Role of Oxidative Metabolism in the Energy Suppply of Active Potassium Transport in Erythrocytes of the Lamprey Lampetra fluviatilis

To study role of glycolysis and oxidative metabolism in providing active transport of monovalent cations, isolated erythrocytes of the lamprey Lampetra fluviatlis were incubated at 20°C in the presence of various metabolic inhibitors. The active (ouabain-sensitive) K⁺ (⁸⁶Rb) influx into erythrocytes did not change after cell incubation for 1–2 h in the absence of glucose or in the presence of 10 mM deoxy-D-glucose or 1 mM monoiodoacetate. Inhibitors of oxidative phosphorylation (antimycin A, rotenone, sodium azide, cyanide) produced a significant decrease (on average, by 74% ) in the active K⁺ transport in the lamprey erythrocytes. All blockers of oxidative phosphorylation produced the same degree of inhibition of the K⁺ transport after the cell pre-incubation with them for 30 and 60 min. In experiments with rotenone, the K⁺ influx was reduced statistically significantly as early as in 5 min of cell incubation and reached a maximal effect after 10–20 min. The intracellular ATP content in erythrocytes decreased by 17, 37, and 45% after 5, 10, and 20 min of cell incubation with rotenone, respectively. The active K⁺ transport in the lamprey erythrocytes is most likely to be closely associated with the intracellular ATP concentration. The data obtained indicate that the energy supply of the Na,K-pump in the lamprey erythrocytes is due exclusively to oxidative phosphorylation processes.     

Characteristics of Membrane Protein Phosphorylation in Plasmodium berghei-Infected Mouse Erythrocytes1

ABSTRACT. Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (³²P)orthophosphate and incubating isolated membrane with (γ-³²P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with M, 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

Altered erythrocyte protein kinase C activity and membrane protein phosphorylation in chronic myelogenous leukemia

The membrane protein kinase C (PKC) content was found to be higher in erythrocytes form patients suffering from chronic myelogenous leukemia (CML) compared to normal erythrocytes. PKC activity was also higher in the cytosol and after translocation to the membrane, as assessed by histone phosphorylation. The increased PKC activity in CML erythrocytes was associated with abnormal phosphorylation of protein 4.1. Since phosphorylation-dephosphorylation mechanisms are likely candidates for controlling membrane protein associations, the altered PKC activity may be one of the factors responsible for altered thermal sensitivity and mechanical stability of CML erythrocytes.     

Insulin activation of NADH ferricyanide reductase in human erythrocytes is mediated by the insulin receptor tyrosine kinase: a comparative study in normal and diabetic states

Summary

We have previously reported that NADH ferricyanide reductase in human erythrocytes is stimulated by insulin. Hormone-stimulated activities are attenuated in the presence of glycolytic inhibitors like vanadate, indicating the involvement of glycolysis in the mechanism by which insulin stimulates ferricyanide reduction. Activation of erythrocyte metabolism in response to insulin could be a result of hormone binding to its receptor, inducing phosphorylation of band 3 (at a site for reversible association of glycolytic enzymes) and/or other membrane proteins like the Na⁺/H⁺ antiport. Activation of the antiporter protein by insulin can stimulate glycolysis by an increase in intracellular pH, an effect which is prevented by amiloride. Evidence for a role for tyrosine phosphorylation in triggering the reductase activation came from studies with protein kinase inhibitors. Genistein, sphingosine and acridine orange have been shown to prevent insulin-stimulated ferricyanide reduction, implicating tyrosine phosphorylation as an important signal for activation of the enzyme by insulin. To evaluate activation of the enzyme by insulin stimulated phosphorylation, a comparative study was done using erythrocytes from healthy and diabetic humans. We measured ferricyanide reductase activities in basal and insulin stimulated states. Basal activities were lower in diabetics than in normal humans. Nevertheless, hormone stimulated activities were similar, despite earlier reports of decreased receptor phosphorylation of exogenous substrates in type 2 diabetics. These observations, together with previous ones, suggest that insulin-receptor kinase interaction may mediate the action of insulin on human erythrocytes by phosphorylation of cellular proteins like band 3 and/or the Na⁺/H⁺ antiport.     

Regulatory differences between Ca(2+)-ATPase in plasma membranes from chicken (nucleated) and pig (anucleated) erythrocytes

Kinetic and regulatory properties of the plasma membrane Ca(2+)-ATPase activity from chicken (nucleated) erythrocytes were studied and compared to those from pig (anucleated) erythrocytes. In the absence of known activators: (1) Ca(2+) affinity for the Ca(2+)-ATPase activity from nucleated erythrocytes was 12-fold higher than that from pig erythrocytes, and thus the enzyme is sensitive to physiological Ca(2+) concentrations; (2) the enzyme from chicken erythrocytes showed two apparent Km values for ATP, as compared to one apparent Km value displayed by pig erythrocytes; (3) Ca(2+)-ATPase inserted in chicken erythrocyte membranes showed a low sensitivity to activation by phosphatidylinositol-4-phosphate; (4) when p-NPP was used as substrate, the activity of chicken erythrocytes was high, similar to that attained by pig erythrocytes, but barely sensitive to activation by dimethylsulfoxide and calmodulin. ATP hydrolysis was 10-fold lower than that displayed by pig erythrocytes and the maximal velocity was activated three-fold by calmodulin. The enzyme was insensitive to alkaline phosphatase treatment and showed a single phosphorylation band in electrophoresis, ruling out the possibility of previous modulation by endogenous kinases and/or by partial proteolysis. The differences may be attributed to some endogenous modulator, to distinct isoforms, or to a difference in the E(1)/E(2) states of the enzyme.     

Protein kinase C of human erythrocytes phosphorylates bands 4.1 and 4.9

Addition of 10 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) to intact human erythrocytes results in rapid phosphorylation of two cytoskeletal components, bands 4.1 and 4.9. The synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, shows a similar effect, while the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, fails to enhance phosphorylation. That TPA and 1-oleoyl-2-acetylglycerol stimulate this phosphorylation suggests that protein kinase C is being activated. In the presence of TPA, bands 4.1 and 4.9 incorporate 1.5 mol Pi/mol protein and 1.2 mol Pi/mol protein, respectively. The pattern and extent of phosphorylation shows that it is not due to cAMP-dependent protein kinases, which also phosphorylate bands 4.1 and 4.9. Ca2+-phospholipid-dependent protein kinase activity is demonstrable in the soluble fraction of erythrocytes, and has been partially purified (2200-fold) from the hemolysate by affinity chromatography (Uchida and Filburn, 1984. J. Biol. Chem. 259, 12311-12314). The affinity purified erythrocyte kinase has a 42 A Stokes' radius and phosphorylates purified bands 4.1 and 4.9 in vitro in a Ca2+- and phospholipid-dependent manner. These results show that human erythrocytes contain protein kinase C, and that band 4.1 and 4.9 are the major endogenous substrates for this kinase.     

Redox modulation of reductase and phosphatase activities in human erythrocytes

Summary We have previously reported that ferricyanide reductase activity in human erythrocytes depended on glycolysis and could be modulated by several compounds including oxidants and hormones like insulin. Insulin could activate glycolysis, probably as a consequence of tyrosine phosphorylation of protein band 3, implicating phosphorylation reactions as an important signal for activation of the reductase by insulin. Reversible phosphorylation of cellular proteins is also believed to play a key role in the action of insulin. Cytosolic acid phosphatase activity has been found in human erythrocytes. To further extend initial reports, we studied the effect of modulators on the cytosolic erythrocyte acid phosphatase. Mild oxidants like ferricyanide (1 mM), vanadate (1 mM), Mn²⁺ (0.5 and 1 mM), and phenylarsine oxide (10 and 100 M) inhibited the phosphatase activity. Similarly, insulin at concentrations that stimulate ferricyanide reduction (500, 1000 IU/ml) inhibited the activity of the phosphatase enzyme. The overall results indicated that oxidants are able to inhibit the acid phosphatase and stimulate the redox enzyme. In addition, a significant negative correlation (r = –0.400; P = 0.006) was observed between phosphatase and reductase activities. The observations discussed here, together with previous ones, emphasize that a close association between reductase and phosphatase enzymes may exist and also suggest a role for redox reactions in tyrosine phosphorylation/dephosphorylation-mediated signal transduction pathways.     

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studied using γ-³²P ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25 kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppresssed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.     

Adenosine uptake, transport, and metabolism in human erythrocytes

Using rapid kinetic techniques, we have determined the kinetics of zero-trans influx and equilibrium exchange of adenosine, and its uptake and in situ phosphorylation at 25 degrees C in human erythrocytes which were pretreated with 2'-deoxycoformycin to inhibit deamination of adenosine. Both the Km and Vmax for adenosine transport were about 300 times higher than those for the in situ phosphorylation of adenosine (Km about 0.2 microM), so that the first order rate constants for both processes were about the same. In contrast, the first order rate constant for adenosine deamination by untreated, intact cells was about 20% of that of adenosine transport or phosphorylation. These kinetic properties of the various steps, in combination with substrate inhibition of adenosine phosphorylation above 1 microM adenosine, assure that, at extracellular concentrations of physiological relevance (less than 1 microM), adenosine is very rapidly and efficiently salvaged by the erythrocytes and converted to ATP, whereas at extracellular concentrations of 10 microM or higher, practically all adenosine transported into the cells is deaminated. When the concentration of adenosine was 0.1 microM, a 10% (v/v) suspension of erythrocytes depleted the extracellular fluid of adenosine within 1 min of incubation at 25 degrees C.     

Protein kinase C phosphorylates a recently identified membrane skeleton-associated calmodulin-binding protein in human erythrocytes

A membrane skeleton-associated protein with calmodulin-binding activity recently has been purified and characterized from human erythrocytes (Gardner, K. and Bennett, V. (1986) J. Biol. Chem. 261, 1339-1348). This new protein (CaM-BP103/97) has now been identified as a major substrate for protein kinase C in erythrocytes since phosphorylation of both of its subunits (Mr = 103,000 and 97,000) is elevated 3-15-fold in the presence of the phorbol ester, 12-O-tetradecanoylphorbol beta-acetate (TPA), under the following conditions: ghost membranes incubated with protein kinase C purified from rat brain, ghost membranes from erythrocytes pretreated with TPA, and intact erythrocytes metabolically labeled with 32PO4 and stimulated by TPA. The sites of phosphorylation of this protein by exogenous and endogenous protein kinase C are identical since two-dimensional 32P-peptide maps of both subunits labeled by either endogenous or exogenous enzyme are indistinguishable. Each subunit of CaM-BP103/97 accepts up to 3 mol of phosphate/polypeptide chain. In the presence of low calcium concentrations and in the absence of cytosol, the phosphorylation of CaM-BP103/97 is, on a molar basis, equal to or greater than that of proteins 4.1 and 4.9. As a target for both calmodulin and protein kinase C, CaM-BP103/97 is likely to play a key role in the effect of calcium on erythrocyte membrane shape and stability.     

Characteristics of membrane protein phosphorylation in Plasmodium berghei-infected mouse erythrocytes

Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (32P)orthophosphate and incubating isolated membrane with (gamma-32P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with Mr 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

Evidence for the participation of cytosolic protein kinases in membrane phosphorylation in intact erythrocytes.

The effects of adenosine 3':5'-monophosphate (cyclic AMP) on the phosphorylation of membrane proteins in intact rabbit and human erythrocytes were investigated. The addition of cyclic AMP to intact human or rabbit erythrocytes results in an increase in the incorporation of ortho[32P]phosphate into several membrane protein components which are known to serve as substrates for the cyclic-AMP-dependent protein kinases. Thus this increase in protein phsophorylation is probably due to the activation of either soluble or membrane-bound cyclic-AMP-dependent protein kinases. Incubation of human erythrocytes in the presence of ortho [32P]phosphate and cyclic AMP also leads to the phosphorylation of a membrane protein component, band 7, which has not been previously detected in the autophosphorylation of isolated ghosts. Since rabbit erythrocyte membranes do not contain any cyclic-AMP-dependent protein kinase, the results suggest that cytoplasmic kinases also play a role in the phosphorylation of membrane proteins in intact cells.     

Immunochemical analysis of histone H1 and H5 from pigeon erythroid cells]

An antiserum with the antibody titer of 1 : 4096 was obtained by immunization of rabbits with the tRNA-histone H5 complex from pigeon erythrocytes. The specificity of the antiserum was studied quantitatively from the reaction of the complement binding to a homologous antigen (histone H5) and its modifications (I, II, III), differing in the degree of phosphorylation. It was shown that phosphorylation of histone H5 increases the ability of the antigen to bind to antibodies, which is especially well-pronounced at the antiserum dilutions as high as 20480. The comparison of the antigenic properties of histones H5 from pigeon and chicken erythrocytes revealed beside structural differences of the proteins the presence of common antigenic determinants. A similar observation was made when histones H5 and H1 from pigeon erythrocytes were compared. Histone H1 from chicken erythrocytes and histone H1 from calf thymus did not produce criss-cross reactions with antiserum H5.     

Effects of manganese on potassium outflow from erythrocytes and on respiration of rat liver mitochondria

In this work, effects of manganese on respiration of rat liver mitochondria and the rate of K⁺ outflow from rat erythrocytes are studied in a broad range of concentrations. It is shown that manganese ions at low concentrations (1 × 10^–7–3 × 10^–5 М) inhibit K⁺ outflow from rat erythrocytes; this can be used to prevent their lysis. At high concentrations (1 × 10^–4–1 × 10^–3 M), manganese activates K⁺ outflow from the erythrocytes but inhibits the valinomycin-induced outflow of the ion from the erythrocytes. This fact is an indication of manganese influence on physicochemical properties of membranes. At low concentrations manganese does not affect parameters of respiration and oxidative phosphorylation of rat liver mitochondria, while at high concentrations it exerts acceleration of the mitochondrial respiration, i.e., uncouples respiration from phosphorilation and, hence, inhibits ATP synthesis.     

Anomeric preference of glucose phosphorylation and glycolysis in human erythrocytes

Lactate output from the alpha and beta anomers of glucose was measured in intact human erythrocytes at 37 degrees C; and glucose anomer phosphorylation, in human erythrocyte homogenates. The rates of both glucose metabolism (lactate output) and phosphorylation were higher in the presence of beta-D-glucose as distinct from alpha-D-glucose at three glucose concentrations used (2, 5, and 10 mM). Thus, the v beta/v alpha ratios of metabolism and phosphorylation of glucose at 2 mM were 1.24 and 1.22, respectively. The results indicate that the beta preference of hexokinase, a rate-limiting enzyme in glycolysis, is reflected in beta-preferential glycolysis.     

大鼠红细胞葡萄糖代谢的异头物选择性

为研究大鼠红细胞对葡萄糖利用的异头物选择性及其作用机制,应用大鼠红细胞,对葡萄糖的两种异头物作了异构化速率、乳酸生成量、内流速度和大鼠红细胞已糖激酶作用下的磷酸化速度等进行了测定．结果指出,３７℃时大鼠红细胞的Ｄ－葡萄糖β－异头物和α－异头物代谢成乳酸的速度分别是０．２７μｍｏｌ／ｇＨｂ（３ｍｉｎ）和０．２１μｍｏｌ／ｇＨｂ（３ｍｉｎ）,即前者快于后者３０％．同时β－Ｄ－葡萄糖向红细胞内转运速度也快于后者：分别是５．０和３．５μｍｏｌ／ｇＨｂ（３ｍｉｎ）．大鼠红细胞已糖激酶的葡萄糖磷酸化速率实验结果指出：β－异头物比α－异头物快３０％;对于该两种异头物已糖激酶的Ｋｍ值均为５３μｍｏｌ／Ｌ．红细胞与α－和β－Ｄ－葡萄糖保温１ｍｉｎ后,其葡萄糖浓度均达到１ｍｍｏｌ／Ｌ左右,说明至少在１ｍｉｎ内对于已糖激酶的磷酸化此两种异头物的葡萄糖浓度均已饱和．这些结果提示,大鼠红细胞葡萄糖利用的β－异头物优选性主要与其磷酸化速度有关,而与其转运速度关系不大．     

Product-precursor relationships amongst inositol polyphosphates. Incorporation of [32P]Pi into myo-inositol 1,3,4,6-tetrakisphosphate, myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 3,4,5,6-tetrakisphosphate and myo-inositol 1,3,4,5,6-pentakisphosphate in intact avian erythrocytes.

Avian erythrocytes were incubated with myo-[3H]inositol for 6-7 h and with [32P]Pi for the final 50-90 min of this period. An acid extract was prepared from the prelabelled erythrocytes, and the specific radioactivities of the gamma-phosphate of ATP and of both the myo-inositol moieties (3H, d.p.m./nmol) and the individual phosphate groups (32P, d.p.m./nmol) of [3H]Ins[32P](1,3,4,6)P4,[3H]Ins[32P](1,3,4,5)P4, [3H]Ins[32P](3,4,5,6)P4 and [3H]Ins[32P](1,3,4,5,6)P5 were determined. The results provide direct confirmation that one of the cellular InsP4 isomers is Ins(1,3,4,5)P4 which is synthesized by sequential phosphorylation of the 1,4,5 and 3 substitution sites of the myo-Ins moiety, precisely as previously deduced [Batty, Nahorski & Irvine (1985) Biochem. J. 232, 211-215; Irvine, Letcher, Heslop & Berridge (1986) Nature (London) 320, 631-634]. This is compatible with the proposed synthetic route from PtdIns via PtdIns4P, PtdIns(4,5)P2 and Ins(1,4,5)P3. The data also suggest that, in avian erythrocytes, the principle precursor of Ins(1,3,4,5,6)P5 is Ins(3,4,5,6)P4. Furthermore, if the gamma- (and/or beta-) phosphate of ATP is the precursor of the phosphate moieties of Ins(3,4,5,6)P4, then this isomer must be derived from the phosphorylation of Ins(3,4,6)P3. If the gamma- (and/or beta-) phosphate of ATP similarly acts as the ultimate precursor to all of the phosphates of Ins(1,3,4,6)P4, then, in intact avian erythrocytes, the main precursor of Ins(1,3,4,6)P4 is Ins(1,4,6)P3. This contrasts with the expectation, based on results with cell-free systems, that Ins(1,3,4,6)P4 is synthesized by the direct phosphorylation of Ins(1,3,4)P3.     

Protein kinase C activity in erythrocytes in primary hypertension: regulation of cell shape and cation transport

Protein kinase C activity in the lysate of erythrocytes of patients with essential hypertension (EH) and spontaneously hypertensive rats (SHR) was found to be increased by 1.6-2.0 times as compared with normotensive controls. Membrane cytoskeleton alterations observed in the erythrocytes of patients with EH and SHR were revealed in decreased average erythrocyte volume, increase of cup-shaped cell formation, and increase of basal phosphorylation of band 4.9 protein. In addition, the rate of Na(+)-H+ exchange in erythrocytes of EH patients and SHR was increased by 1.9-fold. In vitro treatment of erythrocytes of healthy donors and Wistar-Kyoto rats (WKY) with protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate) leads to similar changes of cell shape, cell volume, band 4.9 protein phosphorylation and Na(+)-H+ exchange, as well as to an increase of diS-C3-(5) fluorescence. It may be assumed that alterations of these parameters revealed in primary hypertension are caused by increased activity of protein kinase C.     

The relationship of phosphorylation of membrane proteins with the osmotic fragility and filterability of Plasmodium berghei-infected mouse erythrocytes

Membrane from Plasmodium berghei-infected mouse erythrocytes showed a pattern of protein phosphorylation which was substantially altered from the normal pattern, with an increase in the phosphorylation of the protein with an apparent molecular weight of 43,000 (M 43), which increased from undetectable in uninfected cells to a maximum in the mature trophozoite stage. Phosphorylation levels of this and other minor bands were strongly correlated with osmotic fragility and filterability. The level of M 43 phosphorylation in membranes from cells which remained intact in a hypotonic medium was 3.82 +/- 0.59-times that of lysed cells, compared with the value of 0.76 +/- 0.07 calculated from distribution alone. Results found when intact erythrocytes were phosphorylated by incubation with [32P]Pi prior to partial lysis were similar to those found when membranes from the lysed and unlysed fractions were subsequently phosphorylated with [gamma-32P]ATP. Infected erythrocytes which could pass repeatedly through 3-micron polycarbonate filters had a much higher phosphorylation level for the M 43 region than whole infected cells with similar parasitemia and stage distribution. The phosphorylation change could play a role in the control of osmotic and mechanical properties of the infected erythrocytes during maturation.     

Cyclic GMP-dependent protein kinase stimulates the plasmalemmal Ca2+ pump of smooth muscle via phosphorylation of phosphatidylinositol.

The effect of phosphorylation by cyclic GMP-dependent protein kinase (G-kinase) on the activity of the plasmalemmal Ca2+-transport ATPase was studied on isolated plasma membranes and on the ATPase purified from pig erythrocytes and from the smooth muscle of pig stomach and pig aorta. Incubation with G-kinase resulted, in both smooth-muscle preparations, but not in the erythrocyte ATPase, in a higher Ca2+ affinity and in an increase in the maximal rate of Ca2+ uptake. Cyclic AMP-dependent protein kinase (A-kinase) did not exert such an effect. The stimulation of the (Ca2+ + Mg2+)-dependent ATPase activity of the purified Ca2+ pump reconstituted in liposomes depended on the phospholipid used for reconstitution. The stimulation of the (Ca2+ + Mg2+)-ATPase activity by G-kinase was only observed in the presence of phosphatidylinositol (PI). G-kinase, but not A-kinase, stimulated the phosphorylation of PI to phosphatidylinositol phosphate (PIP) in a preparation of (Ca2+ + Mg2+)-ATPase obtained by calmodulin affinity chromatography from smooth muscle, but not in a similar preparation from erythrocytes. Adenosine inhibited both the phosphorylation of PI and the stimulation of the (Ca2+ + Mg2+)-ATPase by G-kinase. In the absence of G-kinase the (Ca2+ + Mg2+)-ATPase was stimulated by the addition of PIP, but not by PI. In contrast with previous results of Furukawa & Nakamura [(1987) J. Biochem (Tokyo) 101, 287-290], no convincing evidence for a phosphorylation of the (Ca2+ + Mg2+)-ATPase was found. Evidence is presented showing that the apparent phosphorylation occurs in a contaminant protein, possibly myosin light-chain kinase. It is proposed that G-kinase stimulates the plasmalemmal Ca2+ pump of smooth-muscle cells indirectly via the phosphorylation of an associated PI kinase.