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Submitochondrial location of ruthenium red-sensitive calcium-ion transport and evidence for its enrichment in a specific population of rat liver mitochondria.

ABSTRACT: 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.

SUBMITTER: Bygrave FL

PROVIDER: S-EPMC1186007 | biostudies-other | 1978 Sep

REPOSITORIES: biostudies-other

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Project description:The maturation of Ca(2+) transport in mitochondria isolated from rat liver was examined, from 5 days before birth. The mitochondria used were isolated from liver homogenates by centrifugation at 22000g-min. Ca(2+) transport by mitochondria isolated from foetal liver is energy-dependent and Ruthenium Red-sensitive. The transmembrane pH gradient in these mitochondria is higher by about 7mV and the membrane potential lower by about 20mV than in adult mitochondria. The inclusion of 2mm-P(i) in the incubation medium enhances the protonmotive force by approx. 30mV. The rate of Ca(2+) influx in foetal mitochondria measured in buffered KCl plus succinate is low until about 2-3h after birth, when it increases to about 60% of adult values; approx. 24h later it has reached near-adult values. Higher rates of Ca(2+) influx are observed in the presence of 2mm-P(i); 3-5 days before birth the rates are about one-third of adult values and decline slightly as birth approaches. By 2-3h post partum they have reached adult values. The inclusion of 12.5mum-MgATP with the P(i) stimulates further the initial rate of Ca(2+) influx in foetal mitochondria. The rates observed are constant over the prenatal period examined and are 50-60% of those observed in adult mitochondria. Mitochondria isolated from foetal livers 4-5 days before birth retain the accumulated Ca(2+) for about 50min in the presence of 2mm-P(i). In the period 2 days before birth to birth, this ability is largely lost, but by 2-3h after birth Ca(2+) retention is similar to that of adult mitochondria. The presence of 12.5mum-MgATP progressively enhances the Ca(2+) retention time as development proceeds until 2-3h after birth, when it becomes less sensitive to added MgATP. Glucagon administration to older foetuses in utero enhances both the rate of mitochondrial Ca(2+) influx assayed in the presence of 2mm-P(i) and the time for which mitochondria retain accumulated Ca(2+) in the presence of 12.5mum-MgATP and 2mm-P(i). Its administration to neonatal animals leads to an increase in mitochondrial Ca(2+) retention similar to that seen in adult mitochondria. The data provide evidence that the Ruthenium Red-sensitive Ca(2+) transporter is potentially as active in foetal mitochondria 5 days before birth as it is in adult mitochondria. They also show that foetal mitochondria have an ability to retain accumulated Ca(2+) reminiscent of mitochondria from tumour cells and from hormone-challenged rat liver.

Project description: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.

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Submitochondrial location of ruthenium red-sensitive calcium-ion transport and evidence for its enrichment in a specific population of rat liver mitochondria.

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