The association of calmodulin with subcellular fractions isolated from rat liver

Calmodulin associated with rat liver mitochondria has been found to belong to a contaminant membranous fraction which contains different subcellular membranes. The concentration of calmodulin in this fraction is relatively high, about 1.6 micrograms/mg protein, and can not be decreased with EGTA. The calmodulin-rich membranous fraction seems to contain cytoskeletal proteins which could be responsible for the binding of calmodulin.     

Distribution of alkylglycerone-phosphate synthase in subcellular fractions of rat liver

Subcellular fractions of rat liver were isolated by density-gradient centrifugation on a linear Metrizamide gradient and were assayed for marker enzymes of peroxisomes, lysosomes, microsomes and mitochondria. Alkylglycerone-phosphate synthase catalysing the formation of the ether bond in glycerolipids was also determined along the gradient. The enzyme was found to be enriched in the peroxisomal and the microsomal fractions thus, displaying a bimodal distribution pattern. Two reaction-products each, alkylglycerone phosphate and alkylglycerone were obtained in the enzymic assays performed, the ratio of which was clearly dependent upon the fraction employed. Alkylglycerone phosphate was mainly synthesized by the 'peroxisomal synthase', whereas an inverse proportion was observed assaying the microsomal counterpart. Furthermore, comparing the mean specific activities of both the enzymes the microsomal one was shown to be roughly twice as active in metabolizing 1-O-palmitoylglycerone 3-phosphate, simultaneously displaying a somewhat different sensitivity to NaF. These findings provide a first line of evidence, that two separate synthases, one in microsomes and another one in peroxisomes might be engaged in the biosynthesis of 1-O-alkyl-glycerolipids in rat liver.     

Two subcellular locations for peripheral-type benzodiazepine acceptors in rat liver

Subcellular fractionation of rat liver by differential centrifugation showed the mitochondrial fractions to have the greatest enrichment of 'peripheral-type' benzodiazepine acceptor. Two peaks of acceptor sites were found on isopycnic density-gradient centrifugation, one peak (rho = 1.19 g/ml) corresponding to the peak of mitochondria as judged by marker enzyme distribution and by transmission electron microscopy, and the other peak (rho = 1.17 g/ml) which is not mitochondrial as judged by the lack of mitochondrial enzyme markers. Whereas the density of the mitochondrial acceptor was sensitive to sonication and was shown to have an outer-membrane location, the density of the non-mitochondrial acceptor was insensitive to sonication. The non-mitochondrial acceptor was shown not to be associated with Golgi, lysosomes, rough endoplasmic reticular microsomes, peroxisomes, or some types of plasma membranes, as judged by differences in the distribution of marker activities. No enrichment of benzodiazepine acceptor was found in the purified nuclear fraction. Both acceptors were shown to be peripheral-type high-affinity acceptors as judged by ligand specificities and by photoaffinity labelling.     

Characterization of insulin uptake into subcellular fractions of perfused rat liver using two different iodinated tracers

The binding and uptake of insulin in perfused rat liver has been investigated with specifically labelled 125I-A14-tyrosyl insulin as a tracer and compared with a commercially available iodo-insulin preparation. The commercial preparation did not show saturation uptake kinetics and the clearance from the perfusate remained low and constant throughout a wide concentration range. A14 labelled insulin showed saturation kinetics and high clearance at low carrier concentration, falling rapidly with increasing carrier concentration and reaching a steady state value of 1 ml/min. These results emphasize the importance of using specifically labelled insulin in physiological and biochemical studies of hepatic insulin metabolism. Perfusion with A14 tyrosine-labelled insulin at 4 degrees C showed apparent saturation with binding to the plasma membrane fraction. Perfusion at 37 degrees C also showed apparent saturation with uptake predominantly to the ligandosome fraction. These results implicate the plasma membrane-ligandosome pathway in the hepatic uptake of insulin at both physiological and pharmacological concentrations of the hormone.     

Differential induction of peroxisomal populations in subcellular fractions of rat liver

In rat liver, peroxisome proliferators induce profound changes in the number and protein composition of peroxisomes, which upon subcellular fractionation is reflected in heterogeneity in sedimentation properties of peroxisome populations. In this study we have investigated the time course of induction of the peroxisomal proteins catalase, acyl-CoA oxidase (ACO) and the 70 kDa peroxisomal membrane protein (PMP70) in different subcellular fractions. Rats were fed a di(2-ethylhexyl)phthalate (DEHP) containing diet for 8 days and livers were removed at different time-points, fractionated by differential centrifugation into nuclear, heavy and light mitochondrial, microsomal and soluble fractions, and organelle marker enzymes were measured. Catalase was enriched mainly in the light mitochondrial and soluble fractions, while ACO was enriched in the nuclear fraction (about 30%) and in the soluble fraction. PMP70 was found in all fractions except the soluble fraction. DEHP treatment induced ACO, catalase and PMP70 activity and immunoreactive protein, but the time course and extent of induction was markedly different in the various subcellular fractions. All three proteins were induced more rapidly in the nuclear fraction than in the light mitochondrial or microsomal fractions, with catalase and PMP70 being maximally induced in the nuclear fraction already at 2 days of treatment. Refeeding a normal diet quickly normalized most parameters. These results suggest that induction of a heavy peroxisomal compartment is an early event and that induction of 'small peroxisomes', containing PMP70 and ACO, is a late event. These data are compatible with a model where peroxisomes initially proliferate by growth of a heavy, possibly reticular-like, structure rather than formation of peroxisomes by division of pre-existing organelles into small peroxisomes that subsequently grow. The various peroxisome populations that can be separated by subcellular fractionation may represent peroxisomes at different stages of biogenesis.     

Oxidation of reduced glutathione by subcellular fractions of rat liver

1. A new method was used to diminish the autoxidation of GSH. 2. The oxidation of GSH by liver homogenates was studied with regard to concentration of homogenate, concentration of GSH, time, pH and anaerobiosis. 3. GSH was oxidized by recombinations of the supernatant with microsomes and with mitochondria. Each fraction alone caused little oxidation. 4. Proteins in the supernatant were required to obtain the effect, and low-molecular-weight compounds in the same fraction increased its effect. 5. GSH diminished the formation of malonaldehyde in homogenates. 6. GSH prevented a stimulating effect of the supernatant on the formation of malonaldehyde in microsomes and in mitochondria. 7. The malonaldehyde formation in microsomes together with the supernatant did not start until the concentration of endogenous low-molecular-weight thiols had decreased to a low level. 8. It is suggested that part of the oxidation of GSH in homogenates is coupled to a mechanism that counteracts the peroxidation of membrane lipids.     

Preincubation accelerates taurocholate uptake into isolated liver cells

Initial rates of taurocholate uptake into isolated hepatocytes stored at 0°C increased 3-fold during a 25 min preincubation. Concomitantly, $\text{V}$ increased while $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ remained unaffected. There are several possible explanations for the preincubation effects, such as new synthesis of carrier protein, altered fluidity of the membrane or stimulation of the sodium-dependent taurocholate uptake via a change in the cation distribution. The experiments presented strongly favor the latter explanation as the sodium gradient as well as the uptake of the bile acid reach their steady state within 20–30 min and replacement of sodium by potassium in the medium abolished the effect.