Biomedical subjects
Publications and source records attributed to F L Bygrave.
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1. Seven fractions sedimenting at between 3000 and 120000g-min were prepared from a rat liver homogenate by differential centrifugation in buffered iso-osmotic sucrose. The following measurements were carried out on each of these fractions: Ruthenium Red-sensitive Ca(2+) transport in the absence and in the presence of P(i) as well as in the presence of N-ethylmaleimide to prevent P(i) cycling, succinate-supported respiration in the absence and in the presence of ADP, the DeltaE and -59 DeltapH components of the protonmotive force, cytochrome oxidase, uncoupler-stimulated adenosine triphosphatase, alpha-glycerophosphate dehydrogenase, P(i) content and the effect on the ;resting' rate of respiration of repeated additions of a fixed Ca(2+) concentration. 2. Ca(2+) transport either in the presence or in the absence of added P(i) and in the presence of N-ethylmaleimide exhibits significantly higher rates in the fraction sedimenting at 8000g-min. By contrast, respiration in the presence or in the absence of added ADP and the values for DeltaE and -59 DeltapH were similar in those fractions sedimenting between 4000 and 20000g-min, indicating that the driving force for Ca(2+) transport was similar in each of these fractions. 3. Experiments designed to determine the capacity of the individual fractions for Ca(2+), as measured by the effect of repeated additions of Ca(2+) on the resting rate of respiration, showed that fraction 2, i.e. that sedimenting at 8000g-min, also exhibited the greatest tolerance towards the uncoupling action of the ion. 4. Of the three enzyme activity profiles, only that of alpha-glycerophosphate dehydrogenase was similar to that of Ca(2+) transport. Because previous workers have assigned this enzyme to loci in the inner peripheral membrane [Werner & Neupert (1972) Eur. J. Biochem.25, 379-396], it is concluded that the Ruthenium Red-sensitive Ca(2+)- transport system also is located in this domain of the inner membrane. The relation of these findings to the mechanisms of mitochondrial Ca(2+) transport and the biogenesis of mitochondria is discussed.
1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca(2+) transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5'-nucleotidase and Ruthenium Red-sensitive Ca(2+) transport. Initial rates of Ruthenium Red-insensitive Ca(2+) transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca(2+) transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5'-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca(2+) transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca(2+) transport activity, glucose 6-phosphatase and 5'-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca(2+) transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3-4 days, but then rapidly decreases to approach adult values. Maximal activity of 5'-nucleotidase in the microsomal and nuclear fractions is seen about 4-6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca(2+) transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2-3-fold the initial rate of Ruthenium Red-insensitive Ca(2+) transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1mug of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the hormone. 4. Ruthenium Red-insensitive Ca(2+) transport in the post-mitochondrial fraction of foetal liver also responds to the administration in situ of glucagon. The response, which also is prevented by co-administration of puromycin, is maximal in those foetuses nearing term. The suggestion is made that these effects of the hormone on Ruthenium Red-insensitive Ca(2+) transport are an integral part of the physiological network in the liver cell.
Tributyltin in the concentration range 1-4mum failed to stimulate Ca(2+) transport by Lucilia flight-muscle mitochondria in a medium containing KCl and respiratory substrate but devoid of P(i), despite its promotion of a rapid Cl(-)/OH(-) exchange. When 2mm-P(i) was present, concentrations of tributyltin greater than 1mum inhibited the initial rate of Ca(2+) transport and induced efflux of the ion from the mitochondria in Cl(-)- or NO(3) (-)-containing media. Lower concentrations had little effect. Oligomycin added at up to 10mug/mg of mitochondrial protein had no effect on Ca(2+) transport. By contrast, approx. 0.3mum-tributyltin completely inhibited respiration supported by alpha-glycerophosphate in either the presence or absence of added ADP. The data suggest that tributyltin can inhibit Ca(2+) transport in Lucilia flight-muscle mitochondria other than by facilitating a Cl(-)/OH(-) exchange or producing an oligomycin-like effect.
1. Addition of N-ethylmaleimide to rat liver mitochondria respiring with succinate as substrate decreases both the initial rate of Ca(2+) transport and the ability of mitochondria to retain Ca(2+). As a result, Ca(2+) begins to leave the mitochondria soon after it has entered. Half-maximal effects occur at an N-ethylmaleimide concentration of about 100nmol/mg of protein. 2. The efflux of Ca(2+) induced by N-ethylmaleimide is not prevented by Mg(2+) or by Ruthenium Red at concentrations known to prevent Ca(2+) efflux when exogenous phosphate also is present. Swelling of mitochondria does not accompany N-ethylmaleimide-induced Ca(2+) efflux. 3. Addition of Ca(2+) to rat liver mitochondria in the presence of N-ethylmaleimide produces an immediate decrease in DeltaE (membrane potential), which decreases further to only a slight extent over the next 8min. Concomitant with this is an immediate increase and then levelling off of the -59DeltapH (transmembrane pH gradient). 4. Preincubation of rat liver mitochondria with p-chloromercuribenzenesulphonate, which by contrast with N-ethylmaleimide is unable to penetrate the inner mitochondrial membrane, also prevents Ca(2+) retention. The DeltaE and -59DeltapH respond to Ca(2+) addition in a manner similar to that which occurs when N-ethylmaleimide is present. Subsequent addition of mercaptoethanol produces an immediate increase in both DeltaE and -59DeltapH. At the same time Ca(2+) is rapidly accumulated by the organelles. 5. The above data are interpreted as indicating that under the conditions of Ca(2+) efflux seen here, the mitochondria retain their functional integrity. This contrasts with the uncoupling effect of Ca(2+) seen in the presence of P(i), which generally leads to a loss of mitochondrial integrity. We suggest that a unique mechanism of Ca(2+) cycling is able to take place when mitochondria have been treated with N-ethylmaleimide.
1. Ca2+ transport by mitochondria isolated from flight muscle of the sheep blowfly Lucilla cuprina does not occur in the absence of added P1. Maximum rates of transport are attained when about 2.5 mM-phosphate is present. 2. As mitochondria develop, high but not low phosphate concentrations begin to inhibit Ca2+ transport markedly; those isolated from 2-day-old flies for example, are inhibited by about 75% by 20 mM-phosphate. Maximum rates of transport, i.e. those measured in the presence of 2.5 mM-phosphate, begin to decline only when the fly is about 3 days old. 3. Mitochondrial phosphate transport activity does not change during development of the blowfly, but the endogenous concentration of the anion does. At emergence it is about 6nmol/mg of protein, increases to about 17 nmol/mg of protein at 2-3h and then rapidly declines to reach less than 5 nmol/mg of protein after 2 days of adult life. 4. Studies on the effect of phosphate on oxidation of alpha-glycerophosphate in the absence and presence of ADP reveal a lack of inhibition by high phosphate concentrations indicating that the anion does not influence Ca2+ transport by preventing the generation of the proton electrochemical gradient across the inner membrane. 5. It is concluded that the molecular assembly in the inner membrane of Lucilla mitochondria responsible for transporting Ca2+ is fully developed at emergence and remains so for at least 2-3 days of adult life. The possibility exists that Ca2+-transport activity in these mitochondria is controlled at least in part by P1.
Measurements of the initial rate of Ca2+ transport by rat liver microsomal preparations reveal the existence of two phases of transport activity. The first, a phase of rapid transport, is complete by 3-5 min, at which time the second (slower) phase begins; this remains linear for up to at least 40 min. The initial phase is minimal in the absence of MgATP. The initial rate of Ca2+ transport reaches values as high as 25 nmol/min per mg of protein; the Km for Ca2+total is 1-2 micrometer and that for MgATPtotal about 500 micrometer. Ruthenium Red (3-5 nmol/mg of protein) has little effect on the initial rate of transport, whereas tributylin (2 micrometer) inhibits equally in a KC1- or a KNO3-containing medium. Compunds that collapse components of the proton electrochemical gradient in mitochondria (valinomycin and carbonyl cyanide m-chlorophenylhydrazone) each inhibit by 70-80% the initial rate of microsomal Ca2+ transport.
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Choline accumulation and phosphatidylcholine biosynthesis were investigated in the choline-requiring anaerobic protozoon Entodinium caudatum by incubating whole cells or subcellular fractions with [14C] choline, phosphoryl [14C] choline and CDP-[14C] choline. 2. All membrane fractions contained choline kinase (EC 2.7.1.32) and CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), although the specific activities were less in the cell-envelope fraction. Choline phosphate cytidylyltransferase (EC 2.7.7.15) was limited to the supernatant, and this enzyme was rate-limiting for phosphatidylcholine synthesis in the whole cell. 3. Synthesis of phosphatidylcholine from free choline by membranes was only possible in the presence of supernatant. Such reconstituted systems required ATP (2.5 mM), CTP (1 mM) and Mg2+ (5 mM) for maximum synthesis of the phospholipid. CTP and Mg2+ were absolute requirements. 4. Hemicholinium-3 prevented choline uptake by the cells and was strongly inhibitory towards choline kinase; the other enzymes involved in phosphatidylcholine synthesis were minimally affected. 5. Ca2+ ions (0.5 mM) substantially inhibited CDP-choline-1,2-diacylglycerol cholinephosphotransferase in the presence of 15 mM-Mg2+, but choline phosphate cytidylyltransferase and choline kinase were less affected. 6. No free choline could be detected intact cells even after short (10-180s) incubations or at temperatures down to 10 degrees C. The [14C] choline entering was mainly present as phosphorylcholine and to a lesser extent as phosphatidylcholine. 7. It is suggested that choline kinase effectively traps any choline within the cell, thus ensuring a supply of the base for future growth. At low choline concentrations the activity of choline kinase is rate-limiting for choline uptake, and the enzyme might possibly play an active role in the transport phenomenon. Thus the choline uptake by intact cells and choline kinase have similar Km values and show similar responses to temperature and hemicholinium-3.
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1. Rat liver mitochondria were partially depleted of their phospholipids using phospholipase A prepared from porcine pancreas (substrate specificity, cardiolipin greater than phosphatidylethanolamine greater than phosphatidylcholine) or from Crotalus adamanteus venom (substrate specificity, phosphatidylethanolamine = phosphatidylcholine greater than cardiolipin). 2. Removal of only about 1% of the mitochondrial phospholipid with the pancreatic enzyme leads to 50% and 25% losses in ADP and ATP translocation, respectively. Concomitant with the loss in translocation is a decline in the ability of both carbonylcyanide m-chlorophenylhydrazone and Ca2+ to stimulate ATP translocation. 3. To achieve comparable losses in ADP and ATP translocation with the venom enzyme, it is necessary to remove about 8% of the total mitochondrial phospholipid. Following such treatment, carbonylcyanide m-chlorophenylhydrazone and Ca2+ are still capable of stimulating ATP translocation. 4. Control experiments involving treatment of the mitochondria with the products of phospholipase digestion indicate that the effects observed on the translocase reflect a loss of phospholipid from the membrane. 5. Binding studies indicate that the loss in adenine nucleotide translocation following phospholipase treatment cannot be accoundted for by an altered ability to bind adenine nucleotides to atractyloside-sensitive sites. 6. The data are interpreted in terms of a mechanism of adenine nucleotide translocation involving a lipoprotein carrier system, consisting of the translocator protein and phospholipids, possibly cardiolipin and phosphatidylethanolamine.
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This report describes a kinetic analysis of energy-linked Ca2+ transport in rat liver mitochondria, in which a ruthenium red/EGTA [ethanedioxy-bis(ethylamine)-tetraacetic acid] quenching technique has been used to measure rates of 45Ca2+ transport. Accurately known concentrations of free 45Ca2+ were generated with Ca2+/nitrilotriacetic acids buffers for the determination of substrate/velocity relationships. The results show that the initial velocity of transport is a sigmoidal function of Ca2+ concentration (Hill coefficient = 1.7), the Km being 4 muM Ca4 at 0 degrees C and pH 7.4. These values for the Hill coefficient and the Km remain constant in the presence of up to 2 mM phosphate, but with 10 mM acetate both parameters are increased slightly. Both permeant acids increase the maximum velocity to an extent dependent on their concentration. The Ca2+-binding site(s) of the carrier contains a group ionizing at pH approximately 7.5 at 0 degrees C, which is functional in the dissociated state. The stimulatory effect of permeant acids is ascribed to their facilitating the release of Ca2+ from the carrier to the internal phase, an interpretation which is strengthened by the lack of effect of the permeant anion SCN- on Ca2+ transport. Studies on the time-course of Ca2+ uptake and of EFTA-induced Ca2+ efflux from pre-loaded mitochondria demonstrate the reversibility of the carrier in respiring mitochondria and the extent to which this property is influenced by permeant acids. These data are accommodated in a carrier mechanism based on electrophoretic transport of Ca2+ bound to pairs of interacting acidic sites.