Mitochondrial cytochrome c oxidase subunit IV is phosphorylated by an endogenous kinase.

This study was undertaken to identify novel mitochondrial membrane proteins that are potential targets for phosphorylation. Mitochondrial membranes were incubated in the presence of [gamma-32P]ATP and the Triton X-114 extractable protein was subjected to ion-exchange and polyacrylamide gel chromatography to purify a major phosphorylated protein of approximately 17000 Da. The determined peptide sequence of the purified phosphoprotein corresponded to a segment of cytochrome c oxidase subunit IV, an inner membrane protein of 17160 Da. The identity of the phosphoprotein was confirmed by two-dimensional electrophoresis and Western blotting. The results identify mitochondrial cytochrome c oxidase subunit IV as a protein which is phosphorylated by an endogenous kinase.

Alteration of a mitochondrial outer membrane signal anchor sequence that permits its insertion into the inner membrane. Contribution of hydrophobic residues.

Tom70p is targeted and inserted into the mitochondrial outer membrane in the Nin-Ccyto orientation, via an NH2-terminal signal anchor sequence. The signal anchor is comprised of two domains: an NH2-terminal hydrophilic region which is positively charged (amino acids 1-10) followed by the predicted transmembrane segment (amino acids 11-29). Substitution of the NH2-terminal domain with a matrix-targeting signal caused the signal anchor to adopt the reverse orientation in the outer membrane (Ncyto-Cin) or, if presented to mitoplasts, to arrest protein translocation at the inner membrane without insertion. Physically separating the transmembrane segment from the matrix-targeting signal by moving it downstream within the protein resulted in a failure to arrest in either membrane, and consequently the protein was imported to the matrix. However, if the mean hydrophobicity of the Tom70p transmembrane segment was increased in these constructs, the protein inserted into the inner membrane with an Nin-Cout orientation. Therefore we have determined conditions that allow the Tom70p transmembrane domain to insert in either membrane, pass through both membranes, or arrest without insertion in the inner membrane. These results identify the mean hydrophobicity of potential transmembrane domains within bitopic proteins as an important determinant for insertion into the mitochondrial inner membrane.

Purification and characterization of phospholamban phosphatase from cardiac muscle.

A protein phosphatase which dephosphorylates phospholamban was purified from canine cardiac cytosol. Purification involved sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, Mono Q HR 10/10, and Superose 6. The enzyme was composed of three subunits with Mr = 63,000, 55,000, and 38,000, and it could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase. Phospholamban phosphatase activity was enhanced 12-, 9-, and 3-fold by the divalent cations Mg2+, Mn2+, and Ca2+, respectively. The phosphatase was inhibited by PPi, ATP, NaF, and Pi and the degree of inhibition was different with each compound. The substrate specificity of the purified phosphatase for cardiac phosphoproteins was determined using troponin I, phospholamban, and highly enriched sarcolemmal and sarcoplasmic reticulum preparations, phosphorylated by the cAMP-dependent protein kinase. The phosphatase exhibited the highest activity with phospholamban as substrate. Thus, dephosphorylation of phospholamban by this phosphatase may participate in regulation of sarcoplasmic reticulum function in cardiac muscle.

Gas-liquid chromatographic analysis of ethanol and acetaldehyde in blood with minimal artifactual acetaldehyde formation.

A gas-liquid chromatographic procedure utilizing headspace gas analysis is described for the determination of ethanol and its metabolite, acetaldehyde, in a 100-microliters sample of blood from the rat, guinea pig, sheep, or human. Artifactual formation of ethanol-derived acetaldehyde is minimized during sample preparation by using a chemical solution containing perchloric acid and sodium azide in saline, and thiourea. Aqueous standards of ethanol and acetaldehyde are used to calibrate the procedure, and 1-propanol is used as the internal standard of the method. The recovery of ethanol and acetaldehyde from spiked blood samples is quantitative and reproducible, with a within-day coefficient of variation of less than 7% for ethanol and less than 9% for acetaldehyde. The lower limit of quantitative sensitivity is 0.006 mg/ml ethanol and 0.10 microgram/ml acetaldehyde. The instrumental analysis time is less than 3 min, which enables high sample throughput.

An amphiphilic lipid-binding domain influences the topology of a signal-anchor sequence in the mitochondrial outer membrane.

Mas70p is targeted and inserted into the mitochondrial outer membrane in the N(in)-C(cyto) orientation, via an NH2-terminal signal-anchor sequence. The signal-anchor is comprised of two domains: an NH2-terminal hydrophilic region which is positively charged (amino acids 1-10), followed by the predicted transmembrane segment (amino acids 11-29). Substitution of the NH2-terminal hydrophilic domain with a matrix-targeting signal caused the signal-anchor to adopt the reverse orientation in the membrane (N(cyto)-C(in)). This substitution resulted in an increase in the net positive charge of the hydrophilic region, from +4 to +8. In contrast to the endoplasmic reticulum and the bacterial inner membrane, where the net positive charge is an important determinant in conferring protein topology in the lipid bilayer, we show here that the reversal of the Mas70p signal-anchor was not due to differences in the number and positions of basic amino acids in the hydrophilic domain. However, a reduction in the hydrophobic moment of predicted amphiphilic helices containing an arginine, obtained by converting the apolar amino acids flanking the arginine to polar residues, caused the otherwise N(cyto)-C(in) signal-anchor to re-adopt the original N(in)-C(cyto) orientation of Mas70p. The reduced hydrophobic moment at the NH2-terminus significantly reduced the ability of this domain to bind to synthetic liposomes whose lipid composition reflected that of the outer membrane. These results identify amphiphilicity as an important determinant in causing retention of the NH2-terminus of a mitochondrial signal-anchor on the cytosolic side of the outer membrane. In addition to potential interactions between this domain and cytosolic-exposed components of the import machinery, this retention may result as well from interaction of the NH2-terminus with the surrounding membrane surface.

Disposition of ethanol and activity of hepatic and placental alcohol dehydrogenase and aldehyde dehydrogenases in the third-trimester pregnant guinea pig for single and short-term oral ethanol administration.

The disposition of ethanol and its metabolite, acetaldehyde, and the activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenases (ALDH) were determined in the third-trimester pregnant guinea pig following single and 7-day oral administration of ethanol (0.5 g X kg maternal body weight-1 X day-1). Animals were killed at each of selected times after the single and seventh ethanol dose. For both ethanol dosage regimens, the maternal and fetal blood and brain ethanol concentrations were virtually identical during the elimination phase of the time-course study. There was initial slow transfer of ethanol into amniotic fluid, followed by significantly higher ethanol concentration in amniotic fluid relative to maternal and fetal blood during the elimination phase. Acetaldehyde was measurable in maternal blood, maternal brain, and fetal brain at concentrations that were low and variable. For both ethanol dosage regimens, ADH activity was measurable only in maternal liver. Low Km ALDH activity was measurable only in maternal liver and fetal liver. High Km ALDH was measurable in maternal liver, fetal liver, and placenta and was significantly greater in maternal liver. The data indicate that there is bidirectional placental transfer of ethanol in the maternal-fetal unit; the elimination of ethanol from the maternal and fetal compartments is regulated by maternal hepatic biotransformation involving ADH; the amniotic fluid is a reservoir for ethanol in utero; the low Km ALDH in fetal liver protects the fetus from ethanol-derived acetaldehyde in the maternal circulation; and short-term maternal administration of once-daily, low-dose ethanol does not produce major changes in ethanol disposition and the activity of the enzymes involved in ethanol biotransformation.

Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2(+)-ATPase with phospholamban in phospholipid vesicles.

The Ca2(+)-ATPase in cardiac sarcoplasmic reticulum (SR) is under regulation by phospholamban, an oligomeric proteolipid. To determine the molecular mechanism by which phospholamban regulates the Ca2(+)-ATPase, a reconstitution system was developed, using a freeze-thaw sonication procedure. The best rates of Ca2+ uptake (700 nmol/min/mg reconstituted vesicles compared with 800 nmol/min/mg SR vesicles) were observed when cholate and phosphatidylcholine were used at a ratio of cholate/phosphatidylcholine/Ca2(+)-ATPase of 2:80:1. The EC50 values for Ca2+ were 0.05 microM for both Ca2+ uptake and Ca2(+)-ATPase activity in the reconstituted vesicles compared with 0.63 microM Ca2+ in native SR vesicles. Inclusion of phospholamban in the reconstituted vesicles was associated with a significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0. However, phosphorylation of phospholamban by the catalytic subunit of the cAMP-dependent protein kinase reversed the inhibitory effect on the Ca2+ pump. Similar findings were observed when a peptide, corresponding to amino acids 1-25 of phospholamban, was used. These findings indicate that phospholamban is an inhibitor of the Ca2(+)-ATPase in cardiac SR and phosphorylation of phospholamban relieves this inhibition. The mechanism by which phospholamban inhibits the Ca2+ pump is unknown, but our findings with the synthetic peptide suggest that a direct interaction between the Ca2(+)-ATPase and the hydrophilic portion of phospholamban may be one of the mechanisms for regulation.

Differential pharmacokinetics for oral and intraperitoneal administration of ethanol to the pregnant guinea pig.

The disposition of ethanol was studied in third-trimester pregnant guinea pigs (56-59 days gestational age) following maternal administration of ethanol, 0.5 g/kg total body weight, by oral intubation and by intraperitoneal injection. For oral administration, exposure of the fetus to ethanol involved bidirectional placental transfer of ethanol between the maternal and fetal compartments. For ip administration, there was distribution of ethanol from the peritoneal space across the uterus and chorioamniotic membranes into the amniotic fluid in addition to absorption into the maternal blood circulation and subsequent placental transfer into the fetus. This resulted in exposure of the fetus to very high ethanol concentration in the amniotic fluid immediately following ethanol administration. The data indicate that the ip route of ethanol administration does not mimic ingestion of ethanol and should be avoided in future studies of the fetal alcohol syndrome in rodent animal models.

The phospholamban phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme.

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic protein phosphatase activity, which can dephosphorylate phospholamban and regulate calcium transport. This phosphatase has been suggested to be a mixture of both type 1 and type 2 enzymes (E. G. Kranias and J. Di Salvo, 1986, J. Biol. Chem. 261, 10,029-10,032). In the present study the sarcoplasmic reticulum phosphatase activity was solubilized with n-octyl-beta-D-glucopyranoside and purified by sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, and DEAE-Sephadex. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. The partially purified phosphatase could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase(s). Enzymatic activity was inhibited by inhibitor-2 and by okadaic acid (I50 = 10-20 nM), using either phosphorylase a or phospholamban as substrates. The sensitivity of the phosphatase to inhibitor-2 or okadaic acid was similar for the two sites on phospholamban, phosphorylated by the cAMP-dependent and the calcium-calmodulin-dependent protein kinases. Phospholamban phosphatase activity was enhanced (40%) by Mg2+ or Mn2+ (3 mM) while Ca2+ (0.1-10 microM) had no effect. These characteristics suggest that the phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme, and this activity may participate in the regulation of Ca2+ transport through dephosphorylation of phospholamban in cardiac muscle.

Import and insertion of proteins into the mitochondrial outer membrane.

Nuclear-encoded proteins destined for insertion into the mitochondrial outer membrane, follow the same general pathway for import as proteins that are translocated to interior compartments within the organelle. This observation is true both for beta-barrel-type proteins and for proteins that contain hydrophobic alpha-helical transmembrane segments. In this review, we describe what is known about the various steps leading to protein insertion into the outer membrane, and discuss the energetics that favor vectorial translocation into and across this membrane. The selection of the outer membrane during import may involve a lateral release of the translocating polypeptide from the import machinery so that the appropriate domains of the protein become embedded in the lipid bilayer. One type of topogenic domain that can guarantee such selection of the outer membrane is a signal-anchor sequence of the type characterized for the bitopic protein Mas70p. It is suggested that a signal-anchor sequence selective for the mitochondrial outer membrane causes abrogation of polypeptide translocation and triggers the release of the transmembrane segment into the surrounding lipid bilayer, prior to any possibility for the commitment of translocation to the interior of the organelle. Specific structural features of the signal-anchor sequence specify its orientation in the membrane, and can confer on this sequence the ability to form homo-oligomers and hetero-oligomers. Strategies other than a signal-anchor sequence may be employed by other classes of proteins for selection of the outer-membrane. Of note is the ability of the outer-membrane import machinery to catalyze integration of the correct set of proteins into the outer-membrane bilayer, while allowing proteins that are destined for integration into the bilayer of the inner membrane to pass through unimpeded. Again, however, different proteins may employ different strategies. One model proposes that this can be accomplished by a combination of a matrix-targeting signal and a distal stop-transfer sequence. In this model, the formation of contact sites, which is triggered when the matrix-targeting signal engages the import machinery of the inner membrane, may prevent the outer-membrane translocon from recognizing and responding to the downstream stop-transfer domain. This allows the transmembrane segment to pass across the outer-membrane, and subsequently integrate into the inner membrane.

Pharmacokinetics of ethanol and its metabolite, acetaldehyde, and fetolethality in the third-trimester pregnant guinea pig for oral administration of acute, multiple-dose ethanol.

The pharmacokinetics of ethanol and its metabolite, acetaldehyde, were determined in the third-trimester pregnant guinea pig (56-59 days gestation) for oral intubation of four doses of 1 g ethanol/kg maternal body weight, administered at 1-h intervals. Animals (n = 4-7) were sacrificed at each of selected times during the 26-h study. Ethanol and acetaldehyde concentrations were determined by headspace gas-liquid chromatography. The maternal and fetal blood ethanol concentration-time curves were virtually superimposable, which indicated unimpeded bidirectional placental transfer of ethanol in the maternal-fetal unit. The blood and brain ethanol concentrations were similar in each of the maternal and fetal compartments during the study, which indicated rapid equilibrium distribution of ethanol. There was accumulation of ethanol in the amniotic fluid resulting in higher ethanol concentration compared with maternal and fetal blood during the elimination phase, which indicated that the amniotic fluid may serve as a reservoir for ethanol in utero. Acetaldehyde was measurable in all the biological fluids and tissues at concentrations that were at least 1,000-fold less than the respective ethanol concentrations and were variable. There was ethanol-induced fetolethality that was delayed and variable among animals, and was 55% at 23 h. At this time interval, the ethanol concentrations in maternal blood and brain, fetal brain, and amniotic fluid were 35- to 53-fold greater and the acetaldehyde concentrations in maternal blood and fetal brain were four- to five-fold higher in the animals with dead fetuses compared with the guinea pigs with live litters. These data indicated that decreased ethanol elimination from the maternal-fetal unit was related temporally to the fetolethality.

The role of protein kinases and protein phosphatases in the regulation of cardiac sarcoplasmic reticulum function.

Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3',5'-monophosphate (cAMP)-dependent and by calcium.calmodulin-dependent protein kinases on a 27,000 proteolipid, called phospholamban. Both types of phosphorylation are associated with an increase in the initial rates of Ca2+ transport by SR vesicles which reflects an increased turnover of elementary steps of the calcium ATPase reaction sequence. The stimulatory effects of the protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase, which can dephosphorylate both the cAMP-dependent and the calcium.calmodulin-dependent sites on phospholamban. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by protein kinases and protein phosphatases.

A comparative study of the inhibition of hepatic aldehyde dehydrogenases in the rat by methyltetrazolethiol, calcium carbimide, and disulfiram.

Methyltetrazolethiol (1-methyl-5-mercapto-1,2,3,4-tetrazole, MTT) is a heterocyclic substituent of the cephalosporin antibiotics, cefamandole, cefoperazone, and moxalactam. Pretreatment of rats with MTT has been reported to increase blood acetaldehyde concentration after ethanol administration. The time course of MTT-induced inhibition of hepatic aldehyde dehydrogenases (ALDH) was determined in adult, male Sprague-Dawley rats in comparison with the hepatic ALDH inhibition induced by calcium carbimide (calcium cyanamide, CC) and disulfiram (D). The apparent onset of maximal inhibition of hepatic low Km ALDH occurred at 2 h for 50 mg/kg MTT (subcutaneous, s.c.) and 7 mg/kg CC (oral) and at 24 h for 300 mg/kg D (oral). The relative magnitude of maximal inhibition of low Km ALDH was CC greater than D greater than MTT. The relative duration of enzyme inhibition was D greater than MTT greater than CC. High Km ALDH was only inhibited by CC. Hepatic low Km ALDH was selectively inhibited by s.c. and oral administration of 125 mg/kg MTT. For s.c. administration of 125 mg/kg MTT, the magnitude of maximal enzyme inhibition and the duration of inhibition were greater than for the 50 mg/kg dose. Oral administration of 125 mg/kg MTT produced similar inhibition of hepatic low Km ALDH compared with s.c. administration of the same dose. The time course of blood ethanol and acetaldehyde concentrations was determined for the intravenous infusion of two 0.3-g/kg doses of ethanol to rats that were pretreated orally with saline (1 h), MTT (125 mg/kg, 2 h), or CC (7 mg/kg, 1 h). The relative increase in blood acetaldehyde concentration compared with saline pretreatment was CC greater than MTT. The elimination of ethanol from blood was slower in the MTT- and CC-pretreated animals, and this effect was more pronounced for CC pretreatment. Overall, the data demonstrate that the characteristics of hepatic ALDH inhibition for MTT are different from those of the known ALDH inhibitors, CC and D.

Regulation of glycolytically fueled Ca2+ uptake in smooth muscle plasmalemmal vesicles by phosphorylation.

A highly purified plasma membrane vesicular preparation from porcine antrum had an endogenous protein kinase activity with substrates of molecular weights of 11, 15, 20.5, 25, 35, 44, 155, and 230 x 10(3). Phosphorylation of the plasma membranes by the endogenous protein kinase activity resulted in a stimulation of initial rates of Ca2+ uptake into inside-out vesicles, which was associated with an increase in the maximum velocity of the Ca2+ pump with no apparent changes in the half-maximal effective concentration for calcium. Because we have previously reported that a membrane-associated glycolytic system may preferentially provide ATP to fuel the Ca2+ pump (9), we examined the effects of phosphorylation on Ca2+ uptake when glycolysis was the sole source of ATP for the pump. We found that the stimulation of Ca2+ uptake by phosphorylation was more pronounced when Ca2+ uptake was supported by glycolysis rather than 2 mM ATP. When ATP was added at a level similar to that produced by endogenous glycolysis, the stimulation of Ca2+ uptake by phosphorylation was comparable to when glycolysis supported the Ca2+ pump. Our observations suggest that the dynamic range (up to threefold) for regulation of the plasmalemmal Ca2+ pump by phosphorylation is considerably larger than previously reported and thus likely to be of physiological significance.

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.