Rapid separation of particulate components and soluble cytoplasm of isolated rat-liver cells

A method is described for the rapid separation of mitochondria (plus other particulate components) from the soluble cytoplasm of isolated rat-liver cells. The cells were incubated briefly with a low concentration of digitonin. After rapid centrifugation, the pellet contained more than 90% of the total adenylate kinase and glutamate dehydrogenase activities and the supernatant at least 80% of the lactate dehydrogenase activity. About 60% of total adenine nucleotides in hepatocytes were found in the soluble cytoplasm. The ATP/ADP ratio in the particulate fraction 80 s after exposure to digitonin of hepatocytes metabolizing alanine was 2.0–2.4, and that in the soluble cytoplasm 6–19. In the presence of atractyloside, these values were 3.5–4.4 and 1.3–2.2, respectively.     

An alpha-tocopherol binding protein in rat liver cytoplasm.

Gel filtration and sucrose density gradient analysis of rat liver high speed supernatant revealed the presence of a protein capable of binding [³H] α-tocopherol. The protein sedimented with an S value of 3.0. Gel filtration yielded an estimated molecular weight of 31,000. Specificity for α-tocopherol was demonstrated by competition for binding of [³H]α-tocopherol with unlabeled α-tocopherol, but not with α-tocopheryl quinone or α-tocopheryl acetate. Pronase digestion completely abolished binding.     

A small particulate component of the cytoplasm

A particulate component of small dimensions (100 to 150 A) and high density is described in the ground substance of the cytoplasm of mammalian and avian cells. In many cell types that seem to have in common a high degree of differentiation, the new component is preferentially associated with the membrane of the endoplasmic reticulum; whereas in other cell types, characterized by rapid proliferation, it occurs more or less freely distributed in the ground substance of the cytoplasm. In the Discussion an attempt is made to integrate the observations presented in this paper with the already available cytological, histochemical, and cytochemical information.     

Metabolic effects of carbenoxolone in rat liver

The action of carbenoxolone on hepatic energy metabolism was investigated in the perfused rat liver and isolated mitochondria. In perfused livers, carbenoxolone (200-300 microM) increased oxygen consumption, glucose production and glycolysis from endogenous glycogen. Gluconeogenesis from lactate or fructose, an energy-dependent process, was inhibited. This effect was already evident at a concentration of 25 microM. The cellular ATP levels and the adenine nucleotide content were decreased by carbenoxolone, whereas the AMP levels were increased. In isolated mitochondria, carbenoxolone stimulated state IV respiration and decreased the respiratory coefficient with the substrates beta-hydroxybutyrate and succinate. The ATPase of intact mitochondria was stimulated, the ATPase of uncoupled mitochondria was inhibited, and the ATPase of disrupted mitochondria was not altered by carbenoxolone. These results indicate that carbenoxolone acts as an uncoupler of oxidative phosphorylation and, possibly, as an inhibitor of the ATP/ADP exchange system. The inhibitory action of carbenoxolone on mitochondrial energy metabolism could be contributing to induce the mitochondrial permeability transition (MPT), a key phenomenon in apoptosis. The results of the present study can explain, partly at least, the in vivo hepatotoxic actions of carbenoxolone that were found in a previous clinical evaluation.     

Purification of rat liver particulate neutral ribonuclease and comparison of properties with pancreas and serum ribonucleases.

Rat liver particulate neutral ribonuclease (EC 3.1.4.22) was extensively purified (up to 40000-fold). It is shown to be an endonuclease, specific for pyrimidine bases, hydrolysing 5'-phosphate ester bonds. The enzyme specificity, Km, pH optimum, stability in acid medium and thermal stability at high temperature are the same as those of rat pancreatic and serum ribonucleases. Like pancreatic and serum neutral ribonucleases, the hepatic enzyme is sensitive to the liver natural inhibitor. This inhibitor was purified 8000-fold; its association with ribonuclease follows zero-order kinetics. These identical properties for ribonuclease of rat liver, pancreas and serum support the hypothesis [Bartholeyns, Peeters-Joris & Baudhuin (1975) Eur. J. Biochem. 60, 385-393] of an extrahepatic origin for the liver enzyme, the plasma ribonuclease of pancreatic origin being taken up by endocytosis in the liver. Neutral ribonuclease activity was detected in all rat organs investigated; its distribution among tissues is different from the distribution of the natural ribonuclear inhibitor.     

Detergent solubilisation of rat liver particulate cyclic AMP phosphodiesterase

• 1.1. Detergent solubilisation of particulate rat liver low K_m cyclic AMP phosphodiesterase in the presence of protease inhibitors yields a form of the enzyme with a larger molecular weight than the form solubilised by protease treatment.
• 2.2. The detergent solubilised enzyme could be partially purified by anion exchange chromatography.
• 3.3. It displayed a marked tendency to precipitate from solution when detergent was removed.

     

Chain extension of ribonucleic acid by enzymes from rat liver cytoplasm

1. The 105000g supernatant fraction of rat liver catalyses the incorporation of ribonucleotides from ribonucleoside triphosphates into polyribonucleotide material. The reaction requires Mg²⁺ ions and is enhanced by the addition of an ATP-generating system and RNA, ATP, UTP and CTP but not GTP are utilized in this reaction. In the case of UTP, the product is predominantly a homopolymer containing 2–3 uridine residues, and there is evidence that these may be added to the 3′-hydroxyl ends of RNA or oligoribonucleotide primers. 2. The microsome fraction of rat liver incorporates ribonucleotides from ATP, GTP, CTP and UTP into polyribonucleotide material. This reaction requires Mg²⁺ ions and is enhanced slightly by the addition of an ATP-generating system, and by RNA but not DNA. Supplementation of the reaction mixture with the three complementary ribonucleoside 5′-triphosphates greatly increases the utilization of a single labelled ribonucleoside 5′-triphosphate. The optimum pH is in the range 7·0–8·5, and the reaction is strongly inhibited by inorganic pyrophosphate and to a much smaller degree by inorganic orthophosphate. It is not inhibited by actinomycin D or by deoxyribonuclease. In experiments with [³²P]UTP in the absence of ATP, GTP and CTP, 80–90% of ³²P was recovered in UMP-2′ or -3′ after alkaline hydrolysis of the reaction product. When the reaction mixture was supplemented with ATP, GTP and CTP, however, about 40% of the ³²P was recovered in nucleotides other than UMP-2′ or -3′. Although the reactions seem to lead predominantly to the synthesis of homopolymers, the possibility of some formation of some heteropolymer is not completely excluded.     

Binders of intravenously administered 65-zinc in rat liver cytoplasm.

The fate of an i.v. injected trace dose of 65Zn2+ in the rat, was studied over a period of 10 days after injection. Tissue distributions were determined and a special study was made of 65Zn binders in liver cytoplasm. A total of 6 65Zn-binding fractions were observed in liver cytoplasm with apparent molecular weights of about 113,000, 66,400, 47,400, 29,000, 23,000 and 11,400. A time study showed that 4 hours after the injection the most prominent cytoplasmatic 65Zn binders are the 113,000, 66,400 and 23,000 molecular weight fractions. A tentative identification of the main Zn binders in the six 65Zn fractions is given, using the collected data regarding their apparent molecular weight, time dependent prominence and content of stable Zn.     

Metabolic effects of silibinin in the rat liver

The flavonolignan silibinin, which is a mixture of two diastereoisomers, silybin A and silybin B, is a component of the extract obtained from the fruit and seeds of the variegated milk thistle (Silybum marianum (L.) Gaertn. (Asteraceae)), known as silymarin. Among the therapeutic properties credited to silibinin, its antihyperglycaemic action has been extensively explored. Silibinin is structurally related to the flavonoids quercetin and fisetin, which have been previously demonstrated to be very active on liver metabolic processes related to glycaemic regulation. The aim of the present work was to investigate the effects of silibinin on metabolic pathways responsible for the maintenance of glycaemia, particularly glycogenolysis and gluconeogenesis, in the perfused rat liver. The activities of some key enzymes in these pathways and on parameters of energy metabolism in isolated mitochondria were also examined. At a concentration range of 50-300μM, silibinin inhibited gluconeogenesis in the fasted condition and inhibited glycogenolysis and glycolysis in the fed condition. The mechanisms by which silibinin exerted these actions were multiple and complex. It inhibited the activity of glucose 6-phosphatase, inhibited the pyruvate carrier, and reduced the efficiency of mitochondrial energy transduction. It can also act by reducing the supply of NADH for gluconeogenesis and mitochondria through its pro-oxidative actions. In general, the effects and the potency of silibinin were similar to those of quercetin and fisetin. However, silibinin exerted some distinct effects such as the inhibitory effect on oxygen consumption in the fed condition and a change in the energy status of the perfused livers. It can be concluded that the effects of silibinin on liver glucose metabolism may explain its antihyperglycaemic property. However, this effect was, in part, secondary to impairment in cellular energy metabolism, a finding that should be considered in its therapeutic usage.