NADP+-Dependent Sorbitol Dehydrogenase Found in Apple Leaves

An NADP⁺-dependent sorbitol dehydrogenase that catalyzes sorbitoland glucose was found in apple leaves. The partially purifiedenzyme had optimum activity at pH 9.6 and a K_m value of 128mM for sorbitol. Among the polyols studied, this enzyme showedthe most activity for sorbitol. ¹This paper is contribution A-173 of the Fruit Tree ResearchStation. (Received June 4, 1984; Accepted July 31, 1984)     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

Pressure Effects on Catalysis and Control of Catalysis by Liver Fructose Diphosphatase from an Off-Shore Benthic Fish

SYNOPSIS: At low temperature (2°C), in the absence of FDPand Mg²⁺, the enzyme fructose disphosphatase (FDPase), extractedfrom the liver of an off-shore benthic Coryphaenoides species,is inactivated by exposures to relatively low pressures. Thesubstrate, FDP, and the cofactor, Mg²⁺, protect against thisinactivation, so that catalysis per se is not retarded by pressure.In contrast, at alkaline pH, pressure dramatically acceleratesthe catalytic rate when FDP and Mg²⁺ are saturating. The volumechange of activation, V*, for Coryphaenoides FDPase under theseconditions is about –40 cm³/mole. At low concentrationsof FDP and saturating concentrations of cofactor, the reactionrate at alkaline pH is pressure-independent. Similarly, at lowconcentrations of Mg²⁺ but saturating concentration of FDP,the reaction rate is pressure-independent. The K_m for FDP doesnot change measureably with pressure, while the K_a for Mg²⁺increases slightly with pressure. Under conditions of low (probablephysiological) FDP and Mg²⁺ concentrations, it is evident thatthe reaction rate is determined by the kinetic characteristicsof the enzyme and not by its energy-volume relationships, asituation which would appear to be of functional and selectivesignificance to an organism living under constantly high hydrostaticpressure. AMP is a potent specific inhibitor of CoryphaenoidesFDPase. The K₄ for AMP is essentially pressure-independent bothat neutral and alkaline pH, suggesting that efficiency of AMPcontrol of this enzyme is comparable at all pressures likelyto be encountered in nature.     

Purification and some properties of phosphomannomutase from corms of Amorphophallus konjac C. Koch

Phosphomannomutase [Glazer et al.: Biochim. Biophys. Acta 33:522–625 (1959)] was purified 1700-fold in a 39% yieldfrom cell-free extract of konjak (Amorphophallus konjac C. Koch)corms. The molecular weight of the enzyme as determined by gelfiltration was about 62,000. The enzyme required both Mg²+ and-D-glucose-l,6-bisphosphate for activity, although Mg²+ waspartially replaceable by either Co²+ or Ni²+. An apparent equilibriumconstant, K_eq=(mannose-6-phosphate) (mannose-1-phosphate), wasdetermined to be 8.5. Activity was maximal at pH 6.5 to 7.0.Activation energy was 11.1 kcal/mole. The enzyme was the moststable at pH 7.5. The addition of substrate or cofactor markedlyincreased enzyme stability toward heat denaturation. The enzymewas more labile to heat than phosphoglucomutase from konjakcorms. Treatment with various metal ions in Tris buffer inhibited theenzyme. Cu²+ and Zn²+ were the most potent inhibitors amongthe metal ions tested, while Co²+ and Ni²+ were weak. When theenzyme was treated with metal ions in the presence of histidinebuffer, Cu²+ and Zn²+ showed no inhibitory effect on the enzyme,whereas Be²+ inhibited it to an extent similar to that in Trisbuffer. Plots of 1/v versus l/(mannose-l-phosphate) at different fixedconcentrations of glucose-1,6-bisphosphate and 1/v versus 1/(glucose-1,6-bisphosphate)at different fixed concentrations of mannose-1-phosphate wereseries of converging lines. Mannose-1-phosphate at high concentrationswas found to inhibit the enzyme competitively with respect toglucose-l,6-bisphosphate. Apparent K_m and K₁ values for mannose-1-phosphatewere calculated to be 0.2 mM and 1.2 mM, respectively. The K_mvalue for glucose-1,6-bisphosphate was 1.8 µM. ¹This paper constitutes part 5 of a series of studies on konjakmannan biosynthesis. (Received May 24, 1976; )     

Characterization and Induction of the Activity of 1-Aminocyclopropane-1-Carboxylate Oxidase in the Wounded Mesocarp Tissue of Cucurbita maxima

1-Aminocyclopropane-1-carboxylate (ACC) oxidase (ethylene-formingenzyme) was isolated from wounded mesocarp tissue of Cucurbitamaxima (winter squash) fruit, and its enzymatic properties wereinvestigated. The enzyme required Fe²⁺ and ascorbate for itsactivity as well as ACC and O₂ as substrates. The in vitro enzymeactivity was enhanced by CO₂. The apparent K_m value for ACCwas 175 µM under atmospheric conditions. The enzyme activitywas inhibited by sulfhydryl inhibitors and divalent cationssuch as Co²⁺, Cu²⁺, and Zn²⁺. ACC oxidase activity was induced at a rapid rate by woundingin parallel with an increase in the rate of ethylene production.The exposure of excised discs of mesocarp to 2,5-norbornadiene(NBD),an inhibitor of ethylene action, strongly suppressed inductionof the enzyme, and the application of ethylene significantlyaccelerated the induction of the activity of ACC oxidase inthe wounded mesocarp tissue. These results suggests that endogenousethylene produced in response to wounding may function in promotingthe induction of ACC oxidase. (Received January 13, 1993; Accepted April 15, 1993)     

Pharmacological activation of cloned intermediate- and small-conductance Ca(2+)-activated K(+) channels

We previouslycharacterized 1-ethyl-2-benzimidazolinone (1-EBIO), as well as theclinically useful benzoxazoles, chlorzoxazone (CZ), and zoxazolamine(ZOX), as pharmacological activators of the intermediate-conductanceCa²⁺-activated K⁺ channel, hIK1. The mechanismof activation of hIK1, as well as the highly homologoussmall-conductance, Ca²⁺-dependent K⁺ channel,rSK2, was determined following heterologous expression inXenopus oocytes using two-electrode voltage clamp (TEVC) and excised, inside-out patch-clamp techniques. 1-EBIO, CZ, and ZOX activated both hIK1 and rSK2 in TEVC and excised inside-out patch-clamp experiments. In excised, inside-out patches, 1-EBIO and CZ induced aconcentration-dependent activation of hIK1, with half-maximal (K_1/2) values of 84 µM and 98 µM, respectively.Similarly, CZ activated rSK2 with a K_1/2 of 87 µM. In the absence of CZ, the Ca²⁺-dependent activationof hIK1 was best fit with a K_1/2 of 700 nM and aHill coefficient (n) of 2.0. rSK2 was activated byCa²⁺ with a K_1/2 of 700 nM and ann of 2.5. Addition of CZ had no effect on either theK_1/2 or n for Ca²⁺-dependentactivation of either hIK1 or rSK2. Rather, CZ increased channelactivity at all Ca²⁺ concentrations(V_max). Event-duration analysis revealed hIK1 wasminimally described by two open and three closed times. Activation by1-EBIO had no effect on _o1, _o2, or_c1, whereas _c2 and _c3 werereduced from 9.0 and 92.6 ms to 5.0 and 44.1 ms, respectively. Inconclusion, we define 1-EBIO, CZ, and ZOX as the first known activatorsof hIK1 and rSK2. Openers of IK and SK channels may be therapeuticallybeneficial in cystic fibrosis and vascular diseases.

     

Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

These experiments were performed to determine the effects ofreducing Ca²⁺ influx(Ca_in) onK⁺ currents(I_K) inmyocytes from rat small mesenteric arteries by1) adding externalCd²⁺ or2) lowering externalCa²⁺ to 0.2 mM. When measured froma holding potential (HP) of 20 mV(I_K20),decreasing Ca_in decreasedI_K at voltageswhere it was active (>0 mV). When measured from a HP of 60 mV(I_K60),decreasing Ca_in increasedI_K at voltagesbetween 30 and +20 mV but decreased I_K at voltagesabove +40 mV. Difference currents(I_K) weredetermined by digital subtraction of currents recorded under controlconditions from those obtained whenCa_in was decreased. At testvoltages up to 0 mV,I_K60 exhibitedkinetics similar to controlI_K60, with rapidactivation to a peak followed by slow inactivation. At 0 mV, peakI_K60 averaged75 ± 13 pA (n = 8) withCd²⁺ and 120 ± 20 pA(n = 9) with lowCa²⁺ concentration. At testvoltages from 0 to +60 mV,I_K60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20mV. At +60 mV, the initial peakI_K60 averaged115 ± 18 pA with Cd²⁺ and 187 ± 34 pA with low Ca²⁺. With 10 mM pipette BAPTA, Cd²⁺ produced asmall inhibition ofI_K20 but stillincreased I_K60 between 30 and +10 mV. InCa²⁺-free external solution,Cd²⁺ only decreased bothI_K20 andI_K60. In thepresence of iberiotoxin (100 nM) to inhibitCa²⁺-activatedK⁺ channels(K_Ca),Cd²⁺ increasedI_K60 at allvoltages positive to 30 mV while BAY K 8644 (1 µM) decreasedI_K60. Theseresults suggest that Ca_in, through L-type Ca²⁺ channels and perhapsother pathways, increases K_Ca(i.e., I_K20) and decreases voltage-dependent K⁺currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

     

Beta-adrenergic stimulation does not activate Na+/Ca2+ exchange current in guinea pig, mouse, and rat ventricular myocytes

The effect of -adrenergic stimulation on cardiac Na⁺/Ca²⁺ exchange has been controversial. To clarify the effect, we measured Na⁺/Ca²⁺ exchange current (I_NCX) in voltage-clamped guinea pig, mouse, and rat ventricular cells. When I_NCX was defined as a 5 mM Ni²⁺-sensitive current in guinea pig ventricular myocytes, 1 µM isoproterenol apparently augmented I_NCX by 32%. However, this increase was probably due to contamination of the cAMP-dependent Cl^– current (CFTR-Cl^– current, I_CFTR-Cl), because Ni²⁺ inhibited the activation of I_CFTR-Cl by 1 µM isoproterenol with a half-maximum concentration of 0.5 mM under conditions where I_NCX was suppressed. Five or ten millimolar Ni²⁺ did not inhibit I_CFTR-Cl activated by 10 µM forskolin, an activator of adenylate cyclase, suggesting that Ni²⁺ acted upstream of adenylate cyclase in the -adrenergic signaling pathway. Furthermore, in a low-extracellular Cl^– bath solution, 1 µM isoproterenol did not significantly alter the amplitude of Ni²⁺-sensitive I_NCX at +50 mV, which is close to the reversal potential of I_CFTR-Cl. No change in I_NCX amplitude was induced by 10 µM forskolin. When I_NCX was activated by extracellular Ca²⁺, it was not significantly affected by 1 µM isoproterenol in guinea pig, mouse, or rat ventricular cells. We concluded that -adrenergic stimulation does not have significant effects on I_NCX in guinea pig, mouse, or rat ventricular myocytes. cystic fibrosis transmembrane conductance regulator; nickel ion     

Calcium dependence of C-type natriuretic peptide-formed fast K+ channel

The lipid bilayertechnique was used to characterize theCa²⁺ dependence of a fastK⁺ channel formed by a synthetic17-amino acid segment [OaCNP-39-(1-17)] ofa 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchusanatinus) venom (OaV). TheOaCNP-39-(1-17)-formed K⁺ channel was reversiblydependent on1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic)Ca²⁺ concentration([Ca²⁺]_cis).The channel was fully active when[Ca²⁺]_ciswas >10⁴ M andtrans (luminal)Ca²⁺ concentration was 1.0 mM, butnot at low[Ca²⁺]_cis.The open probability of single channels increased from zero at1 × 10⁶ McisCa²⁺ to 0.73 ± 0.17 (n = 22) at10³ McisCa²⁺. Channel openings to themaximum conductance of 38 pS were rapidly and reversibly activated when[Ca²⁺]_cis,but not transCa²⁺ concentration(n = 5), was increased to >5 × 10⁴ M(n = 14). Channel openings to thesubmaximal conductance of 10.5 pS were dominant at5 × 10⁴ MCa²⁺.K⁺ channels did not open whencisMg²⁺ orSr²⁺ concentrations were increasedfrom zero to 10³ M or when[Ca²⁺]_ciswas maintained at 10⁶ M(n = 3 and 2). The Hill coefficientand the inhibition constant were 1 and 0.8 × 10⁴ McisCa²⁺, respectively. Thisdependence of the channel on high[Ca²⁺]_cissuggests that it may become active under1) physiological conditions whereCa²⁺ levels are high, e.g., duringcardiac and skeletal muscle contractions, and2) pathological conditions that leadto a Ca²⁺ overload, e.g., ischemicheart and muscle fatigue. The channel could modify a cascade ofphysiological functions that are dependent on theCa²⁺-activatedK⁺ channels, e.g., vasodilationand salt secretion.

     

Isolated mouse pancreatic beta-cells show cell-specific temporal response pattern

The length of the silent lag time beforeelevation of the cytosolic free Ca²⁺ concentration([Ca²⁺]_i) differs between individualpancreatic -cells. One important question is whether thesedifferences reflect a random phenomenon or whether the length of lagtime is inherent in the individual -cell. We compared the lag times,initial dips, and initial peak heights for[Ca²⁺]_i from two consecutive glucosestimulations (with either 10 or 20 mM glucose) in individualob/ob mouse -cells with the fura 2 technique in amicrofluorimetric system. There was a strong correlation between thelengths of the lag times in each -cell (10 mM glucose:r = 0.94, P < 0.001; 20 mM glucose:r = 0.96, P < 0.001) as well as between theinitial dips in [Ca²⁺]_i (10 mM glucose:r = 0.93, P < 0.001; 20 mM glucose:r = 0.79, P < 0.001) and between theinitial peak heights (10 mM glucose: r = 0.51, P < 0.01; 20 mM glucose: r = 0.77, P < 0.001). These data provide evidence that theresponse pattern, including both the length of the lag time and thedynamics of the subsequent [Ca²⁺]_i, isspecific for the individual -cell.

     

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel

We usedsingle-channel recording techniques to identify and characterize alarge-conductance,Ca²⁺-independentK⁺ channel in the colonicsecretory cell line T84. In symmetric potassium gluconate, this channelhad a linear current-voltage relationship with a single-channelconductance of 161 pS. Channel open probability(P_o) wasincreased at depolarizing potentials. Partial substitution of bathK⁺ withNa⁺ indicated a permeability ratioof K⁺ toNa⁺ of 25:1. ChannelP_o was reduced byextracellular Ba²⁺. Event-durationanalysis suggested a linear kinetic model for channel gating having asingle open state and three closed states: C₃C₂C₁O.Arachidonic acid (AA) increased theP_o of thechannel, with an apparent stimulatory constant(K_s)of 1.39 µM. Neither channel open time (O) nor the fast closed time(C₁) was affected by AA. Incontrast, AA dramatically reduced mean closed time by decreasing bothC₃ andC₂. Thecis-unsaturated fatty acid linoleate increased P_oalso, whereas the saturated fatty acid myristate and thetrans-unsaturated fatty acid elaidatedid not affectP_o. This channelis activated also by negative pressure applied to the pipette duringinside-out recording. Thus we determined the effect of thestretch-activated channel blockers amiloride and Gd³⁺ on theK⁺ channel after activation by AA.Amiloride (2 mM) on the extracellular side reduced single-channelamplitude in a voltage-dependent manner, whereasGd³⁺ (100 µM) had no effect onchannel activity. Activation of this K⁺ channel may be important duringstimulation of Cl secretionby agonists that use AA as a second messenger (e.g., vasoactiveintestinal polypeptide, adenosine) or during the volume regulatoryresponse to cell swelling.

     

Cu2+-induced modification of the kinetics of A beta(1-42) channels

We found that the amyloid peptide A(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu²⁺ or biological redox agents that have been reported to mediate A(1-42) toxicity. The A(1-42)-formed cation channel was inhibited by Cu²⁺ in cis solution ([Cu²⁺]_cis) in a voltage- and concentration-dependent manner between 0 and 250 µM. The [Cu²⁺]_cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 µM [Cu²⁺]_cis and partially reversible at 250 µM [Cu²⁺]_cis. The inhibitory effects of [Cu²⁺]_cis between 50 and 250 µM on the channel could not be reversed with addition of Cu²⁺-chelating agent clioquinol (CQ) at concentrations between 64 and 384 µM applied to the cis chamber. The effects of 200-250 µM [Cu²⁺]_cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 µM [CQ]_cis. The kinetic analysis of the data indicate that Cu²⁺-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu²⁺ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu²⁺-binding site of the A(1-42)-formed channels is modulated with Cu²⁺ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu²⁺ modulation of A and PrP channel proteins linked to neurodegenerative diseases. neurodegenerative diseases; transitional metals; ion channel pathologies; membrane injuries; calcium homeostasis     

ATP stimulation of Na+/Ca2+ exchange in cardiac sarcolemmal vesicles

In cardiacsarcolemmal vesicles, MgATP stimulatesNa⁺/Ca²⁺exchange with the following characteristics:1) increases 10-fold the apparentaffinity for cytosolic Ca²⁺;2) a Michaelis constant for ATP of~500 µM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory;4) not observed in the presence of20 µM eosin alone but reinstated when vanadate is added;5) mimicked by adenosine5'-O-(3-thiotriphosphate), without the need for vanadate, but not by ,-methyleneadenosine 5'-triphosphate; and 6) notaffected by unspecific protein alkaline phosphatase but abolished by aphosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLCeffect is counteracted by phosphatidylinositol. In addition, in theabsence of ATP,L--phosphatidylinositol4,5-bisphosphate (PIP₂) was ableto stimulate the exchanger activity in vesicles pretreated with PI-PLC.This MgATP stimulation is not related to phosphorylation of thecarrier, whereas phosphorylation appeared in the phosphoinositides,mainly PIP₂, thatcoimmunoprecipitate with the exchanger. Vesicles incubated with MgATPand no Ca²⁺ show a markedsynthesis ofL--phosphatidylinositol4-monophosphate (PIP) with little production ofPIP₂; in the presence of 1 µM Ca²⁺, the net synthesis of PIP issmaller, whereas that of PIP₂increases ninefold. These results indicate thatPIP₂ is involved in the MgATPstimulation of the cardiacNa⁺/Ca²⁺exchanger through a fast phosphorylation chain: aCa²⁺-independent PIP formationfollowed by a Ca²⁺-dependentsynthesis of PIP₂.

     

Ca(2+)-dependent activation of Cl(-) currents in Xenopus oocytes is modulated by voltage

Ca²⁺-activatedCl currents (I_Cl,Ca) wereexamined using fluorescence confocal microscopy to monitorintracellular Ca²⁺ liberation evoked by flash photolysis ofcaged inositol 1,4,5-trisphosphate (InsP₃) involtage-clamped Xenopus oocytes. Currents at +40 mV exhibited asteep dependence on InsP₃ concentration([InsP₃]), whereas currents at140 mV exhibited a higher threshold and more graded relationshipwith [InsP₃]. Ca²⁺ levelsrequired to half-maximally activate I_Cl,Ca wereabout 50% larger at 140 mV than at +40 mV, and currents evokedby small Ca²⁺ elevations were reduced >25-fold. Thehalf-decay time of Ca²⁺ signals shortened at increasinglypositive potentials, whereas the decay of I_Cl,Calengthened. The steady-state current-voltage (I-V) relationshipfor I_Cl,Ca exhibited outward rectification withweak photolysis flashes but became more linear with stronger stimuli.Instantaneous I-V relationships were linear with both strongand weak stimuli. Current relaxations following voltage steps duringactivation of I_Cl,Ca decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at 160 mV. We conclude that InsP₃-mediated Ca²⁺liberation activates a single population of Clchannels, which exhibit voltage-dependent Ca²⁺ activationand voltage-independent instantaneous conductance.

     

Effects of Pressure and Temperature on the Catalytic and Regulatory Properties of Muscle Pyruvate Kinase from an Off-Shore Benthic Fish

SYNOPSIS. Muscle pyruvate kinase from an abyssal Coryphaenoidesspecies occurs as a single electrophoretic form with an isoelectricpoint of about pH 6.0. Maximum catalytic rates are dramaticallyreduced by pressure. For catalysis at 3°C, the volume changeof activation, V*, is about 44 cm³/mole (calculated between14.7 and 8000 psi). The value ot V* decreases at higher temperaturesbut is pH independent. The activation energy for rattail musclepyruvate kinase at 14.7 psi is about 13 Kcal/mole and doublesat 12,000 psi. Mg²⁺ saturation kinetics involve positive site-siteinteractions. Hill plots yield n values of about 2.4 and K_avalues of about 2 mM (at 3°C), and these constants are pressureindependent. The K_m values for ADP increase slightly with pressure.PEP saturation curves are complex: at high PEP concentrations,the n values are about 2–2.5, while at low PEP levels,values for the Hill constant are about 1.0. The Hill constantlor PEP is not affected by pressure, but the apparent K_m increasessomewhat with pressure. FDP dramatically activates rattail musclepyruvate kinase (500% activation with 0.1 mM FDP) by (1) reducingthe K_mPEP, (2) increasing the maximum velocity, and (3) overridingnegative ATP modulation of the enzyme. The latter control featureis strictly dependent upon pressure and is not observed at lowpressure. In the presence of FDP, the K_m for PEP decreases athigh pressures, in this way counteracting the inhibitory effectsof pressure. Under low concentrations of substrates, pyruvatekinase activity is probably determined by its kinetic propertiesand not by energy-volume relationships.     

Changes in the Levels of Ribulose-l,5-bisphosphate Carboxylase/Oxygenase (Rubisco) in Tetraedron minimum (Chlorophyta) during Light and Shade Adaptation

Exponentially growing cultures of the chlorophyta Tetraedronminimum were allowed to photoadapt to low (50µmole quantam^–2s^–1) and high (500µmole quanta m^–2–1)irradiance levels. In these cultures, various aspects of theorganization of the photosynthetic apparatus and related differencesin its performance were studied. In this organism, the observed five-fold increase in pigmentationof low-light adapted cells was due to increases in the numbersof PSU's, while their sizes remained constant. Using radioimmunoassay technique, we found that high-light adaptedalgae had over five times more Rubisco per PSU than their low-lightadapted counterparts. The high-light adapted algae also exhibited far higher (x2.3)light saturated photosynthetic rates per chl a. This increasewas the result of a reduction of tau, , the turnover time ofPS II reaction centers. We propose that the increase in Rubisco per PSU in high-lightadapted algae explains the reduction in , which results in thehigher P_max rates per chl a in these algae. The relationship is non linear, since the increase in Rubiscoper PSU was x5.3 whereas that in P_mM per chl a was only x2.3. (Received July 30, 1988; Accepted December 2, 1988)     

Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes

This study examines whether fluid pressure (FP) modulates the L-type Ca²⁺ channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (16 dyn/cm²) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca²⁺ current (I_Ca) and cytosolic Ca²⁺ signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed I_Ca (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of I_Ca. The level of I_Ca suppression by FP depended on the level and duration of pressure. The Ba²⁺ current through the Ca²⁺ channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca²⁺ channel during FP stimulation. The cytosolic Ca²⁺ transients and the basal Ca²⁺ in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the I_Ca and on the Ca²⁺ transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca²⁺ buffer, eliminated the FP-induced acceleration of I_Ca inactivation and reduced the inhibitory effect of the FP on I_Ca by 80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca²⁺ release, eliminated the accelerating effect of FP on the I_Ca inactivation, and they reduced the inhibitory effect of FP on the I_Ca. These results suggest that the fluid pressure indirectly suppresses the Ca²⁺ channel by enhancing the Ca²⁺-induced intracellular Ca²⁺ release in rat ventricular myocytes. L-type Ca²⁺ current; fluid pressure; ventricular myocytes; cytosolic Ca²⁺ transient     

Role of Na+-K+-Cl- cotransport and Na+/Ca2+ exchange in mitochondrial dysfunction in astrocytes following in vitro ischemia

Na⁺-K⁺-Cl^– cotransporter isoform 1 (NKCC1) and reverse mode operation of the Na⁺/Ca²⁺ exchanger (NCX) contribute to intracellular Na⁺ and Ca²⁺ overload in astrocytes following oxygen-glucose deprivation (OGD) and reoxygenation (REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca²⁺ (Ca_m²⁺) overload and dysfunction. OGD/REOX caused a doubling of mitochondrial-releasable Ca²⁺ (P < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial-releasable Ca²⁺ was reduced by 42% (P < 0.05). Genetic ablation of NKCC1 also reduced Ca_m²⁺ accumulation. Moreover, OGD/REOX in NKCC1^+/+ astrocytes caused dissipation of the mitochondrial membrane potential (_m) to 42 ± 3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of _m was attenuated significantly (66 ± 10% of controls, P < 0.05). Cells were also subjected to severe in vitro hypoxia by superfusion with a hypoxic, acidic, ion-shifted Ringer buffer (HAIR). HAIR/REOX triggered a secondary, sustained rise in intracellular Ca²⁺ that was attenuated by reversal NCX inhibitor KB-R7943. The hypoxia-mediated increase in Ca_m²⁺ was accompanied by loss of _m and cytochrome c release in NKCC1^+/+ astrocytes. Bumetanide or genetic ablation of NKCC1 attenuated mitochondrial dysfunction and astrocyte death following ischemia. Our study suggests that NKCC1 acting in concert with NCX causes a perturbation of Ca_m²⁺ homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia. intracellular calcium ion; mitochondrial membrane potential; sodium ion influx; bumetanide; cytochrome c; glial cell death     

Glycogen biosynthesis in Chromatium strain D II. Purification and properties of glycogen synthetase

Glycogen synthetase, ADP-glucose-a (l4) glucan transglucosylase[E.C. 2.4.1.11 [EC] ] from a purple sulfur bacterium, Chromatium,was purified to a homogeneous state and its enzymic propertieswere studied. The molecular weight of the enzyme was 8.6?10⁴dalton as determined by analytical gel filtration on a columnof Sephadex G-100. Since sodium dodecyl sulfate-polyacrylamidegel electrophoresis gave the molecular weight value of 8.4?10⁴to the monomeric form of the enzyme, we concluded that Chromatiumglycogen synthetase is comprised of a single polypeptide chain.The optimal pH of teh transglucosylation reaction was between8.0–8.5. The enzyme molecule utilized only ADP-glucoseas the glucose donor. The k_m value was determined as 3.8?10_-4M by the radioisotopic method of measuring the incorporationof ¹⁴C-glucose into the acceptor glycogen, and 6.1?10^-5M bythe enzyme coupling method. The most effective glucose acceptor(primer) was proved to be a long-chain a (16) branched a (14)polyglucan, e.g. Chromatium and cow glycogen, whereas short-chainmalto-oligosaccharides were much less efficient in the chain-elongationreaction. ¹ Part I of this series is Ref. (9). (Received February 13, 1974; )     

Calcium influx through If channels in rat ventricular myocytes

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I_f)/neuronal (I_h) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I_h channels in neurons. This raises the possibility of Ca²⁺ permeation in I_f, the I_h counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca²⁺ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I_f in rat ventricular myocytes. We observed Ca²⁺ influx when HCN/I_f channels were activated. Ca²⁺ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I_f channel blocker, inhibited I_f and Ca²⁺ influx at the same time. Quantitative analysis revealed that Ca²⁺ flux contributed to 0.5% of current produced by the HCN2 channel or I_f. The associated increase in Ca²⁺ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I_f current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca²⁺ chelator), preactivation of I_f channels significantly reduced the action potential duration, and the effect was blocked by another selective I_f channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I_f channels had no effects on action potential duration. Our data extend our previous discovery of Ca²⁺ influx in I_h channels in neurons to I_f channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels