Repression of the K+ Uptake and Cation-Stimulated ATPase Activity Associated with the Plasma Membrane-Enriched Fraction of Cucumber Roots Due to Ca2+ Starvation

The uptake of K⁺ by cucumber plants decreased markedly duringCa²⁺ starvation. A plasma membrane-enriched fraction, judgedfrom the distribution of marker enzymes, was prepared from controland Ca²⁺-starved roots. The Mg²⁺- and K⁺-Mg²⁺-ATPase activitiesassociated with the plasma membrane-enriched fraction of controlroots were maxima at pH 6.5. Various monovalent cations andpotassium salts of monovalent anions stimulated Mg²⁺-ATPaseactivity. Vanadate, DES and DCCD inhibited K⁺- Mg²⁺-ATPase activity.Of the divalent cations and phosphate esters tested, Mg²⁺ andATP were most effective for the stimulation of ATPase by K⁺,whereas Ca²⁺ was ineffective in replacing Mg²⁺. Mg²⁺- and K⁺-Mg²⁺-ATPase activities associated with the plasmamembrane enriched fraction of Ca²⁺-starved roots were much lowerthan those of control roots. K_m values of K⁺-Mg²⁺-ATPase forATP were comparable for control and Ca²⁺-starved roots. The K⁺-stimulated activity of Mg²⁺-ATPase in Ca²⁺-starved rootswas approximately one fourth that of the control, whereas therate of stimulation was only slightly lower in Ca²⁺-starvedroots. (Received May 9, 1984; Accepted September 17, 1984)     

Repression of Proton extrusion from Intact Cucumber Roots and the Proton Transport Rate of Microsomal Membrane Vesicles of the Roots due to Ca2+ Starvation

Proton extrusion from cucumber roots decreased markedly duringCa²⁺ starvation in the presence of KC1. Vesicles with ATP-dependentproton transport activity were prepared from the microsomalmembrane fraction of control and Ca²⁺-starved roots. The protontransport rate of the vesicles from Ca²⁺-starved roots was repressedto less than half of the vesicles prepared from the controlroots. K⁺-Mg²⁺-ATPase activity associated with the vesiclesprepared from Ca²⁺-starved roots was approximately one-thirdof the activity associated with those prepared from controlroots. K_m values of the proton transport rate and K⁺-Mg²⁺-ATPasefor ATP were much higher in vesicles prepared from Ca²⁺-starvedroots. The repression of proton extrusion linked with K⁺ uptake inthe Ca²⁺-starved roots could be largely caused by the reducedproton pumping activity associated with microsomal membranesin the roots. (Received May 25, 1987; Accepted October 14, 1987)     

Specific Increase in Phosphatase Isozymes in Cucumber Roots Caused by Calcium Deficiency

Phosphatases in cucumber roots, whose production was inducedby Ca^2$ deficiency, were characterized chromatographically usingATP, 2'(3')-AMP and p-nitrophenyl-phosphate (PNPP) as substrates.Ca^2$ deficiency stimulated greater than 10-fold increases inthe activities with these substrates of the non-adsorbed fractionfrom a DEAE-cellulose column. Several fractions associated withthese phosphatase activities were eluted from the column withNaCl solution; their levels increased less with Ca^2$ starvation.When the non-adsorbed fraction from Ca^2$-straved roots was appliedto a Sephadex G-200 column, fractions associated with 2'(3')-AMPase(phosphatase I) and with both ATPase and PNPPase (phosphataseII) were separated. In the control roots, very weak activitiesof phosphatases I and II were observed at the same positionon the gel filtration. The phosphatase I isolated from boththe control and Ca^2$-starved roots was extremely specific tonucleoside 2'(3')-monophosphates, whereas phosphatase II fromboth types of roots had a relatively broad substrate specificity.When phosphatase I from Ca^2$-starved roots was stained with2'(3')-AMP in CaCl₂ after polyacrylamide gel electrophoresis,a single band was obtained. Phosphatase I from control rootsalso showed a single band, with the same Rf value. PhosphataseII from both types of roots contained two isozyme bands whenthe activities were stained with either ATP or PNPP. These resultsindicate that Ca^2$ starvation causes specific increases in thelevel of phosphatases I and II in cucumber roots. (Received October 28, 1981; Accepted January 19, 1982)     

Increase in Cell Wall-Associated Phosphatase Activity in Cucumber Roots during Calcium Starvation: Binding Nature and Properties of the Phosphatase and Cell Wall Analysis

In Ca^2$-starved cucumber roots, about 23% of phosphatase assayedat pH 9.0 (ALPase) in the crude cell walls was solubilized witheither 2 M NaCl or purified endo type polygalacturonase (endo-PG)from yeast culture broth. Coexistence of NaCl and endo-PG hadlittle effect on further release of ALPase, and a small amountof the activity was solubilized from the NaCl-pretreated cellwalls by incubation with endo-PG. Ionically bound ALPase, therefore,seemed to be localized in the fraction which was hydrolyzedby endo-PG in the crude cell walls of Ca^2$-starved cucumberroots. In the control roots, however, ALPase was not effectivelysolubilized by the treatment with endoPG. Ca^2$ starvation reducedthe contents of rhamnose, uronic acids and galactose among non-cellulosicsugars in the cell walls, suggesting that the structure of pecticsubstances, possibly rhamnogalacturonan, is altered during thestarvation. Activities of both ionically and covalently bound ALPases greatlyincreased during Ca^2$ starvation. The increased ALPase in theNaCl-solubilized fraction hydrolyzed most phosphate esters tested,whereas the enzyme from control roots only cleaved nucleoside2'(3')-monophosphates and p-nitrophenylphosphate. Differencesin the properties between both types of roots were also foundwhen the effects of various inhibitors were tested. Profilesof ALPase-isozymes after polyacrylamide gel electrophoresiswere also altered by Ca^2$ starvation. (Received June 2, 1982; Accepted July 20, 1982)     

Functional Reconstitution of Plasma Membrane H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes Prepared with Various Molecular Species of Phospholipids

The activity of solubilized plasma membrane ATPase is affectedby the nature of exogenously added molecular species of phospholipids.To examine the role of the polar head group and of the molecularspecies of phospholipids in H⁺-pumping, the ATPase solubilizedfrom plasma membranes of mung bean (Vigna radiata L.) hypocotylswas reconstituted in liposomes prepared with a variety of phospholipids. The extent of activation of solubilized plasma membrane ATPasedue to the addition of 1-palmitoyl 2-oleoyl-phospholipids (PO-phospholipids)and asolectin decreased in the following order: POPS POPC asolectin POPG > POPE > POPA (see List of Abbreviations). H⁺-pumpinginto proteoliposomes reconstituted with asolectin and plasmamembrane ATPase was demonstrated by quinacrine fluorescencequenching in the presence of ATP-MgSO₄. H⁺-pumping was inhibitedby VO₄ and gramicidin D. When plasma membrane ATPase was reconstitutedin liposomes prepared with various PO-phospholipids, the abilityof PO-phospholipids to support H⁺-pumping into the proteoliposomesdecreased in the following order: POPG POPS > asolectin POPC. POPE and POPA failed to support any H⁺-pumping. A remarkablyhigh rate of H⁺-pumping was observed in proteoliposomes preparedwith 1-saturated 2-unsaturated fatty acids, such as POPC, butH⁺-pumping could hardly be detected in proteoliposomes preparedwith 1-, 2-unsaturated or 1-, 2-saturated fatty acids, suchas PSPC or DLPC. ATPase activity in proteoliposomes was dependenton the species of PO-phospholipids used for reconstitution anddecreased in the following order: POPS > POPG > POPC asolectin > POPA > POPE. DLPC (see List of Abbreviations)which includes a 1-, 2-unsaturated fatty acid supported onlymarkedly depressed activity. Both H⁺-pumping and the hydrolysis of ATP by the plasma membraneATPase are strongly affected by the polar head group and compositionof the fatty acyl chain of phospholipids used to prepare liposomesfor reconstitution of the ATPase. (Received May 31, 1991; Accepted September 18, 1991)     

Stimulation of the Extrusion of Protons and H+-ATPase Activities with the Decline in Pyrophosphatase Activity of the Tonoplast in Intact Mung Bean Roots under High-NaCl Stress and Its Relation to External Levels of Ca2+ Ions

Extrusion of protons as a response to high-NaCl stress in intactmung bean roots was investigated at different external concentrationsof Ca²⁺ ions ([Ca²⁺]_ex). The extrusion of protons was graduallyenhanced in the roots exposed to 100 mM NaCl, and high [Ca²⁺]_exdiminished this enhancement of the extrusion. Vesicles of plasmalemmaand tonoplast were prepared from the roots and the H⁺-translocatingATPase (H⁺-ATPase) activities associated with the two typesof membrane and the H⁺-pyrophosphatase (H⁺-PPase) activity ofthe tonoplast were assayed. The plasmalemma ATPase was stimulatedin parallel with dramatic increases in the intracellular concentrationof Na⁺([Na⁺]_in). High [Ca²⁺]ex prevented the increase in [Na⁺]inand diminished the stimulation of ATPase activity. The tonoplastATPase showed a rapid response to salt stress and was similarlystimulated even at high [Ca²⁺]M. The activities of both ATPaseswere, however, insensitive to concentrations of Na⁺ ions upto 100 HIM. By contrast, H⁺-PPase activity of the tonoplastwas severely inhibited with increasing [Na⁺]in under salt stressand recovered with high [Ca²⁺]ex. These findings suggest thathigh-NaCl stress increases the intracellular concentration ofNa⁺ ions in mung bean roots, which inhibits the tonoplast H⁺-PPase,and the activity of the plasmalemma H⁺-ATPase is thereby stimulatedand regulates the cytoplasmic pH. (Received March 26, 1991; Accepted December 13, 1991)     

Cyclic AMP and calcium modulated ATPase activity in the salivary glands of the lone star tick Amblyomma americanum (L.)

Na⁺,K⁺-activated ATPase activity in tick salivary glands increases during the rapid stage of tick feeding paralleling similar increases in dopamine and cAMP-stimulated fluid secretion. High concentrations of cyclic AMP increase Na⁺,K⁺-ATPase activity in a plasma membrane-enriched fraction from the salivary glands of rapidly feeding ticks. Cyclic AMP-dependent protein kinase inhibitor protein blocks activation of Na⁺,K⁺-ATPase activity at low but not high concentrations of cAMP indicating that both activator and inhibitor modulator phosphoproteins of Na⁺,K⁺-ATPase activity exist in the plasma membrane-enriched fraction.ATPase activity in the plasma membrane-enriched fraction is not measurable in the absence of Mg²⁺, Ca²⁺ and Na⁺. Ca-stimulated nucleotidase activity is highest with ATP serving as the preferred substrate in a series including CTP, UTP, GTP and ADP. Calcium, Mg²⁺ stimulated ATPase activity is activated further by calmodulin and partially inhibited by low concentration of vanadate, trifluoperazine and oligomycin. Results suggest that the plasma membrane-enriched fraction of tick salivary glands contains both Ca²⁺-ATPase activity and oligomycin-sensitive Ca²⁺, Mg²⁺-ATPase activities, the latter likely from a small amount of mitochondria in the partially purified organelle fraction.     

A lipid requirement for the (Ca2+ + Mg2+)-activated ATPase of erythrocyte membranes.

The lipid requirement of the (Ca²⁺ + Mg²⁺)-stimulated ATPase of human erythrocytes has been studied. The enzyme activity was lost after removal of the phospholipids using phospholipase A₂ from Naja naja and serum albumin. Optimal restoration of the (Ca²⁺ + Mg²⁺)-ATPase activity in the partially lipid-depleted membranes was obtained with oleate. The reactivation was not due to the removal of a permeability barrier for ATP, since lysolecithin or cholate did not show latent activity. Reactivation was also obtained with several negatively charged phospholipids. Among the ones normally found in the erythrocyte membranes, only phosphatidyl serine reactivated significantly.     

The Role of Phospholipids in Plasma Membrane ATPase Activity in Vigna radiata L. (Mung Bean) Roots and Hypocotyls

Root and hypocotyl plasma membrane H⁺-ATPases were partially purified from deoxycholate-solubilized fractions of microsomes in mung bean (Vigna radiata L.) plants in the presence of glycerol. Certain properties of the ATPases and the manner in which phospholipids affect their activity were compared. Root ATPase was similar to hypocotyl ATPase with respect to substrate specificity, salt stimulation, pH dependence, K_m for ATP·Mg²⁺ and inhibitor sensitivity, except for inhibition by vanadate. Both purified ATPases required phospholipids for their activation. Optimum concentrations of exogenously added phospholipid mixture (asolectin) to hypocotyl and root ATPase mixture were 0.03% and 1.0%, respectively. Root ATPase activation did not decrease if more than 1.0% asolectin was added. Qualitatively, phosphatidylserine and phosphatidylcholine brought about greater ATPase activation than other phospholipids. The hypocotyl ATPase was activated by phosphatidylinositol, phosphatidylserine and phosphatidylglycerol to a greater extent than the root ATPase. Root, but not hypocotyl ATPase, was slightly inhibited by the addition of phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. The hypocotyl plasma membrane contained phosphatidylinositol + phosphatidylserine, phosphatidylglycerol and phosphatidic acid, and unsaturated fatty acids in greater abundance than the root plasma membrane. The differential activation of the plasma membrane ATPases may arise from these differences.     

A comparison of the properties of ATPase associated with wheat and cauliflower plasma membranes

Plasma membrane-associated ATPase obtained from cauliflower (Brassica oleraceae L.) florets isolated and assayed by several different procedures was stimulated 150 to 400% by K⁺. In contrast, winter wheat (Triticum aestivum L. cv. Kharkov 22 MC) shoot and root ATPase obtained by the same methods exhibited only 10 to 25% stimulation by K⁺. The level of K⁺-stimulation of the wheat enzyme was not significantly increased by purifying the crude microsomal membrane fraction using sucrose density gradients. ATPase associated with density gradient-purified cauliflower membranes was inhibited by Ca²⁺, high ATP concentration in the presence of low Mg²⁺, and by several metabolic inhibitors. In contrast, the wheat enzyme was largely unaffected by all of these treatments. The plasma membranes of intact wheat and cauliflower cells gave a positive reaction with the plasma membrane-specific, phosphotungstic acid-chromic acid stain (PACP). A high proportion of the cauliflower membrane vesicles in the putative plasma membrane-enriched fraction stained with PACP, whereas only a small proportion of the wheat membrane vesicles reacted positively with PACP. These results indicate that a plasma membrane-enriched fraction has been isolated successfully from cauliflower floret tissue, but that none of the procedures used effectively separate plasma membranes from homogenates of wheat shoots and roots.     

Unidirectional Ca2+ Fluxes in Roots of Rye (Secale cereale L). A Comparison of Excised Roots with Roots of Intact Plants

The unidirectional Ca²⁺ fluxes across the plasma membrane andtonoplast were determined in both excised roots and roots ofintact seedlings of rye (Secale cereale L. cv. Rheidol). Theunidirectional Ca²⁺ fluxes across the plasma membrane and tonoplastmeasured in excised roots were of a similar order of magnitudeto those determined in roots of intact plants. Influx and effluxof Ca²⁺ across the root plasma membrane were similar (estimatedto be between 0·7 and 3·4 µmol g     

Changes of Polysomes in Cucumber Plant due to Ca2+ Deficiency

The amounts of monosomes plus polysomes in cucumber plant decreaseddue to Ca²⁺ deficiency. The decrease was more prominent in themembrane-bound forms than free forms. Polysomes in immatureleaves and the proportion of large polysomes in roots decreasedeven at the early stage of Ca²⁺ deficiency. (Received November 28, 1986; Accepted May 22, 1987)     

Increase in Proton-Transport Activity of Tonoplast Vesicles as an Adaptive Response of Barley Roots to NaCl Stress

H⁺-Transport activity of the vesicles prepared from barley rootswas studied at the early phase after application of NaCl stress.The activity reached maximal level at 3 days after the treatmentwith 200 mM NaCl which moderately reduced the growth. This activityincrease could be suppressed in the presence of cycloheximideand actinomycin D. The properties of the membrane vesicles associated with H⁺-transportactivity prepared from both control and NaCl-stressed rootssuggested that it was of tonoplast origin based on the followingfindings: optimal pH at 7.5, strong inhibition by nitrate butnot by vanadate, and stimulation by chloride. The density gradient centrifugation of vesicles with DextranT70 did not show any detectable difference in the distributionpatterns of H⁺-transport activities between control and NaClstressedroots. Furthermore, K_m values for ATP of the H⁺-transport activityof vesicles prepared from control and NaCl-stressed roots werethe same. Therefore, H⁺-transport activity with properties similarto those of the control roots was increased by NaCl stress.The results are discussed in terms of an adaptive mechanismof barley against salt stress. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received April 18, 1988; Accepted July 20, 1988)     

Isolation and characterization of adenosine triphosphatase from Salicornia pacifica var. utahensis

Membrane-bound ATPase associated with plasma membrane and solubleATPase associated with the cytoplasm were prepared from shootsof Salicornia pacifica var. utahensis by sucrose density gradientcentrifugation. The isolated ATPases were tolerant to high concentrationsof NaCl. The Km for membrane-bound ATPase was 1.75 mM and forsoluble ATPase, it was 1.4 mM. The relative effectiveness ofdivalent cations for stimulation of membrane-bound ATPase wasMg²⁺>Fe²⁺>Mn²⁺>Co²⁺>Cu²⁺. Soluble ATPase activitywas stimulated by Ba²⁺>Ca²⁺>Mg²⁺ and was inhibited byCu²⁺, Zn²⁺, Co²⁺ and Fe²⁺. The compounds N,N¹-dicyclohexylcarbodiimide,NaF and ADP, did inhibit the ATPases but ouabain, triphenyltinhydroxide, sodium azide, indoleacetic acid and abscisic aciddid not inhibit the ATPases from s. pacifica var. utahensis. ¹ Present address: Department of Biology, Kong-Ju National College,Kong-Ju, Korea. (Received April 1, 1980; )     

Changes of Some Membrane-Associated Enzyme Activities Degradation of Membrane Phospholipids in Cucumber Roots Due to Ca2+ Starvation

Deprivation of Ca²⁺ from a complete culture medium affectedthe enzyme activities associated with five membrane fractionsof cucmber roots obtained by discontinuous sucrose density gradientcentrifugation. The total activity of K⁺-ATPase, Cyt. c oxidaseand NADPH-Cyt. c reductase of Ca²⁺-deficient roots, starvedfor only 4 days, had decreased to 14, 38 and 60% of the activityof the control roots. In general, loss of enzyme activitieswas accompanied by a shift of activity distribution from theheavier density fractions to lighter ones. The amounts of Ca²⁺ associated with membranes from Ca²⁺-starvedroots decreased to 50–60% of those of the control roots.Both phospholipid and neutral lipid contents in the membranesdecreased markedly while the protein content was not changedby Ca²⁺ deficiency. Phospholipid analysis indicated a drasticdrop in the percent composition of phosphatidylinositol butan increase of phosphatidic acid. Also, phospholipase D activityincreased remarkably during Ca²⁺ starvation, paralleling theappearance of Ca²⁺-deficiency symptoms. Thus, the major effects of Ca²⁺ deficiency appear to be to stimulatephospholipase D activity and a reduction in membrane bound Ca²⁺.These effect may be involved in disorganization of the membranestructure and the changes of enzyme activities associated withthe altered membranes. ¹Rubber Research Institute of Sri Lanka, Dartonfield, Agalwatta,Sri Lanka. (Received July 15, 1985; Accepted November 21, 1985)     

A Novel Phosphorylated Lipid Counteracts Activation of the Renal Plasma Membrane (Ca2+ + Mg2+)ATPase by Endogenous Phosphatidylinositol-4-Phosphate

The plasma membrane (Ca²⁺ + Mg²⁺)ATPase is activated by acidic phospholipids in reconstituted systems. In this report it is shown that reversible phosphorylation of endogenous phosphatidylinositol regulates the renal plasma membrane (Ca²⁺ + Mg²⁺)ATPase, and that a novel phosphorylated lipid that can be isolated from the same membrane strongly counteracts the stimulatory effect of phosphatidylinositol-4-phosphate.     

Reconstitution of Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

Reconstituted proteoliposomes of tonoplast ATPase are formedon solubilization of tonoplast membranes from mung bean (Vignaradiata L.) with deoxycholate (DOC) in the presence of a mixtureof soybean phospholipids (asolectin), after removal of DOC bypassage through a PD-10 column (Pharmacia). This method is idealbecause of its simplicity and rapidity. Selective insertionof sets of tonoplast H⁺-ATPase polypeptides (68 kDa, 60 kDa,16 kDa and several minor polypeptides) into liposomes usingthis method was confirmed by SDS-PAGE and immuno-blotting withantibodies raised against 68-kDa and 60-kDa polypeptides. Pumping of protons across the membranes of the proteoliposomeswas demonstrated by quinacrine-fluorescence quenching in thepresence of ATP-Mg²⁺. ATP-Mg²⁺ was shown to be the preferredsubstrate in both reconstituted and native tonoplast vesicles,and its optimum concentration was 0.75 to 3.0 mM. Quenchingwas completely abolished by a channel-forming ionophore, gramicidinD, and an inhibitor of tonoplast H⁺-ATPase, KNO₃. Antibodiesto 68-kDa and 60-kDa peptides partially inhibited the pumpingof protons. The rate of pumping of protons increased with thenumber of proteoliposomes, the maximal concentration of whichwas equivalent to 250 µg of protein per reaction mixture.The optimum pH for pumping was 6.5 when inside of proteoliposomeswere loaded pH at 7.2. The rate of pumping of protons was reducedwhen proteoliposomes were made using asolectin and cholesterolat 3 : 1 (w/w), as compared with those made with asolectin alone. The ATPase activity in reconstituted proteoliposomes was inhibitedby KNO₃, with half-maximal inhibition at approximately 7 mM.The enzyme actively hydrolyzed ATP in preference to GTP, CTP,UTP, and ADP, but it did not hydrolyze pNPP or AMP. Antibodiesagainst the 60-kDa polypeptide strongly inhibited ATPase activityas compared to antibodies against the 68-kDa polypeptide. Theresults obtained in this study demonstrate directly that functionaltonoplast H⁺-ATPase can be inserted selectively into liposomes. (Received August 31, 1990; Accepted April 18, 1991)     

Reconstitution and Characterization of H+-Translocating ATPase from the Plasma Membrane of Phaseolus mungo L. Roots

Plasma membrane H⁺-translocating ATPase was partially purifiedfrom mung bean (Phaseolus mungo L.) roots and reconstitutedinto soybean phospholipid (asolectin) liposomes by the n-octylglucosidedilution method. The resulting proteoliposomes were mainly unilamellarvesicles ranging in size from 0.05 to 0.2 µm. The existenceof ATP-drived H⁺-pumping across the proteoliposomes was demonstratedby the quenching of quinacrine fluorescence in the presenceof Mg²⁺. The quenching could be abolished by an uncoupler, FCCP,and an inhibitor of H⁺-translocating ATPase, vanadate. The reconstitutedATPase consisted of three major polypeptides of 105 KDa, 67KDa and 57 KDa. Its pH optimum, divalent cation stimulationand vanadate sensitivity were similar to those of partiallypurified ATPase. However, the specificity toward ATP was muchgreater following reconstitution. Also reconstitution reducedthe degree of inhibition by DCCD. Local anesthetics (e.g. dibucaine)had no effect on H⁺-pumping activity but increased the ATPaseactivity when proteoliposomes were reconstituted in their presence. (Received May 2, 1986; Accepted October 17, 1986)     

Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.