Does calcium ameliorate the negative effect of NaCl on melon root water transport by regulating aquaporin activity?

The hydraulic conductance ( L ₀) of detached, exuding root systems from melon ( Cucumis melo cv. Amarillo oro) was measured. All plants received a half-strength Hoagland nutrient solution, and plants stressed either solely with NaCl (50 mM) or with NaCl (50 mM) following treatment (2 d) with CaCl₂ (10 mM) were compared with controls and CaCl₂-treated (10 mM) plants. The L ₀ of NaCl-treated plants was markedly decreased when compared to control and CaCl₂-treated plants, but the decrease was smaller when NaCl was added to plants previously treated with CaCl₂. A similar effect was observed when the flux of Ca²⁺ into the xylem and the Ca²⁺ concentration in the plasma membrane of the root cells were determined. In control, CaCl₂- and NaCl + CaCl₂-treated plants, HgCl₂ treatment (50 μM) caused a sharp decline in L ₀ to values similar to those of NaCl-stressed roots, but L ₀ was restored by treatment with 5 mM DTT. However, in NaCl roots only a slight effect of Hg²⁺ and DTT were observed. The effect of all treatments on L ₀ was similar to that on osmotic water permeability ( P _f) of individual protoplasts isolated from roots. The results suggest that NaCl decreased the passage of water through the membrane and roots by reducing the activity of Hg-sensitive water channels. The ameliorative effect of Ca²⁺ on NaCl stress could be related to water-channel function.     

Alleviation of photoinhibition by calcium supplement in salt-treated Rumex leaves

By analysis of gas exchange and chlorophyll fluorescence, the effects of NaCl treatment and supplemental CaCl₂ on photosynthesis, photosystem II (PSII) photochemistry and photoinhibition were investigated in Rumex leaves. Photosynthesis in Rumex leaves was strongly inhibited by 200 m M NaCl treatment. Such inhibition of photosynthesis was ameliorated by CaCl₂ supplement. Neither NaCl treatment nor CaCl₂ supplement had any significant effects on the PSII primary photochemical reaction in dark-adapted leaves. In light-adapted leaves, however, 200 m M NaCl treatment significantly decreased photochemical quenching (q_p), efficiency of excitation energy capture by open PSII reaction centers (F_V'/F_M') and quantum yield of PSII electron transport (Φ_PSII). These decreases in q_p, F_V'/F_M' and Φ_PSII were mitigated by CaCl₂ supplement with the maximum of its effect appearing at a concentration of 8 m M CaCl₂. A similar mitigating effect was shown in 200 m M NaCl-treated Rumex leaves when susceptibility of PSII to photoinhibition was determined under high irradiance. It is suggested that the mitigation of photoinhibition in NaCl-treated leaves is because of the amelioration of inhibition of photosynthesis.     

Studies of the extracellular proteolytic activity produced by Propionibacterium acnes

Of several commercial media tested, trypticase soya broth containing 0.4% (w/v) D-sorbitol was superior as a growth medium for the production of extracellular proteinase by Propionibacterium acnes (strain P-37). Extracellular proteinase, production of which was shown to be growth-associated by both batch and continuous culture studies, was partially purified by 70% (NH₄)₂SO₄ saturation, Sephadex G-75 chromatography and ion exchange on DEAE-Sephadex A-50. It was shown to be a heterogeneous mixture of at least three molecular species of enzyme. Proteinase I was inhibited by EDTA (10^-3 mol/l) and PMSF (5 millimol/l) and stimulated by CaCl₂ (190% at 10^-3 mol/l). It had a molecular weight of 20 to 30000 and a broad pH optimum from 6.5 to 7.5. Proteinase II was an alkaline proteinase with a molecular weight of 30 to 40000 which was not significantly inhibited by EDTA (10^-2 mol/l) nor stimulated by CaCl₂. Proteinase III represented a minor proportion of the recovered proteolytic activity, had a molecular weight of 20 to 30000 and was most active in the alkaline pH range. This enzyme was inhibited by EDTA (10^-4 mol/l) and PMSF (5 millimol/l), and stimulated by CaCl₂ (250% at 10^-2 mol/l).     

Identification of ricinoleic acid as an inhibitor of Ca2+ signal-mediated cell-cycle regulation in budding yeast

Free fatty acids exhibit diverse biological effects such as the regulation of immune responses in humans and animals. To investigate the biological effect of fatty acids in the model eukaryotic organism yeast, we examined the activity of various fatty acids in a yeast-based drug-screening system designed to detect the small-molecule compounds that inhibit Ca²⁺-signal-mediated cell-cycle regulation. Among the fatty acids examined, ricinoleic acid markedly alleviated the deleterious physiological effects induced by the compelled activation of Ca²⁺ signaling by external CaCl₂, such as the polarized bud growth and the growth arrest in the G₂ phase. In accordance with the physiological consequences induced by ricinoleic acid, it diminished the Ca²⁺-induced phosphorylation of Cdc28p at Tyr-19, concomitant with the decrease in the Ca²⁺-stimulated expression levels of Cln2p and Swe1p.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

Inhibition of Rat Brain Microsomal Na+,K+-ATPase by S-Adenosylmethionine

Abstract: Rat brain microsomes were preincubated with S -adenosylmethionine (SAM), MgCl₂, and CaCl₂, then re-isolated, and the activity of Na⁺,K⁺-ATPase determined. SAM inhibited the Na⁺,K⁺-ATPase activity compared with microsomes subjected to similar treatment in the absence of SAM. A biphasic inhibitory effect was observed with a 50% decrease at a SAM concentration range of 0.4 μ M -3.2 μ M and a 70% reduction at a concentration range above 100 μ M . Inclusion of either S- adenosylhomocysteine or 3-deazaadenosine in the preincubations prevented the SAM inhibition of Na⁺,K⁺-ATPase activity. The inhibition by SAM appeared to be Mg²⁺- or Ca²⁺-dependent.     

Chemical and electroporated transformation of Edwardsiella ictaluri using three different plasmids

Transfer of DNA by conjugation has been the method generally used for genetic manipulation of Edwardsiella ictaluri because, previously, attempts to transform E. ictaluri by the uptake of naked DNA have apparently failed. We report here the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures [conventional calcium chloride (CaCl₂) and 'one-step' (polyethylene glycol, dimethyl sulfoxide and MgSO₄) protocols]. Seven strains of E. ictaluri were transformed using three different plasmids [pZsGreen, pUC18 and pET-30a(+)]. The highest transformation efficiency was achieved by electroporation (5.5±0.2 × 10⁴ transformants ng⁻¹ plasmid DNA) than with the CaCl₂ (8.1±6.1 × 10⁻¹ transformants ng⁻¹ plasmid) and the 'one-step transformation' protocol (2.5±2.7 transformants ng⁻¹ plasmid). An efficient transformation by electroporation required only 0.2 ng of plasmid compared with 200 ng required for the CaCl₂ and one-step protocols. The plasmids were stably maintained in E. ictaluri grown in the presence of antibiotic for 12 or more passages. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for the molecular genetic manipulation of this organism, and is a quicker and easier method than transformation performed by conjugation.     

ACCELERATION OF CHOLINE UPTAKE AFTER DEPOLARIZATION-INDUCED ACETYLCHOLINE RELEASE IN RAT CORTICAL SYNAPTOSOMES

Abstract— Activation of nerve elements in vivo and in vitro is associated with an increased rate of choline uptake by a Na⁺-dependent high affinity transport system. Following the methodology of B arker (1976), rat cortical synaptosomes were depolarized (37°C, 10min) by 25mM-KCl in the presence of CaCl₂ (1 mM) or other divalent cations. After reisolation by centrifugation, the rate of ³H-choline uptake (1.25μM) was measured by Millipore filtration. KCl treatment alone failed to accelerate the rate of uptake in the reisolated synaptosomes. CaCl₂, BaC1₂ or SrCl₂ (but not MgCl₂ or MnCl₂) were necessary (1 mM) to observe the KCl induced acceleration. Moreover, RbCl, but not LiCl or CsCl, also produced the calcium-dependent rate enhancement in the reisolated synaptosomes. The conditions mediating the enhanced rate of choline uptake correlated strongly with those associated with neurotransmitter release. To test this possibility, synaptosomal acetylcholine content was measured in response to the various salt treatments. Treatment with KCI (25 mM) and CaCl₂ (1 mM), but not KCl alone, reduced the synaptosomal acetylcholine content from 154 to 113pmol/mg protein. The respective rates of choline uptake increased about 60%. The increased rate was reversed by incubation with 50 μM-choline followed by synaptosome reisolation. This procedure also normalized the acetylcholine content. In summary, the rate of choline uptake by the high affinity choline uptake system is inversely related to the synaptosomal acetylcholine content.     

Reduced Development of Grey Mould (Botrytis cinerea) in Bean and Tomato Plants by Calcium Nutrition

Fertilization of bean plants grown in perlite with 1 and 3 mM CaCl₂ or Ca(NO₃)₂ reduced severity of grey mould as compared with control plants or plants fertilized with 5 mM of the compounds. Fertilization with Ca(NO₃)₂ reduced severity leaf grey mould and fruit ghost spots of tomato plants grown in perlite by 70 and 45%, respectively. The rate of decrease varied with the position of the fruits on the plants. Leaves from plants treated with calcium or otherwise [KNO₃, (NH₄)₂SO₄] produced less ethylene than leaves of nontreated plants. Rate of growth of B. cinerea was lower on growth medium prepared from washings from leaves of calcium fertilized plants than from leaves from other treatments. The fertilizer combination Ca(H₂PO₄)₂+ CaSO₄ (1 and 3 g/kg soil) applied once to tomato plants grown in soil reduced severity of leaf grey mould by 80 % (significant at P = 0.05) but 1–3 g CaSO₄/kg soil only tended to reduce disease severity (30–40 %, not significant) as compared with the control. The compounds CaCl₂ and Ca(NO₃)₂ increased significantly ( P = 0.05) the growth of B. cinerea on synthetic medium when applied at rates of 1 0–10.0 mM whereas reduction of growth was observed with 0.1 mM of the compounds and of CaSO₄.     

The role of laccase in prostaglandin production by Cryptococcus neoformans

Recently, it has been demonstrated that the opportunistic fungal pathogen Cryptococcus neoformans can synthesize authentic immunomodulatory prostaglandins. The mechanism by which this takes place is unclear as there is no cyclooxygenase homologue in the cryptococcal genome. In this study, we show that cryptococcal production of both PGE₂ and PGF_2α can be chemically inhibited by caffeic acid, resveratrol and nordihydroguaiaretic acid. These polyphenolic molecules are frequently used as inhibitors of lipoxygenase enzymes; however, blast searches of the cryptococcal genome were unable to identify any homologues of mammalian, plant or fungal lipoxygenases. Next we investigated cryptococcal laccase, an enzyme known to bind polyphenols, and found that either antibody depletion or genetic deletion of the primary cryptococcal laccase ( lac1 Δ) resulted in a loss of cryptococcal prostaglandin production. To determine how laccase is involved, we tested recombinant laccase activity on the prostaglandin precursors, arachidonic acid (AA), PGG₂ and PGH₂. Using mass spectroscopy we determined that recombinant Lac1 does not modify AA or PGH₂, but does have a marked activity toward PGG₂ converting it to PGE₂ and 15-keto-PGE₂. These data demonstrate a critical role for laccase in cryptococcal prostaglandin production, and provides insight into a new and unique fungal prostaglandin pathway.     

Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH₄)₂SO₄ precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)^-1 min^-1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn²⁺ or Mg²⁺). In the presence of MnCl₂₊ other divalent cations (Mg²⁺, Ca²⁺, Ba²⁺, Hg²⁺ and Cd²⁺) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The K_m for adenine was 4.5±1.5 μ M , the K_m for PRPP was 0.29±0.06 m M and the K_i for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The K_m for benzyladenine was approximately 0.73±0.06 m M     

Properties of Intracellular Hatching enzyme in the Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

The intracellular hatching enzyme was confirmed to be particulate-bound in the sea urchin, Hemicentrotus pulcherrimus. The enzyme was solubilized most effectively by sonication in buffer containing 12.5 mM CaCl₂, and 0.5 M KCl. The intracellular hatching enzyme is suggested to be activated by an antipain- or elastatinal-susceptible protease(s) on its solubilization. Since the intracellular hatching enzyme solubilized in the absence of protease inhibitors was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, the active hatching enzyme is concluded to be a chymostatin-sensitive serine protease. The enzyme required CaCl₂, and KCl or NaCl for both stability and activity. The preference of the enzyme of anions as sodium salts was as follows: Cl⁻ > NO₃⁻ > I⁻ > SCN⁻. The apparent molecular weights of the intracellular hatching enzyme (IHE) and the hatching enzyme secreted from the blastula with or without the fertilization envelope (SHE or dSHE) were estimated as 89,000, 135,000, 80,000, respectively. On incubations with isolated fertilization envelopes as an enzyme substrate, the apparent molecular weights of dSHE and IHE increased to 128,000 and 105,000, respectively.     

Ca2+ and AtCaM3 are involved in the expression of heat shock protein gene in Arabidopsis

The involvement of calcium and different calmodulin isoforms (Ca²⁺-CaM) in heat shock (HS) signal transduction in Arabidopsis ( Arabidopsis thaliana ) was investigated. Using transgenic Arabidopsis plants which have the AtHsp18.2 promoter/GUS fusion gene, it was found that the level of β -glucuronidase (GUS) activity was up-regulated by the addition of CaCl₂ and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl₃ and verapamil, or the CaM antagonists N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), chlorpromazine (CPZ) and trifluoperazine (TFP). CaCl₂ not only increased the GUS activity after HS, but also up-regulated the GUS activity under non-HS conditions. These results provide additional support for the involvement of the Ca²⁺-CaM signalling system in HSP gene expression. The expression of nine CaM genes (AtCaM1–9) from Arabidopsis was differentially regulated by HS at 37 °C. The expression of AtCaM3 and AtCaM7 genes increased during HS. The temporal expression of the AtCaM3, AtCaM7 and hsp18.2 genes demonstrated that up-regulation of AtCaM3 expression occurred earlier than that of AtCaM7 or hsp18.2 .     

Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.     

DHPOSITION OF LIPIDS IN THE DEVELOPING CENTRAL AND PERIPHERAL NERVOUS SYSTEMS OF THE CHICKEN

Abstract— The lipid composition of chick brain and sciatic nerve was determined during development. It was confirmed that the addition of CaCl₂ to solvents during the extraction of lipids from brain results in much higher yields of diphosphoinositides particularly from unmyelinated embryo brain. Unlike the earlier report for rat brain, the recovery of triphosphoinositides was also Substantially increased. The amount of CaCl₂, required to achieve optimal recoveries decreased with increasing age and addition of more than this optimal amount depressed the yields of polyphosphoinositides, particularly triphosphoinositides. CaCl₂, addition did not improve the yield of diphosphoinositides from sciatic nerve of any age but drastically reduced recovery of triphosphoinositidcs. Differenccs in the effect of CaCl₂ were not the result of variation in the tissue concentrations of calcium or magnesium.
The lipid composition of sciatic nerve closely reflected that of the myelin. Both polyphosphoinositides were absent initially and their accumulation paralleled that of cerebrosides and sulfatides. The concentration of diphosphoinositides remained constant after the period of most active myelination while triphosphoinositides and the galactolipids continued to increase suggesting maturational changes in the myelin composition. The pattern of deposition in chick brain was similar except for the much greater contribution of non-myelin structures. Both polyphosphoinositides were present in equimolar amounts in pre-myelination embryonic tissue. The concentration of diphosphoinositides increased during active myelination only while triphosphoinositides continued to increase thereafter.     

Photoactivation of the latent water-oxidizing complex in photosystem II membranes isolated from dark-grown spruce seedlings

Photosystem II membranes (D-PSII) were isolated from dark-grown spruce seedlings. All major PSII proteins except the 17- and 23-kDa extrinsic proteins were present in D-PSII. O₂ evolution and Mn content in D-PSII were negligible, while PSII-donor activity showed a value comparable to that of NH₂OH-treated PSII membranes (NH₂OH-L-PSII) from light-grown seedlings. Light incubation of D-PSII with 1 m M MnCl₂, 50 m M CaCl₂ and 100 μ M DCIP at pH 5.3 resulted in activation of the latent water-oxidizing complex. Accomplishment of photoactivation of PSII membranes from dark-grown spruce seedlings clearly indicates that only ligation of Mn²⁺ to the apo-water oxidizing complex is required for expression of O₂ evolution, and that protein synthesis is not involved in the photoactivation process. There was no essential difference between 'photoactivation' of naturally Mn-free PSII membranes and 'photoreactivation' of artificially Mn-depleted PSII membranes on kinetics, pH dependence, Mn²⁺-concentration dependence. However, kinetics and pH dependence of photoactivation were appreciably different in spruce PSII membranes and in PSII membranes of angiosperms such as wheat and spinach.     

The controlling influence of cell-surface electrical potential on the uptake and toxicity of selenate (SeO42–)

Root elongation in wheat seedlings ( Triticum aestivum L. cv. Atlas 66) was inhibited by micromolar activities of SeO₄^2–. SeO₄^2– inhibition was enhanced by supplementation of the rooting medium with CaCl₂, MgCl₂, SrCl₂, or the reduction of pH. These solute treatments, as well as the addition of tris (ethylenediamine)cobalt³⁺, enhanced the uptake of Se by the roots. The results are interpreted to reflect an elevated PM-surface activity of SeO₄^2– caused by solute-induced reductions of plasma membrane (PM) surface negativity. (PM-surface electrical potential is sometimes measured electrophoretically as the zeta potential.) This study complements an extensive literature documenting the suitability of an electrostatic model (Gouy-Chapman-Stern), based almost entirely upon experiments with cations rather than anions. The close correspondence among uptake, intoxication, and model-computed SeO₄^2– activity at the PM surface adds credibility to the model and its evaluated parameters. The model may be useful for the interpretation of other plant-anion interactions, and phosphate and sulphate nutrition in acidic soils are considered as examples.     

Ca2+-dependent in vitro phosphorylation of soluble proteins from germinating wheat (Triticum turgidum) endosperm

A 40000 g supernatant fraction from extracts of germinating wheat ( Triticum turgidum Desf. cv. Edmore) endosperm contains protein kinase activity that phosphorylates several endogenous proteins. In vitro incorporation of radiolabel from [³²P]-ATP into phosphoproteins was maximal in the presence of 1 m M CaCl₂ and 5 m M MgCl₂Ca²⁺ at micromolar concentrations greatly stimulated the phosphorylation of 49 and 47 kDa polypeptides and also inhibited the phosphorylation of a few specific polypeptides. The phosphorylation of the 49 and 47 kDa polypeptides was present at 2 days after seed germination and was maximal at 8 days. Quantitative protein changes were also detected during the seed germination, but differences could not be correlated with changes in protein phosphorylation. Phosphoamino acid analysis by two dimensional thin-layer electrophoresis showed that the Ca²⁺-dependent protein kinase phosphorylates a serine residue of the 47 kDa polypeptide. Ca²⁺-dependent protein kinase phosphorylates a serine residue of the 47 KDa polypeptide. Ca²⁺ dependent protein phosphorylktion was inhibited by phenothiazine-derived drugs. Addition of S-adenosylmethionine to the in vitro phosphorylation reaction specifically inhibited the Ca²⁺-dependent protein phosphorylation.     

Characterization of Membrane-Bound and Soluble Catechol-O-Methyltransferase from Human Frontal Cortex

Abstract: Catechol- O -methyltransferase (COMT; E.C. 2.1.1.6) from human frontal cortex occurs in both a soluble and membrane-bound form. Attempts to solubilize the membrane-bound transferase by repeated washing or by extraction into solutions of high ionic strength were unsuccessful. The finding that Triton X-100 was capable of solubilizing membrane-bound COMT suggested that the membrane-bound transferase is an integral membrane protein. The membrane-bound and soluble enzymes did not differ in their requirements for magnesium ions or in their pH-activity profiles; both enzymes showed an optimum near pH 8.0 when assayed in phosphate buffer. In addition, the two enzymes did not differ in the degree of inhibition caused by CaCl₂, both enzymes displaying 65% inhibition at 2.5 m M CaCl₂. The competitive inhibitors tropolone and nordihydroguaiaretic acid displayed K _i values for the membrane-bound transferase five- to 10-fold lower than those observed for the soluble transferase. Solubilization of membrane-bound COMT in Triton X-100 resulted in an increase in the apparent K _m value of the membrane-bound transferase for dopamine. The increase in K _m appeared to be due to apparent competitive inhibition by Triton X-100 and reached a limiting value of approximately 80 μM. These results confirm that membrane-bound COMT is an integral membrane protein that may be structurally distinct from soluble COMT.     

Calcium taste preferences: genetic analysis and genome screen of C57BL/6J x PWK/PhJ hybrid mice

To characterize the genetic basis of voluntary calcium consumption, we tested C57BL/6J mice (B6; with low avidity for calcium), PWK/PhJ mice (PWK; with high avidity for calcium) and their F₁ and F₂ hybrids. All mice received a series of 96-h two-bottle preference tests with a choice between water and the following: 50 m m CaCl₂, 50 m m calcium lactate, 50 m m MgCl₂, 100 m m KCl, 100 m m NH₄Cl, 100 m m NaCl, 5 m m citric acid, 30 μ m quinine hydrochloride and 2 m m saccharin. Most frequency distributions of the parental and F₁ but not F₂ groups were normally distributed, and there were few sex differences. Reciprocal cross analysis showed that B6 × PWK F₁ mice had a non-specific elevation of fluid intake relative to PWK × B6 F₁ mice. In the F₂ mice, trait correlations were clustered among the divalent salts and the monovalent chlorides. A genome screen involving 116 markers showed 30 quantitative trait loci (QTLs), of which six involved consumption of calcium chloride or lactate. The results show pleiotropic controls of calcium and magnesium consumption that are distinct from those controlling consumption of monovalent chlorides or exemplars of the primary taste qualities.