Effects of delta9-tetrahydrocannabinol on ATPases in mouse brain subcellular fractions.

The effect of (?)-Δ⁹-THC on the activities of Mg²⁺?, Na⁺?K⁺? and Mg²⁺Ca²⁺-ATPases were studied in mouse brain subcellular fractions. $\text{In vitro}$treatment with Δ⁹-THC produced a dose dependent stimulation of Mg²⁺ ATPase in the crude mitochondrial fraction and its subfractions and a dose-related inhibition of this activity in the microsomal fraction. Na⁺-K⁺- and Mg²⁺-Ca²⁺-ATPase activities were inhibited in a dose-related manner in all subcellular fractions studied.     

Effects of glucagon, insulin and cyclic-AMP on mitochondrial calcium uptake in the liver.

The effects of glucagon and insulin administration $\text{in vivo}$ on hepatic mitochondrial Ca²⁺ uptake were compared with the effects of these hormones when they were added directly to the perfused liver. Glucagon administration increased mitochondrial calcium uptake both $\text{in vivo}$ and in the perfused liver. In contrast, while injection of insulin into rats stimulated, addition of insulin to the perfusate, inhibited Ca²⁺ uptake. Cyclic AMP, when added to the perfusate, also increased the uptake of Ca²⁺ by mitochondria, subsequently isolated. The possible implications of the results are discussed.     

On the existence of two populations of mitochondria in a single organ. Respiration, calcium transport and enzyme activities.

Mitochondria isolated from rat heart and kidney cortex by Polytron treatment of the tissues exhibit lower state 3 rates of respiration than mitochondria isolated by Nagarse method. Addition of cytochrome $\text{c}$ to Polytron mitochondria isolated from heart, but not from kidney, increases oxygen uptake to values approaching those of Nagarse-treated preparations. Similar results were observed for Ca²⁺ uptake. Kidney Polytron mitochondria exhibited lower mitochondrial, but higher non-mitochondrial enzyme activities compared to kidney Nagarse mitochondria. Enzyme activities were the same in Polytron and Nagarse mitochondria from heart. The differences between Polytron and Nagarse mitochondria appear to be mainly due to lower cytochrome $\text{c}$ content of Polytron mitochondria from heart and higher contamination of Polytron mitochondria from kidney.     

The role of the sites for ATP of the Ca2+-ATPase from human red cell membranes during Ca2+-phosphatase activity

1. In the presence of ATP, the Ca²⁺ pump of human red cell membranes catalyzes the hydrolysis of $\text{p-}\text{nitrophenyl}$ phosphate. The requirement for ATP of the Ca²⁺-$\text{p-}\text{nitrophenylphosphatase}$ activity was studied in relation to the two classes of site for ATP that are apparent during Ca²⁺ -ATPase activity. 2. (a) The $\text{K}{}_{0.5}$ for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ extrapolated at 0 mM PNPP is equal to the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the Ca²⁺ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca²⁺ -ATPase or as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 4. At ATP concentrations that almost saturate the high-affinity site, Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity increases as a function of PNPP along an S-shaped curve, while Ca²⁺ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca²⁺ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca²⁺ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca²⁺ -ATPase activity at the high-affinity size persists during Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity.     

Defective calcium pump in the sarcoplasmic reticulum of the hypertrophied rabbit heart.

To evaluate Ca²⁺-uptake in sarcoplasmic reticulum in the hypertrophied rabbit heart, microsomes were prepared from myocardium of rabbits with experimentally induced aortic stenosis. A significant reduction of microsomal Ca²⁺-uptake was observed in hypertrophied left ventricle, 195±10 compared to 280±18 nmol/mg found in control animals. A similar pattern was observed for the Ca²⁺-stimulated ATPase (30±9 and 59±10 nmol/min/mg resp.). A minimal activity difference of the microsomal marker enzyme rotenone-insensitive NADPH cyt. $\text{c}$ reductase was found (7.77±0.05 and 8.17±0.11 nmol/min/mg resp.). The specific activity of the latter enzyme was 5–6 fold increased in microsomes compared to homogenates in both animal groups, which excludes the possibility of increased amounts of contaminant or nonfunctional protein in sarcoplasmic reticulum prepared from hypertrophied myocardium. In addition the yield of microsomal protein did not differ significantly. Maximal phosphorylation by exogenous cyclic AMP and protein kinase increased Ca²⁺-uptake in both microsomal preparations (to 287±27 and 375±26 nmol/mg resp. for hypertrophied and control hearts), but Ca²⁺-transport rate found in pathological hearts remained lower than in controls. These findings indicate that impairment of Ca²⁺-metabolism in the hypertrophied heart is based on a defective Ca²⁺-pump.     

Vanadate inhibition of sarcoplasmic reticulum Ca2+-ATPase and other ATPases.

Vanadate is a potent inhibitor of the Ca²⁺-ATPase activity of sarcoplasmic reticulum in the presence of A-23187. The purified enzyme is sensitive to vanadate even in the absence of the ionophore. Ca²⁺ and norepinephrine protect the enzyme against inhibition of vanadate. The nonspecificity of vanadate is emphasized by the finding of inhibition of several other ATPases including the Ca²⁺Mg²⁺-ATPases of the ascites and human red cell plasma membranes, Mg²⁺-ATPase of the ascites plasma membrane, and the K⁺-ATPases of $\text{E.}\text{coli}$ and hog gastric mucosal cell membranes. The ascites plasma membrane Ca²⁺-ATPase (an ecto ATPase) and mitochondrial ATPase are not inhibited by vanadate.     

Effect of long chain unsaturated fatty acids on the calcium transport of sarcoplasmic reticulum

The effect of archidonic, oleic and linoleic acid on calcium uptake and release by sarcoplasmic reticulum isolated from longissimus dorsi muscle was investigated using a Ca²⁺ electrode. All three long chain fatty acids stimulated the release of Ca²⁺ from sacroplasmic reticulum when added after exogenous Ca²⁺ was accumulated by the vesicles, and also inhibited Ca²⁺ uptake when added before Ca²⁺. This inhibitory effect on the calcium transport by arachidonic, oleic and linoleic acid was prevented by bovine serum albumin through its ability to bind with the fatty acid. The order of effectiveness of the fatty acids in inhibiting calcium transport by isolated sarcoplasmic reticulum was $\text{arachidonic acid> oleic acid >}$ linoleic acid. Similar inhibition of calcium uptake and induction of calcium release by arachidonic acid was observed in muscle homogenate sarcoplasmic reticulum preparations. Both arachidonic and oleic acid stimulated the (Ca²⁺ + Mg²⁺)-ATPase activity of sarcoplasmic reticulum at low concentrations, but inhibited the (Ca²⁺ + Mg²⁺)-ATPase activity at high concentrations. The maximal (Ca²⁺ + Mg²⁺-ATPase activity observed with arachidonic acid was twice that obtained with oleic acid, but the concentration of arachidonic acid required was 3–4-times greater than that of oleic acid. The concentration of arachidonic acid required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity was 3.6-times greater than that needed for complete inhibition of calcium accumulation by the sacroplasmic reticulum. With oleic acid, however, the concentration required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity inhibited the sarcoplasmic reticulum Ca²⁺ accumulation by 72%. The present data support our hypothesis that, in porcine malignant hyperthermia, unsaturated fatty acids from mitochondrial membranes released by endogenous phospholipase A₂ would induce the sarcoplasmic reticulum to release calcium (Cheah K.S. and Cheah, A.M. (1981) Biochim. Biophys. Acta 634, 70–84).     

Increase in calcium content and Ca2+-ATPase activity in the brain of fasted rats: Comparison with different ages

The effect of fasting on calcium content and Ca²⁺-ATPase activity in the brain tissues of 5 weeks and 50 weeks old rats was investigated. Brain calcium content and Ca²⁺-ATPase activity in the microsomal and mitochondrial fractions of the brain homogenate from young and elderly rats were significantly increased by overnight–fasting. These increases were appreciably restored by a single oral administration of glucose solution (400 mg/100 g body weight) to fasted rats. In comparison with young and elderly rats, brain calcium content and microsomal Ca²⁺-ATPase activity were significantly elevated by increasing ages. The effect of ageing was not seen in the brain mitochondrial Ca²⁺-ATPase activity. When calcium (50 mg/100 g) was orally administered to young and elderly rats, brain calcium content was significantly elevated. The calcium administration–induced increase in brain calcium content was greater in elderly r crease in Ca²⁺-ATPase activity in the microsomal and mitochondrial fractions of brain homogenates from young rats. In aged rats, the microsomal Ca²⁺-ATPase activity was not further enhanced by calcium administration, although the mitochondrial enzyme activity was significantly raised. The present study demonstrates that the fasting–induced increase in brain calcium content is involved in Ca²⁺-ATPase activity raised in the brain microsomes and mitochondria of rats with different ages, supporting a energy–dependent mechanism in brain calcium accumulation.     

Dissociation between Ca2+-ATPase and alkaline phosphatase activities in plasma membranes of rat duodenum

The presence of Ca²⁺-ATPase activities with high-affinity sites for Ca²⁺ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802–803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca²⁺, the substrate specificity of Ca²⁺-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca²⁺-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca²⁺-dependent ATP-hydrolysis was greater than that of ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ at Ca²⁺ concentrations below 25 μM. At 0.2 mM Ca²⁺ the rates of ATP, ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca²⁺, but at 0.2 mM Ca²⁺, Ca²⁺-induced hydrolysis of ADP and AMP was greater than either ATP or $\text{p-}\text{nitrophenylphosphate}$. Alkaline phosphatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca²⁺-stimulated ATP hydrolysis at 1 μM Ca²⁺ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca²⁺, Ca²⁺-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca²⁺-stimulated ATP hydrolysis, either at low or at high Ca²⁺ concentrations. Chlorpromazine fully inhibited Ca²⁺-stimulated ATP hydrolysis in basolateral fragments at 5 μM Ca²⁺, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca²⁺-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca²⁺-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium, is stimulated slightly by low Ca²⁺ concentrations, but this Ca²⁺-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.     

Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca²⁺-activated Mg²⁺-ATPase and Ca²⁺ transport activities. Membrane vesicles that were exposed to oxalate as a Ca²⁺-trapping agent accumulated Ca²⁺ in the presence of Mg²⁺ and ATP. The $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for Ca²⁺ uptake was 33 nmol/mg protein per h, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for Ca²⁺ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca²⁺ ionophore A23187, added initially, completely inhibited net Ca²⁺ uptake and, if added later, caused the release of Ca²⁺ previously accumulated. A Ca²⁺-activated ATPase was present in the same membrane vesicles which had a $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 1.5 μmol/mg protein per h at free Ca²⁺ concentration of 10 μM. This Ca²⁺-ATPase had $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 4.5 μM and 110 μM for Ca²⁺ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca²⁺ by the vesicles. The Ca²⁺-ATPase activity was insensitive to ouabain. Both Ca²⁺ transport and Ca²⁺-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca²⁺ transport activity and a Ca²⁺-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.     

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 $\text{2 : 1}$.     

Relationship between calcium ion transport and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca²⁺ transport in this preparation were characterized and compared to those of (Ca²⁺ + Mg²⁺)-ATPase activity. The time course of Ca²⁺ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8–11.5 nmol Ca²⁺/mg protein after 3–4 min of incubation. The rate of Ca²⁺ transport was increased by higher oxalate concentrations with a near linear rate of uptake at 20 mM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca²⁺/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 100 μM and 7 μM for ATP concentration ranges above and below 50 μM, respectively. The apparent $\text{K}\text{m}$ values for Mg²⁺ and Ca²⁺ were 132 μM and 0.36–0.67 μM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$)-ATPase. The presence of potassium was required for maximum Ca²⁺ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca²⁺ transport and ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{-}\text{ATPase}$ activity was $\text{K}{}^{\mathrm{+}}\text{>}\text{Na}{}^{\mathrm{+}}\text{=}\text{NH}{}_4{}^{\mathrm{+}}\text{>}\text{Li}{}^{\mathrm{+}}\text{.}\text{Ca}{}^{\mathrm{2+}}$ transport and ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ activity were both inhibited 10–20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 μM $\text{p-}\text{chloromercuribenzene}$ sulfonate. The results indicate that Ca²⁺ transport in adipocyte endoplasmic reticulum is mediated by a $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ and suggest an important role for endoplasmic reticulum in control of intracellular Ca²⁺ distribution.     

Modulation of lyso-platelet-activating factor:acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions

The enzyme lyso-platelet-activating factor:acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca²⁺ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca²⁺. The effect of Ca²⁺ was on the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the enzyme for the substrate acetyl-CoA without showing any significant effect on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca²⁺ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca²⁺, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca²⁺ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.     

Localization of (Ca2+ + Mg2+)-ATPase,Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1·10^?4 M. The sarcolemmal markers, ouabain-sensitive (Na⁺ + K⁺)-ATPase and Na⁺/Ca²⁺-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na⁺ pump (K⁺-p-nitrophosphatase) were not affected. Almost 20% of the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27–39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca²⁺ + Mg²⁺-ATPase and Ca²⁺ pump compared to control hearts. (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for inhibition approx. 1.5 μM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg²⁺-ATPase and Ca²⁺-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

Evidence for an endogenous protein inhibitor of sarcoplasmic reticulum calcium pump in heart muscle

A cytosolic protein fraction, termed CPF-I, derived by (NH₄)₂ SO₄ fractionation of rabbit heart cytosol caused marked inhibition (up to 95%) of ATP-dependent Ca²⁺ uptake by cardiac sarcoplasmic reticulum. The inhibitory effect of CPF-I was concentration-dependent (50% inhibition with ～ 80–100 μg CPF-I) and heat labile. The inhibitor reduced the velocity of Ca²⁺ uptake without altering the apparent affinity of the transport system for Ca²⁺. Concomitant with the inhibition of Ca²⁺ uptake, Ca²⁺-sensitive ATP hydrolysis was also inhibited by CPF-I. The inhibitor did not cause release of Ca²⁺ from Ca²⁺-preloaded membrane vesicles. The inhibitor activity of CPF-I could be adsorbed to a DEAE cellulose column and could be eluted with a linear gradient of KCl. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum calcium pump in cardiac muscle and raises the intriguing possibility of its participation in the regulation of calcium pump $\text{in}\text{vivo}$.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+-transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca²⁺ + Mg²⁺)-ATPase activity and in Ca²⁺ efflux at 37°C, depending on the side of the membrane at which the monovalent cations K⁺ and Na⁺ were placed. Under the conditions used, (Ca²⁺ + Mg²⁺)-ATPase activity and Ca²⁺ efflux was highest when K⁺ ($\text{35 ± 0.5}\text{mM (± S.E.)}$, mean of four experiments) was at the inside and Na⁺ (130 mM) at the outside of the ghost membrane.     

The effect of inorganic phosphate on calcium influx into rat heart mitochondria

The effect of inorganic phosphate on the accumulation of Ca²⁺ by heart mitochondria has been reinvestigated. Inorganic phosphate has no effect on the initial rate of Ca²⁺ uptake supported by respiration on either ascorbate $\text{plus}$ tetramethylenephenylene diamine, pyruvate $\text{plus}$ malate, or glutamate $\text{plus}$ malate, although it does increase the final amount of Ca²⁺ accumulated; evidence suggests that the latter phenomenon requires phosphate influx via the phosphate carrier. It is concluded that the earlier reports that phosphate augments the initial rate of Ca²⁺ influx reflects an effect of phosphate on succinate oxidation, which was employed in the previous studies, rather than an Ca²⁺ transport itself.     

Effect of phospholipid methylation on calcium transport and (Ca2+ + Mg2+)-ATPase activity in kidney cortex basolateral membranes

Basolateral membranes isolated from hog kidney cortex, enriched 12- to 15-fold in (Na⁺ + K⁺)-ATPase activity, were 80% oriented inside-out as determined by assay of oubain-sensitive (Na⁺ + K⁺)-ATPase activity before and after opening of the membrane vesicle preparation with a mixture of deoxycholate and EDTA. In these membrane preparations 80% of total phosphatidylethanolamine was accessible to trinitrophenylation by trinitrobenzenesulfonic acid at 4°C, while at 37°C all of phosphatidylethanolamine fraction was chemically modified. Phospholipase C treatment resulted in hydrolysis of 80% phosphatidylethanolamine, 40% phosphatidylcholine and 35% of phosphatidylserine. Sphingomyelinase treatment resulted in 20% hydrolysis of sphingomyelin, presumably derived from right-side-out oriented vesicles. Results indicate that phosphatidylethanolamine is oriented exclusively on the outer leaflet of the lipid bilayer of inside-out oriented vesicles. Methylation of phospholipids in basolateral membranes with $\text{S-}\text{adenosyl}\text{[methyl-}{}^3\text{H}\text{]}\text{methionine}$ resulted in the three successive methylation of ethanolamine moiety of phosphatidylethanolamine to phosphatidylcholine. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for $\text{S-}\text{adenosylmethionine}$ was 1·10^?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg²⁺ was without any effect between pH 5 and 10. Basolateral membranes incubated in the presence of methyl donor, $\text{S-}\text{adenosylmethionine}$, exhibited increased (12–15%) (Ca²⁺ + Mg²⁺)-ATPase activity and increased ATP-dependent uptake of calcium. ATP-dependent calcium uptake in these vesicles was insensitive to oligomycin and ouabain but was abolished completely by 50 μM vanadate. The increase in ATP-dependent calcium uptake was due to an increase in $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ and not due to a change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Ca²⁺. Preincubation of membranes with $\text{S-}\text{adenosylhomocysteine}$, a methyltransferase inhibitor, abolished the stimulatory effect of phospholipid methylation on calcium uptake. Phospholipid methylation at both low and high pH did not result in a change in bulk membrane fluidity as determined by the fluorescence polarization of diphenylhexatriene. These results suggest that phospholipid methylation may regulate transepithelial calcium flux in vivo.