Ca2+-free calmodulin and calmodulin damaged by in vitro aging are selectively degraded by 26 S proteasomes without ubiquitination.

The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca(2+)-binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca(2+)). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca(2+) stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca(2+)-free (apo) CaM without ubiquitination. Addition of Ca(2+) dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca(2+) binding, native CaM assumes a non-degradable conformation, which most of the age-damaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.

Dynamic changes in sarcoplasmic reticulum function in cardiac hypertrophy and failure.

Previous studies have demonstrated that cardiac function changes with development of pressure overload-induced hypertrophy. The present study was undertaken to discover the basis for the changes in sarcoplasmic reticulum (SR) functions: uptake, (as related to the SR Ca2+ pump properties) and release in isolated, perfused hypertrophied rat hearts. Our results demonstrated significant prolongation of the time-to-90%-relaxation, both during the period of compensation (8 weeks after banding the ascending aorta, group HR1), when systolic function was preserved, and later with progressive hypertrophy (20 weeks after banding, group HR2) and contractile failure (20-22 weeks after banding, group F). The initial rates of the oxalate-supported SR Ca2+ uptake and the maximum transport rate (Vmax) of the SR Ca2+ pump, measured in the left ventricular homogenates, during blockade of the SR Ca2+ release channels with ruthenium red, were preserved in group HR1. To correlate early relaxation abnormalities with SR function, the [Ca2+] required for half-maximal pump activation (EC50) was examined and increased significantly in HRI vs. Sham1 (0.95+/-0.06 vs. 0.81+/-0.04 microM, P<0.05) indicating that the affinity of the SR Ca2+ pump for Ca2+ was reduced. The same tendency was demonstrated in groups HR2 (0.94+/-0.06 vs. 0.79+/-0.05) and F (0.89+/-0.05 vs. 0.78+/-0.05). In addition, with progression of hypertrophy we observed a significant decline in the amount of SR Ca2+ pump, as assessed by the Vmax, from 31.22+/-1.20 (Sham2) to 26.47+/-1.58 HR2) nmol/mg protein per min (P<0.05), and from 33.81+/-1.23 (Sham3) to 25.15+/-1.57 (F) nmol/mg protein per min, (P<0.01). This decrease was accompanied by a parallel reduction in the number of SR Ca2+ release channels by 14% (HR2) and 23% (F), as determined by maximum [3H] ryanodine binding (Bmax). These results suggest that pressure overload-induced changes in SR Ca2+ uptake (as reflected by Vmax and EC50) and SR Ca2+ release (as reflected by Bmax), both leading to diminished Ca2+ sequestration, may contribute to impaired cardiac relaxation with compensatory hypertrophy and failure.

Altered phosphorylation of sarcoplasmic reticulum contributes to the diminished contractile response to isoproterenol in hypertrophied rat hearts.

We tested the hypothesis that changes in phosphorylation of the sarcoplasmic reticulum (SR) protein, phospholamban (PIB) and myofibrillar proteins troponin I (TnI) and C protein are responsible for the decreased relaxant response to isoproterenol in cardiac hypertrophy and failure induced by ascending aortic banding in rats. In isolated perfused heart preparations under maximal isoproterenol stimulation, the capacity for in vitro cAMP-dependent phosphorylation of PIB was significantly increased at the compensatory stage of hypertrophy (126-130%, P<0.001), but decreased with failure (70-76%, P<0.001). Phosphorylation of TnI also decreased in the failing hearts, however to a lesser extent (80-83%, P<0.05). No significant hypertrophy-related difference was evident in isoproterenol-induced phosphorylation of C protein. The relative tissue level of PIB was increased (150-168%, P<0.001) in hypertrophied and decreased (71-83.8%, P<0.05) in failing hearts compared with the respective age-matched sham-operated controls (100%). As a percentage above baseline, the maximal isoproterenol-induced increase in the EC50 of the SR Ca2+ pump in response to phosphorylation of PIB was 38.5+/-1.1% for sham-operated rats, and 26.0+/-3.8% and 15.4+/-4.2% for hypertrophied and failing hearts respectively. As a consequence, linear correlation was observed between the maximal increase in EC50 and the maximal rate of relaxation [(-dP/dt)/DevP] upon isoproterenol stimulation, declining with progressive hypertrophy to failure. These data suggest that hypertrophy-induced alterations in PIB phosphorylation and protein level contribute to the diminished relaxant response of the hypertrophied and failing heart to adrenergic agonists.

Carboxyl methylation of deamidated calmodulin increases its stability in Xenopus oocyte cytoplasm. Implications for protein repair.

The widely distributed protein-L-isoaspartate(D-aspartate) O-methyltransferase (PIMT; EC 2.1.1.77) is postulated to play a role in the repair or metabolism of damaged cellular proteins containing L-isoaspartyl residues derived primarily from the spontaneous deamidation of protein asparaginyl residues. To evaluate the functional consequence of PIMT-catalyzed methylation on the stability of isoaspartyl-containing proteins in cells, Xenopus laevis oocytes were microinjected with both deamidated and nondeamidated forms of recombinant chicken calmodulin (CaM) containing a hemagglutinin (HA) epitope at its N terminus. Processing of HA-CaM was monitored by electrophoretic analysis and Western blotting of oocyte extracts. The experiments indicate that deamidated HA-CaM is degraded after microinjection, while nondeamidated HA-CaM is stable. Kinetic analysis is consistent with the entry of microinjected HA-CaM into two intracellular pools with distinct hydrolytic stabilities. The larger, more stable pool may consist of HA-CaM bound to the heterogeneous pool of oocyte CaM binding proteins detected by an overlay procedure. Enzymatic methylation of deamidated HA-CaM with purified PIMT prior to injection results in its stabilization. Conversely, inhibition of endogenous oocyte PIMT with sinefungin, a nonhydrolyzable analog of S-adenosylhomocysteine, increases the rate of deamidated HA-CaM degradation. These results are consistent with a role for PIMT-catalyzed methylation in the repair of damaged cellular proteins.

Inhibitory effect of free radicals derived from organic hydroperoxide on progesterone synthesis in human term placental mitochondria.

Different natural and synthetic organic hydroperoxides have been found to stimulate TBARS formation in human term placental mitochondria. The levels of TBARS were lower than arising from NADPH-dependent lipid peroxidation. BHT, Mn2+ and DMPO counteracted TBARS formation in the presence of cumene hydroperoxide implicating involvement of free radicals in this process. On the other hand superoxide dismutase, catalase and EDTA while being inhibitory in NADPH-dependent lipid peroxidation did not inhibit cumene hydroperoxide-dependent TBARS formation. Amphenone B and SKF-525A, inhibitors of cytochrome P-450, strongly inhibit both NADPH- and cumene hydroperoxide-dependent lipid peroxidation. These data provide evidence that cytochrome P-450SCC is involved in both these processes. However NADPH-dependent lipid peroxidation and the cumene hydroperoxide have been found to inactivate placental mitochondrial cytochrome P-450SCC. The presence of cumene hydroperoxide resulted in a more rapid inactivation of cytochrome P-450SCC and consequently inhibited NADPH-dependent lipid peroxidation. It has been observed for the first time that progesterone biosynthesis can be inhibited by cumene hydroperoxide. Protective effect of Mn2+ and DMPO on progesterone biosynthesis indicates the importance of free radicals as transient products of cytochrome P-450SCC-dependent cumene hydroperoxide metabolism. In contrast to progesterone formation from cholesterol, the conversion of pregnenolone to progesterone was not affected by cumene hydroperoxide. This suggests that inhibition of progesterone synthesis from cholesterol by hydroperoxide may be ascribed to its effect on the desmolase activity of cytochrome P-450SCC in placental mitochondria. On the basis of the results obtained, we propose that the inhibition of progesterone biosynthesis by naturally occurring hydroperoxides may contribute to the development of preeclampsia.

Construction of an epitope-tagged calmodulin useful for the analysis of calmodulin-binding proteins: addition of a hemagglutinin epitope does not affect calmodulin-dependent activation of smooth muscle myosin light chain kinase.

An epitope-tagged calmodulin (CaM), capable of interacting with CaM-binding proteins in cellular extracts, would be a valuable tool for identifying proteins in signal transduction pathways involving calcium. A bacterial overexpression vector for epitope-tagged CaM has been constructed by inserting the coding sequence for a nine amino acid portion of the influenza virus hemagglutinin (HA) protein into the initiation site of an overexpression vector for chicken CaM. The HA-CaM fusion produced in bacteria was compared to native CaM for its ability to activate smooth muscle myosin light chain kinase (MLCK), one of the best understood CaM-dependent enzymes. MLCK activity was tested in both a purified system and a CaM-depleted "native actomyosin" preparation maintaining many of the regulatory properties of the intact smooth muscle. HA-CaM behaves identically to unmodified CaM in both systems, indicating that the HA epitope does not adversely affect CaM function. The recombinant HA-CaM was used to sensitively detect CaM interactions with smooth muscle proteins in a modified gel overlay assay, using a monoclonal antibody against the HA epitope as the secondary reagent. Enzymatically active complexes of HA-CaM and MLCK could be immunoprecipitated from actomyosin preparations using the same monoclonal antibody and protein G-Sepharose beads.

Helicobacter pylori in kidney allograft recipients: high prevalence of colonization and low incidence of active inflammatory lesions.

Since kidney transplant recipients are at enhanced risk for developing severe upper gastrointestinal disease and Helicobacter pylori (Hp) is an important pathogen in active gastritis and peptic ulcer, we performed gastroduodenoscopic examination, coupled with assessment of Hp colonization in 29 renal allograft recipients complaining of recurrent dyspepsia. Results were compared with those of 25 chronically hemodialyzed patients and 16 subjects free from renal disease, also suffering from upper gastrointestinal symptoms of similar severity. We found that while transplant recipients have had a high prevalence of Hp infection (62 vs. 34.6% in dialysis and 43.6% in control dyspeptic patients), active gastritis was clearly less frequently seen in these patients than in control subjects (transplant group: 6.9%, dialysis 3.8%, control 31.3%) and peptic ulceration was totally absent. Prevalence of Hp colonization was even higher in renal graft recipients on triple posttransplant immunosuppression (82%). In dyspeptic transplant and dialysis patients, colonization with Hp did not account for development of active inflammatory lesions, an association frequently seen in subjects free from renal disease and immunosuppressive therapy.

Validation of different methods to compare isovolumic cardiac function in isolated hearts of varying sizes.

Functional comparison of isolated hearts with different sizes has been difficult because function varies at different ventricular volumes. To date, no standard volume has been established. To determine the most accurate experimental approach, we tested five different methods to standardize volume in control hearts with different sizes but similar papillary muscle function and in hearts with concentric hypertrophy: intracardiac balloon volume (VB) = 120 microliters (M1). VB at diastolic pressure = 10 mmHg (M2) or diastolic wall stress = 4 kdyn/cm2 (M3), V1 = 25 microliters/100 g body weight (M4) and VB = 50% of volume at peak developed pressure (Vmax; M5). Systolic and diastolic functions of control groups were different using M1 and comparable using M2 or M5. M3 and M4 showed borderline significant differences. We concluded that M5 and M2 were suitable to compare function among hearts of different sizes. If diastolic compliance is of interest, as in concentric hypertrophy, parameter-volume curves should be normalized by Vmax to compare function at corresponding points of the Frank-Starling curve (e.g., at 50% of Vmax, M5).

Effects of dantrolene sodium on intracellular Ca2(+)-handling in normal and Ca2(+)-overloaded cardiac muscle.

We investigated the effects of dantrolene sodium on intracellular Ca2+ homeostasis in normal and Ca2+ overloaded rat cardiac muscle. In isometrically contracting rat papillary muscles loaded with the Ca2+ indicator aequorin, dantrolene (50 microM) produced a mild negative inotropic effect (28 +/- 1.8 to 21 +/- 1.1 mN/mm2; mean +/- S.E.; n = 6; P < 0.01), which was paralleled by a decrease in peak systolic [Ca2+]i (0.81 +/- 0.04 to 0.67 +/- 0.04 microM; P < 0.01). In isolated cardiac sarcoplasmic reticulum, dantrolene (50 microM) increased the initial Ca2+ uptake rate by 23% as compared to control preparations (at pCa 6.2: 46.9 +/- 1.6 to 61.1 +/- 2.2 nmol/mg per min; n = 4; P < 0.001). Intracellular Ca2+ overload was provoked in isoproterenol-pretreated (100 microM) preparations with [Ca2+]o = 5.0 mM at a stimulation rate of 1.0 Hz (n = 12). Diastolic Ca2+ oscillations and aftercontractions increased mean diastolic [Ca2+]i (0.33 +/- 0.1 to 0.56 +/- 0.1 microM) and tension (9.5 +/- 1.8 to 15.3 +/- 2.1 mN/mm2), respectively. Addition of dantrolene (50 microM) reduced the amplitude of Ca2+ oscillations and aftercontractions; mean diastolic [Ca2+]i decreased to 0.44 +/- 0.1 microM and diastolic tension to 13.5 +/- 2.2 mN/mm2. We conclude, therefore, that dantrolene sodium modifies Ca2+ handling by the myocardial sarcoplasmic reticulum, an effect that might be useful in cardiac disorders with impaired [Ca2+]i homeostasis.

Alterations in sarcoplasmic reticulum calcium uptake, relaxation parameters and their responses to beta-adrenergic agonists in the developing rabbit heart.

Developmental changes in cardiac sarcoplasmic reticulum function, which may reflect alterations in the myocardial rate of relaxation and its responses to beta-adrenergic stimulation, were assessed using fetal, 4-day-old, 21-day-old and adult rabbit hearts. The fetal hearts exhibited the slowest rate of relaxation (-dP/dt) and the lowest Vmax and EC50 of the sarcoplasmic reticulum Ca(2+)-pump for Ca2+ compared to the other age groups. These parameters were similar among the 4-day-old, 21-day-old and adult hearts. The low physiological and biochemical parameters in the fetal hearts reflected reduced levels of expression of the sarcoplasmic reticulum Ca(2+)-pump and its inhibitor, phospholamban, assessed by quantitative immunoblotting. Isoproterenol perfusion of fetal hearts had no significant effect on their relaxation parameters or on the EC50 of the Ca(2+)-pump for Ca2+, consistent with the low relative levels of phospholamban expressed in these hearts. However, perfusion of the 4-day-old, 21-day-old and adult hearts with isoproterenol resulted in significant increases in the rates of relaxation of each group. The increases in relaxation parameters were associated with decreases in the EC50 of the cardiac sarcoplasmic reticulum Ca(2+)-pump for Ca2+, suggesting a phosphorylation-mediated relief of the phospholamban inhibitory effects. These findings indicate that developmental regulation of the levels of the activity of the cardiac sarcoplasmic reticulum Ca(2+)-pump may reflect alterations in cardiac relaxation parameters and their modulation by beta-adrenergic agonists.

Regulation of the skeletal sarcoplasmic reticulum Ca(2+)-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles.

The Ca(2+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca(2+)-uptake and Ca(2+)-ATPase activities were significantly increased (2-3 fold). The highest activation was obtained at a 50:50 molar ratio of PS:PC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca(2+)-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca(2+)-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca(2+)-ATPase were similar in either PC or PC:PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca(2+)-ATPase. Regulation of the Ca(2+)-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1-25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca(2+)-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.

Effect of hyper- and hypothyroidism on phospholipid fatty acid composition and phospholipases activity in sarcolemma of rabbit cardiac muscle.

Lipid content and composition of fatty acids esterified to phospholipids of cardiac sarcolemma isolated from hyperthyroid, hypothyroid and control rabbits were analysed. Hyperthyroidism resulted in a significant reduction of the cholesterol to phospholipid molar ratio as compared to control animals, while hypothyroidism exerted the opposite effect. Complex changes in composition of phospholipid fatty acids observed in hyperthyroid state led to an elevation of the fatty acid unsaturation index over the control value. The unsaturation index value was, however, not affected in the hypothyroid state. Thyroxine hormone administration increased phospholipase A1 and decreased phospholipase A2 activity. The opposite effect was observed in thyreodectomized animals. The effect of changes in sarcolemmal bulk phospholipids upon thyroxine administration or deficiency on regulation of activity of membrane-bound enzymes is discussed.

Reconstitution of the skeletal sarcoplasmic reticulum Ca2(+)-pump: influence of negatively charged phospholipids.

The Ca2(+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in the presence of phosphatidyl choline using the freeze-thaw sonication technique. The effect of incorporation of negatively charged phospholipids, phosphatidylserine and phosphatidylinositol phosphate, into the phosphatidylcholine proteoliposomes was investigated. Various ratios of phosphatidylserine or phosphatidylinositol phosphate to phosphatidylcholine were used, while the total amount of phospholipid in the reconstituted vesicles was kept constant. Enrichment of phosphatidylcholine proteoliposomes by phosphatidylserine or phosphatidylinositol phosphate was associated with activation of Ca2(+)-uptake and Ca2(+)-ATPase activities. The highest activation was obtained at a 50:50 molar ratio of phosphatidylserine:phosphatidylcholine and at a 10:90 molar ratio of phosphatidylinositol phosphate:phosphatidylcholine. The initial rates of Ca2(+)-uptake obtained at 1 microM Ca2+ were 2.6 +/- 0.1 mumol/min per mg of phosphatidylserine:phosphatidylcholine proteoliposomes and 1.5 +/- 0.1 mumol/min per mg of phosphatidylinositol phosphate:phosphatidylcholine proteoliposomes, compared to 0.9 +/- 0.05 mumol/min per mg of phosphatidylcholine proteoliposomes. These findings suggest that negatively charged phospholipids may be involved in the activation of the reconstituted skeletal muscle sarcoplasmic reticulum Ca2(+)-pump.

The role of phospholamban in the regulation of calcium transport by cardiac sarcoplasmic reticulum.

The calcium transport mechanism of cardiac sarcoplasmic reticulum (SR) is (SR) is regulated by a phosphoregulatory mechanism involving the phosphorylation-dephosphorylation of an integral membrane component, termed phospholamban. Phospholamban, a 27,000 Da proteolipid, contains phosphorylation sites for three independent protein kinases: 1) cAMP-dependent, 2) Ca2(+)-calmodulin-dependent, and 3) Ca2(+)-phospholipid-dependent. Phosphorylation of phospholamban by any one of these kinases is associated with stimulation of the calcium transport rates in isolated SR vesicles. Dephosphorylation of phosphorylated phospholamban results in the reversal of the stimulatory effects produced by the protein kinases. Studies conducted on perfused hearts have shown that during exposure to beta-adrenergic agents, a good correlation exists between the in situ phosphorylation of phospholamban and the relaxation of the left ventricle. Phosphorylation of phospholamban in situ is associated with stimulation of calcium transport rates by cardiac SR, similar to in vitro findings. Removal of beta-adrenergic agents results in the reversal of the inotropic response and this is associated with dephosphorylation of phospholamban. These findings indicate that a phospho-regulatory mechanism involving phospholamban may provide at least one of the controls for regulation of the contractile properties of the myocardium.

Regulation of the skeletal sarcoplasmic reticulum Ca2+ pump by phospholamban in reconstituted phospholipid vesicles.

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR). The mechanism of regulation appears to involve inhibition by dephosphorylated phospholamban, and phosphorylation may relieve this inhibition. Fast-twitch skeletal muscle SR does not contain phospholamban, and it is not known whether the Ca(2+)-ATPase isoform from this muscle may be also subject to regulation by phospholamban in a similar manner as the cardiac isoform. To determine this we reconstituted the skeletal isoform of the SR Ca(2+)-ATPase with phospholamban in phosphatidylcholine proteoliposomes. Inclusion of phospholamban was associated with significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0, and phosphorylation of phospholamban by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects on the Ca2+ pump. Similar effects of phospholamban were also observed using phosphatidylcholine:phosphatidylserine proteoliposomes, in which the Ca2+ pump was activated by the negatively charged phospholipids (24). Regulation of the Ca(2+)-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by cross-linking experiments, using a synthetic peptide that corresponded to amino acids 1-25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca(2+)-ATPase may be also regulated by phospholamban, although this regulator is not expressed in fast-twitch skeletal muscles.

The membrane lipid and fatty acid composition of erythrocyte ghosts from three patients with paramyotonia congenita.

The membrane lipid and fatty acid composition of red blood cell ghosts from three paramyotonia patients was investigated. Cholesterol and total phospholipid contents were not different from the controls, but the sphingomyelin content was reduced, and this was compensated for by an increase in phosphatidylcholine. Thus, the molar ratios of phosphatidylcholine/sphingomyelin and phophatidylcholine/phosphatidylethanolamine were greater than normal. The saturated fatty acids in the total phospholipids were increased so that the ratio of saturated/unsaturated fatty acids was 1.4-1.6 versus 1.1-1.2 in the controls. The polyunsaturated fatty acids comprised only 22-26% of the fatty acids versus 31-32% in controls. The reduction in content of unsaturated fatty acids concerned all phospholipid classes in one patient and only the choline phospholipids in the tow other patients who were related to each other. The pattern of the fatty acids in the C2-position of the glycerophospholipids reflected the finding in the total phospholipids. Thus, an alteration of the activity of the acyl-CoA: 1-mono-acylphosphoglyceride-acyltransferase seems unlikely. The results support the notion of a generalized membrane defect in paramyotonia congenita, although the degree of abnormality in the fatty acid pattern was not correlated with the severity of the clinical symptoms.

Properties and topology of enzymes methylating phosphatidylethanolamine to phosphatidylcholine in sarcoplasmic reticulum.

1. The synthesis of phosphatidylcholine (PC) by stepwise methylation of phosphatidylethanolamine (PE) is carried out by two enzymes in sarcoplasmic reticulum (SR) membrane of rabbit fast-twitch skeletal muscles. 2. Two methyltransferases (Met I and Met II) have a different pH optimum and affinity for methyl donor--S-adenosyl-L-methionine (SAM). 3. Met I is an integral SR membrane protein which active site faces the cytoplasmic surface of the membrane. 4. Met II is a peripheral, loosely bound protein, localized mainly on the extracytoplasmic (luminal) part of the SR membrane.

Hyperthyroidism affects the activity of a cAMP-dependent protein kinase and protein phosphorylation in heart sarcolemma.

We have investigated the effect of thyroxine administration on the cAMP related systems in heart sarcolemma. Hyperthyroidism produces an increase in the number of beta-receptors and enhances in situ the phosphorylation of some sarcolemmal proteins, in particular the 55 kDa peptide. On the other hand, the activity of a membrane-bound cAMP-dependent protein kinase is considerably lower in sarcolemma obtained from animals receiving the hormone. We suggest that the thyroxine-induced alteration in beta-receptors elevates the content of cAMP in the cell which, in turn, evokes a transient dissociation of the catalytic subunit of a cAMP-dependent kinase from the regulatory subunit embeded in the membrane.

The lipid composition of erythrocyte ghosts from a patient with congenital paramyotonia.

The membrane lipid and fatty acid compositions of red blood cells from a paramyotonia patient were investigated. Cholesterol and total phospholipid contents in paramyotonia were not different from control. Only the sphingomyelin content was lower, and thus the molar ratio of phosphatidylcholine/sphingomyelin was higher than normal. The major abnormality concerned the fatty acid pattern. In all the phospholipid classes saturated fatty acids were increased and unsaturated fatty acids were decreased. The overall ratio of saturated/unsaturated fatty acids was 2.1 vs 1.6 in controls. Similar findings have been reported for the sarcolemma from paramyotonia patients. Thus, the results indicate that the membrane defect in this disease may be generalized.