PERMEABILITY OF MICROSOMAL MEMBRANES ISOLATED FROM RAT LIVER

Water compartments, permeability, and the possible active translocation of various substances in rat liver microsomes were studied by using radioactive compounds and ultracentrifugation. The total water of the microsomal pellet, 3.4 µl/mg dry weight, is the sum of water in the extramicrosomal and intramicrosomal spaces, or 56 and 44%, respectively. Sucrose space accounts for 77% of the intramicrosomal water and the hydration water ~ 14%, leaving almost no sucrose-impermeable space when using the ultracentrifugation approach. With increasing sucrose concentration, microsomes do not show an osmotic response. The intramicrosomal water decreases greatly in the presence of Cs⁺ and Mg⁺⁺ in rough but not in smooth microsomes. Uncharged substances of molecular weight of up to at least 600 freely penetrate microsomal membranes, which already become impermeable to charged substances at a molecular weight of 90. These substances also induce an osmotic response. The vesicles can be made permeable to charged substances after water treatment and cooling, which, however, does not increase glucose-6-phosphatase and inosine diphosphatase (IDPase) activities, and these enzymes can still be activated by deoxycholate. IDPase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation reactions, performed in vitro, also disproved the hypothesis of an accumulation of charged substances inside of vesicles of being a major pathway. The products of the enzymic reactions as well as the glucuronidated form of a hydroxylated product can be recovered on the cytoplasmic side of membranes, and little accumulation occurs in the intravesicular compartment.     

HEXAGONAL ARRAY OF SUBUNITS IN TIGHT JUNCTIONS SEPARATED FROM ISOLATED RAT LIVER PLASMA MEMBRANES

Solubilization of isolated rat liver plasma membranes in 1% deoxycholate and centrifugation yielded a fraction (pellet) that consisted mainly of tight junctions (zonulae occludentes). An hexagonal array of subunits similar to that previously found in a number of the unfractionated plasma membranes was demonstrated in all the membrane sheets of these preparations by negative staining. It is concluded that the hexagonal subunit pattern is present in the tight junctions, and that this structural differentiation may be related to the intercellular diffusion afforded by the junctional membrane.     

EFFECT OF AN INHIBITING FACTOR ISOLATED FROM RAT LIVER ON DNA POLYMERASES IN REGENERATING RAT LIVER

We partially purified an inhibitory factor (LIF), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in vitro cell multiplication by blocking the G₁—S transition, and reduced in vivo [³H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10 hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in vivo treatment prevented α DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on α DNA polymerase was detected. LIF did not affect β DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.     

ENZYMATIC PROPERTIES OF THE INNER AND OUTER MEMBRANES OF RAT LIVER MITOCHONDRIA

Treatment of rat liver mitochondria with digitonin followed by differential centrifugation was used to resolve the intramitochondrial localization of both soluble and particulate enzymes. Rat liver mitochondria were separated into three fractions: inner membrane plus matrix, outer membrane, and a soluble fraction containing enzymes localized between the membranes plus some solublized outer membrane. Monoamine oxidase, kynurenine hydroxylase, and rotenone-insensitive NADH-cytochrome c reductase were found primarily in the outer membrane fraction. Succinate-cytochrome c reductase, succinate dehydrogenase, cytochrome oxidase, β-hydroxybutyrate dehydrogenase, α-ketoglutarate dehydrogenase, lipoamide dehydrogenase, NAD- and NADH-isocitrate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and ornithine transcarbamoylase were found in the inner membrane-matrix fraction. Nucleoside diphosphokinase was found in both the outer membrane and soluble fractions; this suggests a dual localization. Adenylate kinase was found entirely in the soluble fraction and was released at a lower digitonin concentration than was the outer membrane; this suggests that this enzyme is localized between the two membranes. The inner membrane-matrix fraction was separated into inner membrane and matrix by treatment with the nonionic detergent Lubrol, and this separation was used as a basis for calculating the relative protein content of the mitochondrial components. The inner membrane-matrix fraction retained a high degree of morphological and biochemical integrity and exhibited a high respiratory rate and respiratory control when assayed in a sucrose-mannitol medium containing EDTA.     

OBSERVATIONS ON THE BASEMENT MEMBRANES IN RAT KIDNEY

Basement membranes in the kidney are made up of a homogeneous matrix. In argyria, silver passes from the blood in the ionic form and diffuses into the kidney basement membranes in which it is precipitated. X-ray diffraction studies of "silver-stained" rat kidneys show that most of the silver in the kidneys is combined with some form of sulfur. Histochemical staining for sulfhydryls and disulfides demonstrates the presence of these groups in basement membranes. It appears that silver ions combine with either or both the sulfhydryl or disulfide groups in the basement membranes and also in mitochondria (when the silver diffuses into a cell).     

ON THE MECHANISM OF DRUG HYDROXYLATION IN RAT LIVER MICROSOMES

The TPNH- and O₂-dependent drug hydroxylation system of liver microsomes has been studied using normal rats and rats in which the drug-hydroxylating activity has been enhanced by repeated injections of phenobarbital. The oxidative demethylation of aminopyrine is employed as an assay. Optimal conditions for the assay with regard to the concentrations of TPNH and aminopyrine are established. TPN inhibits the reaction in a competitive manner, similarly to its effect on the microsomal TPNH-cytochrome c reductase. Drug hydroxylation, but not the "TPNH oxidase," TPNH-cytochrome c, -2,6-dichlorophenolindophenol, or -neotetrazolium reductase reaction, or the TPNH-dependent lipid peroxidation, is blocked by carbon monoxide. Microsomes from phenobarbital-treated rats exhibit increased activities of the various TPNH-linked reductase reactions, parallel to the increased drug hydroxylation activity, whereas the "TPNH oxidase" activity does not change appreciably. Measurements with microsomes from drug-treated animals reveal a 1:1:1 stoichiometry of aminopyrine-dependent oxygen uptake, TPNH oxidation, and formaldehyde formation. Attempts to solubilize the drug-hydroxylating enzyme system are also presented. It is concluded that the drug-hydroxylating enzyme system involves the microsomal TPNH-cytochrome c reductase and CO-binding pigment, and a hypothetic reaction scheme accounting for the data presented is proposed.     

大鼠肝细胞膜高密度脂蛋白受体的研究

以 ¹²⁵碘标记不含apoE的人HDL₃为配体,采用简便的聚乙二醇沉淀分离法,建立了纯化的大鼠肝细胞膜HDL受体分析法,并对膜HDL受体的性质进行了研究。     

山莨菪碱对人红细胞膜蛋白及膜脂运动的影响

本文采用自旋标记顺磁共振波谱技术,研究了山茛菪碱对人红细胞膜蛋白和膜脂运动的影响.结果表明:用马来酰亚胺标记的人红细胞膜,加入山茛菪碱后,其顺磁共振波谱中强、弱固定化作用谱的峰值比增大,膜蛋白的运动受到限制.山茛菪碱对红细胞膜脂的作用部位主要在极性头部,并影响膜脂的流动性.本文还对山茛菪碱与红细胞膜作用的可能机制进行了讨论.     

STUDIES ON SOLUBLE PROTEINS RELEASED FROM THE SYNAPTIC VESICLES OF RAT BRAIN CORTEX

Abstract— The soluble proteins released from the synaptic vesicles of rat cerebral cortex were studied. One fraction (D₄) of these proteins was released in parallel with release of acetylcholine when synaptic vesicles were incubated at 37°C for 10 min in isotonic medium. Another fraction (Dj) was liberated from synaptic vesicles when their membranes were ruptured by mild treatment under hyposmotic conditions and freeze-thawing after release of D₁ fraction. Fractions D₁ and D₂ contained 12 and 9 per cent, respectively, of the total protein in the synaptic vesicles. Some properties of these fractions were investigated by zone electrophoresis and ultracentrifugation, and by measuring their binding capacities for [¹⁴C]acetylcholine and various enzyme activities related to acetylcholine metabolism.     

肝炎平对D－氨基半乳精所致肝损害保护作用的酶组织化学研究

本实验采用Ｄ－氨基半乳糖（Ｄ－ＧａｌＮ）诱导的大鼠急性肝损伤模型,观察大鼠肝脏组织化学的变化,探讨肝炎平对急性肝损伤的保护作用。实验分为四组,即正常对照组、模型组、肝炎平及肝得健保护组。结果表明：肝炎平对肝细胞膜系统有一定的保护作用。肝炎平组和肝得健组ＳＤＨ、ＣＣＯ及ＣｈＥ活性明显高于模型组,且与正常对照组相近。本实验模型组ＡＣＰ的活性明显高于正常组,而肝炎平组ＡＣＰ的活性明显低于模型组,与正常对照组无显著性差异。提示：肝炎平可显著改善因Ｄ－氨基半乳糖所致肝损害的作用。且其对肝细胞的保护作用与肝得健一致。     

致癌大鼠肝细胞内蛋白磷酸化

本实验观察到二乙基亚硝胺(DENA)致癌大鼠肝细胞液及颗粒提取液中P21及P42磷酸化都比正常肝高,增高的程度似与肝细胞癌变相关。新生一天及四天大鼠肝中P21及P42磷酸化也有增高,但再生肝中不增高。磷酸化21的磷酸根接在丝氨酸羟基,PP21不含磷酸酪氨酸或磷酸苏氨酸,或含量极微。     

HETEROGENEITY OF ROUGH-SURFACED LIVER MICROSOMAL MEMBRANES OF ADULT,PHENOBARBITAL-TREATED,AND NEWBORN RATS

Rough microsomes from the livers of adult, phenobarbital-treated, and newborn rats were subfractionated on a continuous sucrose gradient. Among the subfractions a marked heterogeneity in the distribution patterns of some enzyme activities appears. The isopycnic density of the various fractions in aqueous sucrose ranges from 1.17 to 1.25. The sedimentation coefficients (s₀) in 0.25 M sucrose lie between 0.4 x 10³ S and 1.2 x 10³ S. In adult animals, the NADH- and NADPH-cytochrome c reductase as well as the G6Pase activities are much higher in the slower sedimenting fractions than in the pellet. The increase in the level of G6Pase induced by fasting as well as the phenobarbital-induced changes are most prominent in the slowly sedimenting fractions. Three injections of phenobarbital have no effect on the specific NADPH-cytochrome c reductase activity in the pellet, but cause a significant increase of this enzyme activity in the light fractions. In the newborn animal, the NADH-ferricyanide reductase and NADPH-cytochrome c reductase activities are highest in the light fractions. On the other hand, the amount of cytochrome b ₅ is evenly distributed in all cases. Short-term incorporation of leucine-¹⁴C and glycerol-³H in vivo after phenobarbital treatment shows contrasting results, as the former is increased and the latter is decreased in the slowly sedimenting fractions. Leucine-¹⁴C incorporation into isolated, total membrane proteins is greater in both phenobarbital-treated and newborn animals than in untreated adults. The data support a multistep model for membrane biogenesis and indicate dynamic and individual behavior of the different parts of the rough-surfaced endoplasmic reticulum.     

THE PHOSPHORYLATION OF NUCLEAR PROTEINS IN THE REGENERATING AND PREMALIGNANT RAT LIVER AND ITS SIGNIFICANCE FOR CELL PROLIFERATION

In liver regeneration or neoplastic transformation, phosphorylation of nuclear proteins is stimulated. In the regenerating liver all main histone fractions are involved in this process. The type of histone phosphorylated seems to be dependent on the position of the partially synchronized cells within the generation cycle. At a time when most cells are exhibiting maximum HnRNA-synthesis, histone F2a2 belongs to those fractions with highly stimulated phosphate incorporation. Phosphorylation of this fraction alone is stimulated by cyclic AMP in parallel to a stimulation of HnRNA-synthesis. The preneoplastic liver is characterized by oscillating phosphorylation and dephosphorylation reactions of nearly all histone fractions during the first days of N-nitroso-diethylamine administration. After 2 months of carcinogen feeding a 50–150% stimulation of the phosphorylation of Fl subfractions is observed. The phosphate content of the other histones, however, has returned to the original level. A series of further proteins, isolated together with the histones, show very similar phosphorylation characteristics. These proteins are mostly of non-histone origin. It is suggested that some of them are responsible for the transport of RNA with messenger properties within the cell.     

IN VITRO STIMULATION OF ENZYME SECRETION AND THE SYNTHESIS OF MICROSOMAL MEMBRANES IN THE PANCREAS OF THE GUINEA PIG

Several mechanisms have been suggested to explain how secretory cells remove from the plasmalemma the excess membrane resulting from the insertion of granule membrane during exocytosis: intact patches of membrane may be internalized and then reutilized within the cell; alternatively these membranes may be either disassembled to subunits or degraded. In the latter case new membranes should be synthetized at other sites of the cell, probably in the rough-surfaced endoplasmic reticulum (RER) and the Golgi complex. In the present research, membrane subfractions were obtained from rough microsomes (derived from fragmented and resealed RER cisternae) and from smooth microsomes (primarily contributed by Golgi stacks and vesicles) of the guinea pig pancreas by incubation at 4°C for 4 hr in 0.0005 M puromycin at high ionic strength followed by mild (pH 7.8) alkaline extraction with 0.2 M NaHCO₃. Such treatments release the majority of nonmembrane components of both microsomal fractions (i.e., contained secretory enzymes, ribosomes, and absorbed proteins of the cell sap) and allow the membranes to be recovered by centrifugation. The effect of in vitro stimulation of enzyme secretion (brought about in pancreas slices by 0.0001 M carbamoyl choline) on the rate of synthesis of the phospholipid (PLP) and protein of these membranes was then investigated. In agreement with previous data, we observed that in stimulated slices the synthesis of microsomal PLP was greatly increased. In contrast, the synthesis of microsomal membrane proteins was unchanged. These results suggest that exocytosis is not coupled with an increased rate of synthesis of complete ER and Golgi membranes and are, therefore, consistent with the view that excess plasma membrane is preserved and reutilized, either as discrete membrane patches or as membrane macromolecules, throughout the secretory cycle.     

AXONAL TRANSPORT OF PROTEINS IN THE HYPOTHALAMO-NEUROHYPOPHYSIAL SYSTEM OF THE RAT

—Axonal transport of proteins in the hypothalamo-neurohypophysial system of the rat was studied after a local injection of [³⁵S]cysteine in the region of the supraoptic nucleus. The migration of labelled proteins was followed by measuring the specific radioactivity of the proteins in various parts of the hypothalamo-neurohypophysial tract. Between 2 and 4 h after the isotope injection there was a sharp increase in the protein-bound specific radioactivity of the posterior pituitary lobe, demonstrating that a transport of ³⁵S-labelled proteins had occurred from the supraoptic nucleus to the neurohypophysis. The rate of the transport was 2-3 mm/h. During the first 24 h after the injection a continuous accumulation of labelled material occurred in the neural lobe. Considerable radioactivity could still be recovered 6 days after the isotope injection. Fractionation of the neurohypophysial proteins by polyacrylamide gel electrophoresis revealed that approximately 90 per cent of the radioactivity of the soluble proteins was recovered in a single protein fraction. Labelling of this fraction was not observed until 2 h after isotope injection. The radioactivity increased markedly up to 4 h. It is suggested that this protein component is involved in the neurohypophysial response to osmotic stress since the protein disappeared from the posterior lobe upon dehydration of the rat.     

THE ISOLATION OF A CELL MEMBRANE FRACTION FROM RAT LIVER

A procedure is described for isolating cell membranes from rat liver homogenates. 20 gm. of rat liver was homogenized in a Dounce homogenizer in ice cold water buffered to pH 7.5 with NaHCO₃, rupturing all of the cells and most nuclei. The diluted homogenate was filtered through cheesecloth to remove precipitated nucleoprotein and centrifuged at 1500 g, 10 minutes, to sediment a crude membrane fraction. The membrane containing sediment was recentrifuged 3 times in conical tubes (1220 g, 10 minutes), the top layer of the 2-layered sediment being retained. Flotation in a sucrose solution d = 1.22 freed the preparation from contaminating cell fragments and nuclear membranes not previously disintegrated. The floating material ~0.4 ml. was quite homogeneous and consisted of thin amorphous membranes. Electron micrographs revealed numerous double profiles similar in shape and dimensions to apposed liver cell membranes in intact tissue.     

ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER : I. Biochemical Methods

The series introduced by this paper reports the results of a detailed analysis of the microsomal fraction from rat liver by density gradient centrifugation. The biochemical methods used throughout this work for the determination of monoamine oxidase, NADH cytochrome c reductase, NADPH cytochrome c reductase, cytochrome oxidase, catalase, aminopyrine demethylase, cytochromes b₅ and P 450, glucuronyltransferase, galactosyltransferase, esterase, alkaline and acid phosphatases, 5'-nucleotidase, glucose 6-phosphatase, alkaline phosphodiesterase I, N-acetyl-β-glucosaminidase, β-glucuronidase, nucleoside diphosphatase, aldolase, fumarase, glutamine synthetase, protein, phospholipid, cholesterol, and RNA are described and justified when necessary.