate,Halocynthia roretzi Drashe, was studied under voltage-clamp. The noise spectrum of the steady-state current recorded at hyperpolarized potentials was measured in solutions in which Na, Cs, Hydrazine, or Sr caused inactivation of the current. The unitary conductance estimated was independent of which cation caused inactivation. From the relation between the concentration of cations which caused inactivation and the extent of inactivation at fixed potentials, the binding of one inactivator to a channel was found to cause inactivation, and the potency of inactivation was Cs⁺>Hydrazine⁺>Na⁺>Li⁺, and Ba²⁺>Sr²⁺. The inactivation caused by Na⁺ was increased by K⁺ when [K] _o was lower than 20mm, but was decreased by K⁺ in higher K-ASW (artificial sea water). One K⁺ was found to inactivate the channel cooperatively with one Na⁺. Increase of inactivation by K⁺ was a dominant effect in Cs-ASW. The inactivation was explained quantitatively by a model assuming cooperative plugging by a monovalent inactivator and a K⁺.     

Cooperative binding of calcium to glycerinated skeletal muscle fibers

The binding of ⁴⁵Ca²⁺ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg²⁺ (no ATP) binding was half-maximal at 1.4 · 10^?6M Ca²⁺ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 10⁶ M^?1) and the Hill coefficient was 1.0. In the absence of Mg²⁺, binding was half-maximal at 3 · 10^?7 M Ca²⁺ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 10⁷ and 2 · 10⁶ M^?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg²⁺ binding to about half of the total Ca²⁺ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites.     

Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state

Summary The membrane potential of mitochondria was estimated from the accumulation of tetraphenyl phosphonium (TPP⁺), which was determined with the TPP⁺-selective electrode developed in the present study. The preparation and some operational parameters of the electrode were described. The kinetics for uptake by mitochondria of TPP⁺ and DDA⁺ (dibenzyldimethyl ammonium) were analyzed, and it was found that TPP⁺ permeated the mitochondrial membrane about 15 times faster than DDA⁺. The final amounts of accumulation of TPP⁺ and DDA⁺ by mitochondria were approximately equal. For the state-4 mitochondria, the membrane potential was about 180 mV (interior negative). Simulataneous measurements of TPP⁺-uptake and oxygen consumption showed that the transition between states 3 and 4 was detectable by use of the TPP⁺-electrode. After the TPP⁺-electrode showed that state-4 was reached, the extramitochondrial phosphorylation potential was measured. The difference in pH across the membrane was measured from the distribution of permeant anion, acetate, so as to calculate the proton electrochemical potential. The ratio of extra-mitochondrial phosphorylation potential to proton electro-chemical potential,n was close to 3. This value ofn was also found to be 3 when ATP was hydrolyzed under the condition that the respiratory chain was arrested. The implication thatn=3 was discussed.     

Loading of lipophorin particles with phospholipids at the midgut of Rhodnius prolixus

³²P-Labelled midguts (³²P-midguts) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release of radioactivity to the medium was analysed. The radioactivity found in the medium was associated with lipophorin phospholipids. When the ³²P-midguts were incubated in the absence of lipophorin, no ³²P-phospholipids were found in the medium. Comparative analysis by thin-layer chromatography of ³²P-phospholipids derived from metabolically labelled ³²P-midgut or lipophorin particles after incubation with ³²P-midgut showed some differences, revealing a possible selectivity in the process of phospholipids transfer. The transfer of phospholipids to lipophorin was linear with time up to 45 min, was saturable with respect to the concentration of lipophorin, and was half-maximal at about 5 mg/ml. The binding of ³²P-lipophorin to the midgut at O°C reached the equilibrium at about 1 h of incubation. The binding of ³²P-lipophorin was inhibited by an excess of nonradioactive lipophorin, which suggests a specific receptor for lipophorin. The capacity of midguts and fat bodies to transfer phospholipids to lipophorin varied during the days following the meal. When lipophorin enzymatically depleted of phospholipids by treatment with phospholipase A² was incubated with ³²P-midguts, the same amount of phospholipids was transferred, indicating a net gain of phospholipids by the particle. © 1995 Wiley-Liss, Inc.     

Intracellular Ca<Superscript>2+</Superscript> Pools and Fluxes in Cardiac Muscle-Derived H9c2 Cells

Relevant Ca²⁺ pools and fluxes in H9c2 cells have been studied using fluorescent indicators and Ca²⁺-mobilizing agents. Vasopressin produced a cytoplasmic Ca²⁺ peak with half-maximal effective concentration of 6 nM, whereas thapsigargin-induced Ca²⁺ increase showed half-maximal effect at 3 nM. Depolarization of the mitochondrial inner membrane by protonophore was also associated with an increase in cytoplasmic Ca²⁺. Ionomycin induced a small and sustained depolarization, while thapsigargin had a small but transient effect. The thapsigargin-sensitive Ca²⁺ pool was also sensitive to ionomycin, whereas the protonophore-sensitive Ca²⁺ pool was not. The vasopressin-induced cytoplasmic Ca²⁺ signal, which caused a reversible discharge of the sarco-endoplasmic reticulum Ca²⁺ pool, was sensed as a mitochondrial Ca²⁺ peak but was unaffected by the permeability transition pore inhibitor cyclosporin A. The mitochondrial Ca²⁺ peak was affected by cyclosporin A when the Ca²⁺ signal was induced by irreversible discharge of the intracellular Ca²⁺ pool, i.e., adding thapsigargin. These observations indicate that the mitochondria interpret the cytoplasmic Ca²⁺ signals generated in the reticular store.     

Regulation of sodium transport in erythrocytes

The kinetic response of swine erythrocyte (Na + K)-ATPase to Na⁺ concentration was hyperbolic in low KCl (5–25 mm) but became increasingly sigmoidal (n = 2.2) as KCl was increased to 150 mm. The addition of 150 mm LiCl did not cause an increase in sigmoidicity although it decreased the apparent affinity for Na⁺. The dependence of ouabain-inhibited efflux of Na⁺ on internal Na⁺ concentration was measured in intact cells with intracellular cation concentrations altered by incubation in p-ehloromercuriphenyl sulfonate. The response to Na⁺ was sigmoidal (n = 2.2) in cells containing high K⁺ but hyperbolic in preparations in which most of the intracellular K⁺ was replaced by Li⁺, even in the presence of 150 mm external KCl. The data are consistent with a model in which internal K⁺ is an allosteric (feedback) inhibitor of Na⁺ efflux and there are three Na⁺ sites which interact cooperatively.     

Immobilized antibody-based flow type enzyme immunosensor for determination of human serum albumin

A flow-type enzyme immunosensor was prepared for the electrochemical determination of human serum albumin (HSA). The immunosensor was constructed from the immobilized antibody (anti-HSA IgG) reactor and an oxygen electrode. The immunochemical reaction of catalase-labelled antibody with HSA was completed with 30 min. After the immunochemical reaction, hydrogen peroxide solution was injected into the system and a peak current was obtained within 2 min. A linear relationship was observed between the current increase and the logarithm of HSA concentration in the range 10⁻⁸-10⁻⁶ g ml⁻¹. The minimum measurable concentration was 10⁻⁸ g ml⁻¹. The current increase was reproducible with 10% of the relative errors when a sample solution containing 10⁻⁷ g ml⁻¹ of HSA was used. The minimum measurable concentration increased to 10⁻⁹ g ml⁻¹ when hydrogen peroxide was recycled for 5 min in the reactor system. The immobilized antibody reactor could be reused. HSA in human serum was determined by the system proposed.     

Basic physico-chemical parameters of milt from sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss)

The aim of the study was to compare the physico‐chemical parameters of milt from sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss). Milt was collected by stripping and spermatozoa concentrations, were determined and compared with sperm motility and spermatocrit values along with seminal plasma indices (pH, osmolality, sodium, potassium, chlorine, calcium, magnesium, glucose and protein concentrations). The highest spermatozoa concentration of 22.3 ± 6.7 × 10⁹ ml^?1 was found in the sea trout milt, and was significantly different of those observed in brook trout (11.9 ± 3.3 × 10⁹ ml^?1) and rainbow trout (10.7 ± 4.4 × 10⁹ ml^?1). The values for pH and K⁺ did not differ significantly among species. The mean pH was 8.0 in the milt of each species and the K⁺ concentrations ranged from 24.8 ± 7.2 to 30.5 ± 7.6 mm L^?1. Considerable differences were determined for the Ca²⁺ ions concentrations. The highest value was found in sea trout (1.7 ± 0.3 mm L^?1), while in the rainbow trout it was 0.7 ± 0.5 and in the brook trout 0.4 ± 0.1 mm L^?1. The most pronounced differences were found in the glucose concentration cause of its unnaturally low concentration in rainbow trout of the mean value of 6.0 ± 15.2 mg L^?1. The mean value in sea trout and brook trout was 185.0 ± 172.4 and 231.2 ± 148.4 mg L^?1 respectively. For all species, protein mean values were below 1.3 g L^?1. The mean osmolality was between 230.6 ± 98.6 and 272.0 ± 26.4 mOsm kg^?1 in the species studied. No correlation was found between any components determined in milt and the spermatozoa motility (P > 0.05). The sperm concentration was positively correlated with the protein content in the milt of the three species studied, other less exhibited correlation was found.     

Effects of cations on Methanosarcina thermophila TM-1 growing on moderate concentrations of acetate: production of single cells

Summary The effects of different concentrations of Mg²⁺, Ca²⁺, or Na⁺ on the morphology and growth of Methanosarcina thermophila TM-1 growing on acetate at concentrations comparable with those found in anaerobic digestors was studied. At 30 mm Mg²⁺ or less, M. thermophila grew as large aggregates that settled rapidly. At 100 mm Mg²⁺ or more, the bacteria grew as single cells or a mixture of single cells and small aggregates is suspended culture. Mg²⁺ was necessary for growth and could not be substituted by addition of either Ca²⁺ or Na⁺. The optimal Mg²⁺ concentration was 30 mm and no growth was observed at 400 mm Mg²⁺. Cultures could be adapted to 300 mm Mg²⁺ without a change in growth rate. Added Ca²⁺ was not required for growth and had no effect on cell morphology. Inhibition by Na⁺ was directly related to the Mg²⁺ concentration. When the Mg²⁺ was 0.05 mm or less, 0.35 m Na⁺ completely inhibited growth. However, more Na⁺ was required for inhibition at higher Mg²⁺ concentrations. The same inhibitory effect of Na⁺ was observed when the temperature was 52°C or 45°C. The potential for disaggregation of Methanosarcina aggregates in anaerobic digestor environments was discussed. Offprint requests to: B. K. Ahring     

Involvement of cation channels and NH4+-sensitive K+ transporters in Na+ uptake by cowpea roots under salinity

Na⁺ accumulation was investigated in the roots of 11-d-old cowpea [Vigna unguiculata (L.) Walp.] plants. The relative contribution of different membrane transporters on Na⁺ uptake was estimated by applying Ca²⁺, K⁺, NH₄ ⁺, and pharmacological inhibitors. Na⁺ accumulation into the root symplast was decreased by half in the presence of 1 mM Ca²⁺ and it was almost abolished by 100 mM K⁺. The inhibitory effect of external NH⁴⁺ on Na⁺ accumulation was more pronounced in the roots of NH₄ ⁺-free growing plants. Na⁺ accumulation was reduced about 73 % by 0.1 mM flufenamate and it was almost blocked by 2 mM quinine. In addition, 20 mM tetraethylammonium and 1.0 mM Cs⁺ decreased Na⁺ accumulation by 28 and 30 %, respectively. These results evidenced that low-affinity Na⁺ uptake by cowpea roots depends on Ca²⁺-sensitive and Ca²⁺-insensitive pathways. The Ca²⁺-sensitive pathway is probably mediated by nonselective cation channels and the Ca²⁺-insensitive one may involve K⁺ channels and to a lesser extent NH₄ ⁺-sensitive K⁺ transporters.     

Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells

Ca²⁺-transport and its energy consumption were studied in intact human red cells loaded with Ca²⁺ by the aid of the ionophore A23187.After the complete elimination of the ionophore the passive Ca²⁺-permeability of the membrane returned to its normal low value, except when the intracellular Ca²⁺-concentration was higher than 3 mM or the ATP level fell below 100 μM. Within these limits the rate of Ca²⁺-extrusion was independent of the cellular ATP content but was greatly enhanced by increasing [Ca²⁺]_i and reached a plateau at about 1 mM intracellular Ca²⁺-concentration. The maximum rate of Ca²⁺-efflux was about 85 μmol/l of cells per min at 37°C, pH 7.4. The activation energy of active Ca²⁺-extrusion was found to be 15 200 cal/mol, and the optimum pH in the suspension was 7.7.Ca²⁺-efflux was not connected with the counter-transport of cations.The Ca²⁺-pump was not affected by ouabain or oligomycin and only partial inhibition could be achieved by the SH-reagents: ethacrynic acid, $\text{N-}\text{ethylmaleimide}$ and $\text{p-}\text{chloromercuribenzoate}$ or with propranolol and ruthenium red. An 80 to 95% inhibition of the active Ca²⁺-extrusion was brought about by 50–250 μM lanthanum, which in the above concentrations caused no aggregation or haemolysis. The inhibition of the Ca²⁺-pump by lanthanum was found to be reversible, the site of inhibition being at the external surface of the cell membrane.To examine the energy consumption of the Ca²⁺-extrusion, ATPase activity was assessed by measuring inorganic phosphate liberation in Ca²⁺-loaded red cells the metabolism of which was inhibited by iodoacetamide + Na⁺-tetrathionate. Ca²⁺-activated ATPase activity connected with the Ca²⁺-pump was distinguished from other Ca²⁺-ATPase by using the non-penetrating inhibitor, lanthanum. The molar ratio of Ca²⁺-transported per ATP split was found to be $\text{2 : 1}$.     

Properties and substrate specificities of an extracellular lipase purified from Ophiostoma piceae

An extracellular lipase produced by the sapstaining fungus Ophiostoma piceae 387N in a liquid medium was purified to homogeneity using ammonium sulphate and acetone fractionation, hydrophobic interaction and anion exchange chromatography. The overall purification based on lipase activity was 5200-fold with a yield of 26%. The molecular mass of the lipase was 35kDa, as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and 37 kDa, as measured by size exclusion chromatography. The purified enzyme was resolved as three bands at pI values of 4.3, 4.1 and 3.8 in IEF (isoelectric focusing) gels. Lipolytic stain demonstrated that all three bands were lipolytically active. The N-terminal amino acid sequence was determined asD¹-V²-S³-V⁴-T⁵-T⁶-T⁷-D⁸-I⁹-D¹⁰-A¹¹-L¹²-A¹³-F¹⁴-F¹⁵-T¹⁶-Q¹⁷-W¹⁸-A¹⁹-G²⁰ . The lipase was shown to be glycosylated, containing 10.1% carbohydrate. The lipase was stable between pH 4 and pH 8 and at temperatures below 40°C. The lipase activity had a pH optimum of approximately 5 and a temperature optimum of 30°C. The enzyme activity was not influenced by N-ethylmaleimide, -mercaptoethanol or dithiothreitol, was enhanced by Ca²⁺ or Mn²⁺, but was severely inhibited by Hg²⁺, Fe³⁺, butyric acid, caproic acid, diethyl pyrocarbonate, and diethyl p-nitrophenyl phosphate. The lipase hydrolysed mainly triglycerides, although some activity was measured on waxes and cholesteryl esters. It belongs to a group of 1 (3) positional specific lipases. It showed little activity for substrates with short chain fatty acids (C2–C6), but demonstrated high specificity for substrates with intermediate and long chain fatty acid residues (C10–C18).     

Calcium dependent action potentials in skeletal muscle fibres of a beetle larva, Xylotrupes dichotomus

The ionic requirement for generating action potentials in ventral longitudinal muscle fibers dissected from beetle larvae was examined by conventional electrophysiological techniques. Muscle fibers that generated only graded responses in physiological saline were able to generate an all-or-none action potential when the potassium permeability of the membrane was inhibited by tetraethylammonium⁺ added to the saline. The peak of the action potential thus elicited was intimately related to the external Ca⁺⁺ concentration. The action potential was blocked by Co⁺⁺ which is known as a competitive inhibitor of Ca-spikes. Neither tetrodotoxin (3 μM) nor a Na-free condition effectively blocked the generation of the action potential. Mg⁺⁺ induced a shift in the peak of the action potential; this was, however, due to the stabilizing action of Mg⁺⁺ but not due to the penetration of Mg⁺⁺ through the muscle membrane. No action potential was elicited in the muscle fiber when immersed in a Ca-free, EGTA saline even when a high concentration of either Mg⁺⁺, Na⁺, or tetraethylammonium⁺ was present. The action potential of the larval muscle fiber was thus concluded to be a Ca-spike, through the channel of which Na⁺ or Mg⁺⁺ did not penetrate.