Isolated Membrane Fractions Research Articles

A novel CoA-independent transacetylase produces the ethanolamine plasmalogen and acyl analogs of platelet-activating factor (PAF) with PAF as the acetate donor in HL-60 cells.

In this study, we demonstrate the presence of a unique membrane-associated transacetylase that transfers the acetate group from platelet-activating factor (PAF) to lysoplasmalogen (in the presence of EDTA and sodium acetate) with the formation of 1-alk-1-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alk-1-enylacetyl-GPE). The identity of alk-1-enylacetyl-GPE was confirmed by acid hydrolysis, phospholipases A2 or C treatment and derivatization by fluorodinitrobenzene. The transacetylase has no requirement for Ca2+, Mg2+, or CoA and a broad pH optimum (7.0-8.0) with Km values of 12.0 microM for PAF and 106.4 microM for lysoplasmalogens. The enzyme activity from the isolated membrane fraction is not changed when whole cells are supplemented with 20:4, induced to differentiate into granulocytes, or treated with ionophore A23187. Radyllyso-sn-glycero-3-phosphocholine (GPC), radyllyso-GPE, acyllyso-sn-glycero-3-phosphoserine (GPS), acyllyso-sn-glycero-3-phosphoinositol (GPI), alkyllyso-sn-glycero-3-phosphate (GP), acyllyso-GP, or cis-9-octadecen-1-ol can also serve as acetate acceptors, whereas alkylglycerol, acylglycerol, or cholesterol are inactive. Differences in substrate acceptor specificity, sensitivity toward phenylmethylsulfonyl fluoride, and response to temperature suggest that the CoA-independent transacetylase and the CoA-independent transacylase that transfers long-chain acyl moieties are two separate enzymes. With intact differentiated HL-60 cells, [3H]acetate from [3H]PAF can be incorporated into alk-1-enylacetyl-GPE in the presence of ionophore A23187, but not in its absence. Moreover, phospholipase A2 inhibitors (p-bromophenacyl bromide and mepacrine) block the transacetylation process in whole cell system. These results indicate the production of alk-1-enyllyso-GPE is a rate-limiting factor for the subsequent transacetylation step during cell activation. We conclude that the transacetylase may participate in the biosynthesis of ethanolamine plasmalogen and acyl analogs of PAF, in vivo, fine-tuning of PAF biological responses, and cross-talk between de novo and remodeling pathways of PAF biosynthesis.

In vitro activation of dinitrogenase reductase from the cyanobacterium Anabaena variabilis (ATCC 29413).

Nitrogenase of the heterocystous cyanobacterium Anabaena variabilis was inactivated in vivo (S. Reich, H. Almon, and P. Böger, FEMS Microbiol. Lett. 34:53-56, 1986). Partially purified and modified (inactivated) dinitrogenase reductase (Fe-protein) of such cells was reactivated by isolated membrane fractions of A. variabilis or of Rhodospirillum rubrum, and acetylene reduction was measured. Reactivation requires ATP, Mg2+, and Mn2+. The activating principle is localized in the heterocyst and was found effective only when prepared from cells exhibiting active nitrogenase. It also restores the activity of modified Fe-protein from R. rubrum.

Eicosatetraynoic and arachidonic acid-induced changes in cell membrane fluidity consonant with differences in computer-aided design-structures

ETYA (5,8,11,14-eicosatetraynoic acid), a competitive analogue of arachidonic acid (AA), inhibits the proliferation of U937 (human monoblastoid) and PC3 (human prostate) cancer cells, without the overt cytotoxicity associated with AA at similar concentrations. The mechanism of inhibition is not established. ETYA at 100 μM acutely increased whole cell and isolated microsomal membrane fluidity of both cell lines to a greater extent than arachidonic acid. PC3 cells incubated with ETYA for 72 h evidenced increased membrane fluidity. This was measured by the fluorescence polarization parameter, R, using the probes TMA-DPH and DPH for whole cell and isolated membrane fractions, respectively. Compared with whole cells, isolated membranes yielded a 10–20-fold increase in fluorescence intensity. The intramolecular conformational profiles of both ETYA and AA were explored using a combination of molecular mechanics energy minimization and molecular dynamics simulation. While it is possible that not all of the low energy conformational states of either molecule were sampled, the large number of low-energy conformers determined for ETYA correspond to kink deformed conformers relative to the family of AA conformers. These kinks make the molecular cross sections of ETYA larger than AA and arise from the four alkyne bond geometries. This structural finding is consistent with ETYA's greater effect on membrane fluidity. Dissociation between the extent of change in membrane fluidity due to ETYA or AA and inhibition of DNA synthesis can suggest that either (A) increased fluidity and inhibition of DNA synthesis are independent, or as we believe more likely, (B) greater membrane fluidity evoked by ETYA is important for inhibiting DNA synthesis, while changes induced by AA are insufficient or differ qualitatively from those required to initiate and sustain these nonlethal events.

Binding sites for 1,4-dihydropyridine Ca2+-channel modulators in rat intestinal smooth muscle

The contractile response of intestinal smooth muscle to depolarization is characterized by a phasic and a tonic component which are differently sensitive to blockade by 1,4-dihydropyridines. As this difference in sensitivity could be related to different binding sites associated with distinct calcium channels, we analyzed the binding of the calciumantagonist 1,4-dihydropyridine (+)PN 200-110 [isopropyl-4-(2,1,3-benzodiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- methoxycarbonyl-pyridine-3-carboxylate] in longitudinal smooth muscle of the rat ileum. We carried out saturation binding experiments on intact tissue exposed to physiological and depolarizing (100 mmol l-1 K+) solution, and on different membrane fractions: the total microsomal fraction, the light microsomal fraction (enriched with plasma membranes) and the mitochondrial fraction. Binding of 3H(+)PN 200-110 to the intact longitudinal smooth muscle of rat ileum appeared to be voltage-dependent, KD decreased in depolarized tissue whereas Bmax was unchanged (change in membrane potential was assessed by measuring the distribution of 3H-tetraphenylphosphonium bromide). In membrane fractions two binding sites were detected, a high-affinity site associated with plasma membrane and a low-affinity site presumably associated with mitochondria (abundant in the fractions where the cytochrome c oxidase activity was high, and undetectable in the light microsomes poor in cytochrome c oxidase activity). The KD value of the high-affinity binding in isolated membrane fractions was similar to the KD value measured in intact depolarized tissue. The low affinity binding increased at high ionic strength and did not display any stereoselectivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional repression of the GLUT4 and C/EBP genes in 3T3-L1 adipocytes by tumor necrosis factor-alpha.

Fully differentiated 3T3-L1 adipocytes were chronically exposed to 5 nM tumor necrosis factor-alpha (TNF). This resulted in the development of an insulin resistance based on the inability of insulin to stimulate hexose uptake. Western blot analysis for glucose transporter protein in isolated membrane fractions indicated a total depletion of GLUT4 protein (insulin-responsive glucose transporter) in cells chronically treated with TNF. Plasma membrane content of GLUT1 protein (growth-related glucose transporter) was similar in both control and TNF-treated cells; however, the GLUT1 content of the intracellular membrane compartment had decreased markedly after TNF treatment. Continuous exposure to TNF resulted in an 85-90% decrease in the mRNA content for both GLUT4 and 422 (aP2, a lipid binding protein) genes relative to age matched controls, whereas insulin receptor mRNA levels declined by at least 50%. This was preceded by a marked decrease in mRNA accumulation for C/EBP, a transcription factor proposed to control expression of both GLUT4 and 422. The specificity of these observations was demonstrated by the lack of an effect of the chronic TNF treatment on either beta-actin or lipoprotein lipase mRNA content. The decreased content of GLUT4 and C/EBP mRNA was judged to be regulated at least in part at the level of transcription, based on the results of transcription run-on assays. Thus, the lack of response to insulin appeared due to a suppression of GLUT4 expression as well as a decreased intracellular content of GLUT1.

Energy dependence of O-antigen synthesis in Salmonella typhimurium

The uncoupler 2,4-dinitrophenol prevents in vivo synthesis of O antigen in Salmonella typhimurium by inhibiting the first reaction of the pathway, formation of galactosyl-pyrophosphoryl-undecaprenol. Inhibition was observed only in intact cells; dinitrophenol had no effect on activity of the synthase enzyme in isolated membrane fractions. In vivo inhibition could not be explained by changes in intracellular nucleotide pools or a shift in the equilibrium of the reaction and appeared to be specific for the first step in the pathway. Neither the subsequent mannosyl transferase, which catalyzes formation of the trisaccharide-lipid intermediate, mannosyl-rhamnosyl-galactosyl-pyrophosphoryl-undecaprenol, nor O-antigen polymerase was inhibited. In addition, incorporation of galactose into core lipopolysaccharide was only modestly inhibited under conditions in which O-antigen synthesis was abolished. The results suggest that maintenance of proton motive force is required for access of substrate, UDP-galactose and/or undecaprenyl phosphate, to the active site of the galactosyl-pyrophosphoryl-undecaprenol synthase enzyme.

Normalization of Na+-K+-ATPase Activity in Isolated Membrane Fraction From Sciatic Nerves of Streptozocin-Induced Diabetic Rats by Dietary myo-Inositol Supplementation in Vivo or Protein Kinase C Agonists in Vitro

A myo-inositol-related defect in nerve Na(+)-K(+)-ATPase in experimental diabetes has been invoked in the pathogenesis of diabetic neuropathy, but the mechanism linking altered myo-inositol metabolism and Na(+)-K(+)-ATPase regulation in diabetic nerve is uncertain. Decreased Na(+)-K(+)-ATPase in diabetic rat nerve is normalized by aldose reductase inhibitors or dietary myo-inositol, which preserve normal nerve myo-inositol content in vivo. Decreased Na(+)-K(+)-ATPase in diabetic rabbit nerve is acutely reversed by exposure to protein kinase C agonists in vitro. This study explored the relationship between the myo-inositol-sensitive and protein kinase C-agonist-sensitive Na(+)-K(+)-ATPase defects in diabetic rat nerve. Ouabain-sensitive ATPase activity was measured in an enriched membrane fraction isolated from nondiabetic, streptozocin-induced diabetic, and myo-inositol-supplemented streptozocin-induced diabetic rats before and after the membranes were exposed to protein kinase C agonists in vitro. The decreased ouabain-sensitive ATPase activity in plasma membranes from untreated diabetic rats was increased after exposure to two structurally unrelated protein kinase C agonists; the normal ouabain-sensitive ATPase in plasma membranes from myo-inositol-supplemented diabetic rats was unaffected by protein kinase C agonists. The nonadditivity and implied equivalence of the Na(+)-K(+)-ATPase defect corrected by myo-inositol in vivo and by protein kinase C agonists in vitro are consistent with the postulated existence of a deficient myo-inositol-dependent phospholipid-derived protein kinase C agonist (presumably diacylglycerol) in diabetic nerve that regulates nerve Na(+)-K(+)-ATPase either directly or via a protein kinase C mechanism.

Evidence for energy-dependent transposition of core lipopolysaccharide across the inner membrane of Salmonella typhimurium

The uncoupler 2,4-dinitrophenol blocks the final step of lipopolysaccharide assembly--transfer of O antigen from undecaprenyl pyrophosphate to core lipopolysaccharide--in intact Salmonella typhimurium but not in isolated membrane fractions. The O-antigen ligase enzyme is not inhibited by dinitrophenol in vitro, and core lipopolysaccharide synthesized in the presence of uncoupler in vivo is functional as acceptor of O antigen in vitro. The evidence strongly suggests that maintenance of proton motive force is required for transmembrane transposition of core lipopolysaccharide to the active site of O-antigen ligase at the periplasmic face of the inner membrane.

Normalization of Na(+)-K(+)-ATPase activity in isolated membrane fraction from sciatic nerves of streptozocin-induced diabetic rats by dietary myo-inositol supplementation in vivo or protein kinase C agonists in vitro

Normalization of Na(+)-K(+)-ATPase activity in isolated membrane fraction from sciatic nerves of streptozocin-induced diabetic rats by dietary myo-inositol supplementation in vivo or protein kinase C agonists in vitro

Metabolism of platelet-activating factor in human haematopoietic cell lines. Differences between myeloid and lymphoid cells

The binding and metabolism of platelet-activating factor (PAF) was studied in human cell lines resembling myeloid cells (HL60 and U937) and B and T lymphocytes (Daudi and Jurkat). All of the cell lines were found to bind and catabolize exogenous [3H]PAF in a time- and temperature-dependent manner. PAF binding could also be demonstrated in isolated membrane fractions, which provides further evidence of the existence of true membrane receptors. Myeloid cell lines contained numbers of receptors at least 10-fold higher than in lymphoid cell lines. Biosynthesis of PAF upon challenge by ionophore A23187 could be demonstrated in HL60 and U937 cells. In contrast, lymphoid cell lines were unable to produce PAF. Incubation with [14C]acetate showed incorporation of the label into three main fractions: neutral lipids, phosphatidylcholine and PAF, but the distribution of the label varied depending on the cell line. Significant incorporation into phosphatidylcholine was observed in uninduced myeloid cell lines. A phospholipase A2 acting on 1-O-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine and an acetyl-CoA:lyso-PAF acetyltransferase were expressed in the HL60 cell line and showed variations in specific activity with granulocytic differentiation. In contrast, these enzyme activities were not expressed in Daudi and Jurkat cell lines. These data indicate (1) the occurrence of PAF binding and catabolism in both myeloid and lymphoid cell lines; (2) the restriction of PAF biosynthesis to myeloid cell lines, especially HL60 cells; (3) the occurrence of differentiation-elicited changes in the specific activities of the enzymes involved in PAF biosynthesis by the remodelling pathway; and (4) the central role played by the disposal of lyso-PAF, a product of the phospholipase A2 reaction, in PAF biosynthesis.

Evidence that Human Chorionic Gonadotropin/Luteinizing Hormone Receptor Down-Regulation Involves Decreased Levels of Receptor Messenger Ribonucleic Acid*

Injection of pseudopregnant rats with pharmacological doses of hCG leads to a characteristic decrease in LH/hCG binding by the isolated luteal cells. The steady state levels of LH/hCG receptor mRNA were determined in rat ovaries during hCG-induced down-regulation of the receptor. Northern blots were performed using a 20-mer probe corresponding to a guanine cytosine-rich carboxyl-terminal untranslated region of the LH/hCG receptor cDNA. The hybridization of the probe to LH/hCG receptor mRNA was highly specific, since the probe hybridized only to rat luteal cell RNA fraction, with no signal detected in nontarget tissues. The LH/hCG receptor level was quantitated by [125I]hCG binding to the isolated membrane fractions from the corresponding treatment and control groups. Examination of mRNA levels of the receptor during hCG-induced down-regulation showed a steady decrease from 0-24 h, followed by a gradual increase to control levels from 24-72 h corresponding to days 8-9 of pseudopregnancy. The [125I]hCG-binding activity during down-regulation paralleled the mRNA profile in both the experimental and control groups. Examination of the levels of mRNA for alpha-actin showed no change during this period, suggesting that the loss of LH/hCG receptor mRNA at 24 h was not due to a general loss of mRNA in luteal cells. These results suggest that hCG-induced down-regulation of the LH/hCG receptor in luteal cells involves regulation of the receptors at the message level.

A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction.

The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.

Localization of penicillin-binding protein 1b in Escherichia coli: immunoelectron microscopy and immunotransfer studies

We report the localization of penicillin-binding protein 1b (PBP 1b) in Escherichia coli KN126 and in an overproducing construct containing plasmid pHK231. We used PBP 1b-specific antiserum for the immunoelectron microscopy of ultrathin sections of whole cells and for immunoelectrophoresis of cytoplasm and isolated membrane fractions. We studied ultrathin sections of both glutaraldehyde-fixed cells that had been embedded after progressively lowering the temperature and cryofixed cells that had been freeze-substituted in Lowicryl K4M and HM20. Most of the PBP 1b-specific label was observed in the inner membrane (IM) and the adjacent cytoplasm, much less was observed in the outer membrane (OM); appreciable amounts were also seen in the bulk cytoplasm. Distribution and intensity of label were both temperature dependent: temperature shift-up to 37 degrees C, causing PBP 1b overproduction in the construct, showed a statistically highly significant increase in label of the IM, including a cytoplasmic zone (of at least 30 nm in depth) adjacent to the IM, a zone we termed the membrane-associated area. Concomitant with the temperature shift-up, a decrease in label density was observed in the bulk cytoplasm. Increased label was also found in IM-OM contact areas (zones of membrane adhesion). The periplasm did not show significant label. Western blotting (immunoblotting) revealed PBP 1b in most of the isolated membrane fractions; however, the highest label density was found in membrane fractions of intermediate density, supporting the suggestion of an increased concentration of PBP 1b in the membrane adhesion zones. In summarizing, we propose that PBP 1b is present in the membrane-associated area of the cytoplasm, from where proteins (such as PBP 1b or thioredoxin) gain access to their specific insertion sites in the envelope. The use of several methods of immunoelectron microscopy provided the first unequivocal evidence for localization of PBP 1b at membrane adhesion sites. Since such sites are specifically labeled with anti-PBP 1b serum, we hypothesize that they contain parts of the machinery for assembly and growth of the murein layer.

Advantages of the use of intact plant tissues in freeze-fracture electron microscopy.

The use of whole, intact plant tissue for freeze-fracture electron microscopy provides important information that cannot be obtained from the use of isolated biological membranes or of artificial (phospholipid) membrane preparations. This is not to imply that these examinations of such preparations are not useful, since it would be difficult to interpret our observations of intact cells and tissues without the analysis of these model systems. Analysis of intact tissue and cells reveals the relative densities of membrane proteins of the different membranes within a cell; the three-dimensional organization of various organelles, especially the endoplasmic reticulum; changes in intramembranous particle (IMP) distribution due to stress or injury; and, in conjunction with the use of filipin, membrane sterol content and relative distribution. It is our intention that this survey of freeze-fracture images of intact plant tissues will illustrate the uniqueness of the information gained from an analysis of whole plant tissues compared to isolated membrane fractions.

Immunocytochemical Localization of the Vacuolar H+-ATPase in Maize Root Tip Cells

The vacuolar H(+)-ATPase of maize (Zea mays L.) root tip cells has been localized at the EM level using rabbit polyclonal antibodies to the 69 kilodalton subunit and protein A-colloidal gold. Intracellular gold particles were detected mainly on the tonoplast and Golgi membranes. Only about 27% of the vacuoles were labeled above background. The absence of gold particles on the majority of vacuoles suggests either that the tonoplast H(+)-ATPase is degraded during tissue preparation or that the small vacuoles of root tip cells are specialized with respect to H(+)-ATP ase activity. The pattern of gold particles on the labeled vacuoles ranged from uniform to patchy. Virtually all of the Golgi bodies were labeled by the antibody, but the particle densities were too low to determine whether the H(+)-ATPase was associated with specific regions, such as the trans-face. Cell wall-labeling was also observed which could be partially prevented by the inclusion of gelatin as a blocking agent. The immunocytochemical results confirm previous biochemical studies with isolated membrane fractions (A Chanson, L Taiz 1985 Plant Physiol 78: 232-240).

Na+ pump in renal tubular cells is regulated by endogenous Na+-K+-ATPase inhibitor from hypothalamus.

Bovine hypothalamus contains a high affinity, specific, reversible inhibitor of mammalian Na+-K+-ATPase. Kinetic analysis using isolated membrane fractions showed binding and dissociation rates of the hypothalamic factor (HF) to be (like ouabain) relatively long (off rate = 60 min). To determine whether the kinetics of inhibition in intact cells might be more consistent with regulation of physiological processes in vivo, binding and dissociation reactions of HF in intact renal epithelial cells (LLC-PK1) were studied using 86Rb+ uptake and [3H]ouabain binding. As with membranes, a 60-min incubation with HF inhibited Na+-K+-ATPase in LLC-PK1 cells. In contrast to membrane studies, no prolonged incubation with LLC-PK1 was needed to observe inhibition of Na+-K+-ATPase. HF caused a 33% inhibition of ouabain-sensitive 86Rb+ influx within 10 min. Incubation of cells with HF followed by washout showed rapid reversal of pump inhibition and a doubling of pump activity. The dose-response curve for HF inhibition of LLC-PK1 86Rb+ uptake showed a sigmoidal shape consistent with an allosteric binding reaction. Thus HF is a potent regulator of Na+-K+-ATPase activity in intact renal cells, with binding and dissociation reactions consistent with relevant physiological processes.

Oxidation-induced increase in activity of angiotensin converting enzyme in the rat kidney

The activity of angiotensin converting enzyme(ACE) in crude extracts of the rat renal cortex was increased when the oxidizing agent diamide was added to the extract. The maximal activity was obtained at concentrations over 1 mM, and the value was twice or more the activity in the absence of the pretreatment. The activity of ACE was also increased by the diamide-pretreatment of the isolated membrane fraction of the renal cortex, thereby indicating that the increase in activity was not due to oxidation of endogenous glutathione (GSH) that may lower the ACE activity, but rather that ACE itself was oxidized. When O2 was included in the extract for 2 h, the ACE activity also increased to about twice the original activity. Lineweaver-Burk plots analysis demonstrated that, after oxidation with diamide and O2, the Vmax was increased but the Km remained unchanged. We conclude that the action of ACE in the kidney functions may differ in relation to oxidation of the tissue.

Lectin binding sites of the sarcolemma and the surrounding interstitial tissue

Binding properties of lectins to the membrane of isolated adult human and rat cardiocytes and to the outer layers of the sarcolemma and the interstitial tissue were investigated by indirect immunofluorescence studies. Specific binding sites were determined with isolated membrane fractions from human and rat myocardium by Western blot analysis.

The major metabolite of doxorubicin is a potent inhibitor of membrane-associated ion pumps. A correlative study of cardiac muscle with isolated membrane fractions.

Doxorubicin (adriamycin) is a highly effective cancer chemotherapeutic drug but its clinical utility is limited by its cardiotoxicity. Doxorubicinol, the major metabolite of doxorubicin, is up to 10 times more potent than doxorubicin at inhibiting isometric contraction of the papillary muscle isolated from the right ventricle of rabbit heart. Doxorubicinol also increases resting tension of isolated cardiac muscle indicative of incomplete relaxation between contractions, a characteristic of doxorubicinol but not of doxorubicin. This study assesses the effect(s) of doxorubicinol on a variety of ion pumps which may explain, in part, the action of the metabolite in the intact muscle. We find the doxorubicinol is a potent inhibitor (IC50 less than 5 micrograms/ml) of calcium-stimulated ATPase activity of sarcoplasmic reticulum from canine heart and rabbit skeletal muscle. At comparable levels, doxorubicinol is also a potent inhibitor of (Na + K)-ATPase of cardiac sarcolemma and the Mg-dependent ATPase activity referable to the F0F1 proton pump of mitochondria. For each of these ion pumps, doxorubicinol is at least 80 times more potent an inhibitor than doxorubicin. Doxorubicinol, between 10 and 50 micrograms/ml, increases resting tension up to 4-fold in isolated papillary muscles cyclically contracting at 30 times/min. Resting stress is relatively insensitive to doxorubicin. Thus, doxorubicinol is a potent inhibitor of several key cationic pumps that directly or indirectly regulate cell calcium and inhibits relaxation in the isolated fiber preparation. These observations add a new dimension to understanding the cardiotoxicity of doxorubicin.

The Role of Microsomal ATPase Activity in Light-induced Protoplast Swelling in Wheat

A microsomal ATPase activity isolated from etiolated wheat leaf protoplasts required Mg 2+ and was stimulated by K + . The activity showed a pH optimum of about 6.5 with Mg 2+ , while the K + -stimulated activity was optimal at pH 5.5–6.0. The ATPase was inhibited by 50 μM vanadate. Vanadate inhibited the swelling response of protoplasts to red light, and also the reduction in size following transfer of protoplasts from 0.5 M to 0.6 M sorbitol. However no consistent effects of red light on microsomal ATPase could be detected, either when light treatment was applied to the protoplasts before membrane isolation or to the isolated membrane fraction.