Heparinase activity in rat liver.

Heparin was degraded to oligosaccharides by an endoglycosidase present in rat liver lysosomes. Inorganic sulfate equivalent to approximately one sulfamide bond cleaved per heparin chain was also released in incubations of N-[³⁵S]heparin with crude lysosomal preparations. There was no evidence of exoglycosidase or further sulfamidase activity although oligosaccharides approaching the size of tetrasaccharide were produced. The endoglycosidase has a broad pH-dependence with optimum activity observed at pH 4.4 and intermediate activity at pH 5.5 and 3.8.     

Hydroperoxide-metabolizing systems in rat liver.

1. Metabolism of added hydroperoxides was studied in hemoglobin-free perfused rat liver and in isolated rat hepatocytes as well as microsomal and mitochondrial fractions. 2. Perfused liver is capable of removing organic hydroperoxides [cumene and tert-butyl hydroperoxide] at rates up to 3--4 mumol X min-1 X gram liver-1. Concomitantly, there is a release of glutathione disulfide (GSSG) into the extracellular space in a relationship approx. linear with hydroperoxide infusion rates. About 30 nmol GSSG are released per mumol hydroperoxide added per min per gram liver. GSSG release is interpreted to indicate GSH peroxidase activity. 3. GSSG release is observed also with added H2O2. At rates of H2O2 infusion of about 1.5 mumol X min-1 X gram liver-1 a maximum of GSSG release is attained which, however, can be increased by inhibition of catalase with 3-amino-1,2,4-aminotriazole. 4. A contribution of the endoplasmic reticulum in addition to glutathione peroxidase in organic hydroperoxide removal is demonstrated (a) by comparison of perfused livers from untreated and phenobarbital-pretreated rats and (b) in isolated microsomal fractions, and a possible involvement of reactive iron species (e.g. cytochrome P-450-linked peroxidase activity) is discussed. 5. Hydroperoxide addition to microsomes leads to rapid and substantial lipid peroxidation as evidenced by formation of thiobarbituric-acid-reactive material (presumably malondialdehyde) and by O2 uptake. Like in other types of induction of lipid peroxidation, malondialdehyde/O2 ratios of 1/20 are observed. Cumene hydroperoxide (0.6 mM) gives rise to 4-fold higher rates of malondialdehyde formation than tert-butyl hydroperoxide (1 mM). Ethylenediamine tetraacetate does not inhibit this type of lipid peroxidation. 6. Lipid peroxidation in isolated hepatocytes upon hydroperoxide addition is much lower than in isolated microsomes or mitochondria, consistent with the presence of effective hydroperoxide-reducing systems. However, when NADPH is oxidized to the maximal extent as evidenced by dual-wavelength spectrophotometry, lipid peroxidation occurs at large amounts. 7. A dependence of hydroperoxide removal rates upon flux through the pentose phosphate pathway is suggested by a stimulatory effect of glucose in hepatocytes from fasted rats and by an increased rate of 14CO2 release from [1-14C]glucose during hydroperoxide metabolism in perfused liver.     

Nonexistence of N-acetylseryl-tRNA in rat liver.

Reports of the existence of N-acetylseryl-tRNA in rat liver were reinvestigated. Methods were developed to permit recovery of N-acetylserine in a yield of 30--40% from N-acetylseryl-tRNA added to liver homogenates and cell-free incubations. [14C]Serine and [3H]acetate were injected into rats pretreated with iron and into rats after partial hepatectomy, and aminoacyl-tRNA was isolated from their livers. The amount of radioactivity associated with N-acetylserine in the amino acids released by hydrolysis from the aminoacyl-tRNA was negligible. No formation of N-acetylseryl-tRNA could be observed in incubations of acetyl CoA and seryl-tRNA or tRNA with enzyme fractions from liver of rats pretreated with iron. It is concluded that previous reports of the existence of N-acetylseryl-tRNA in rat liver are erroneous.     

Turnover of metallothioneins in rat liver.

Two electrophoretically distinguishable metallothioneins were isolated from the livers of Cd2+-treated rats and had thiol group/metal ratios of 3:1, a total metal content, in each of these proteins, of 3.6 atoms of Cd2+ + 2.4 atoms of Zn2+/molecule and 4.2 atoms of Cd2+ + 2.8 atoms of Zn2+/molecule and respective apoprotein mol.wts. of 5844 and 6251. Studies with 1 h pulse labels of [3H]cysteine, given after a single injection of ZnCl2 or CdCl2, showed that these metals stimulated radioactive isotope incorporation into the metallothioneins over the control value by 10- and 15-fold respectively. This stimulation was maximal at 4 h after a single CdCl2 injection and decreased to control values by 16 h, suggesting that either a translational event is responding to free intracellular Cd2+ or a short-lived mRNA is being produced or stabilized in response to the metal treatment. In rats chronically exposed to CdCl2, the metallothioneins increased to 0.2% of the liver wet weight from a control value of 2--4 mumol/kg of liver, with a maximum rate of accumulation of 2--3 mumol/h per kg of liver. The turnover of these proteins in control animals was 0.3--0.6 mumoles/h per kg of liver, measured by the rate of disappearance of 203Hg2+, which binds irreversibly to the metallothioneins. Pretreatment with CdCl2 completely stopped the rapid 203Hg turnover observed in untreated animals. Unlike CdCl2, treatment with ZnCl2 increased the concentration of metallothioneins to a new steady-state pool, 11 mumole/kg of liver, after 10 h. The increase in the zinc-thionein pool by exposure to ZnCl2 in vivo was determined to be primarily due to a stimulation of metallothionein biosynthesis.     

Compartmentation of NADPH in rat liver.

Rat liver slices were incubated with specifically ³H-labeled glucoses and [2-³H]sorbitol, and the incorporations of ³H into fatty acids and cholesterol were determined. Incorporation of ³H from [1-³H]glucose relative to that from [3-³H]glucose via NADPH formed in the pentose cycle was similar into fatty acids and cholesterol. This indicates (1) the presence of a common pool of NADPH formed via the pentose cycle, from which is derived the reductive hydrogens for fatty acid and cholesterol synthesis; (2) the absence of a major separate pool of NADPH formed from glucose by microsomal glucose dehydrogenase (EC 1.1.1.47) catalysis for use in cholesterol synthesis. ³H from [4-³H]glucose and from [2-³H]sorbitol was incorporated into cholesterol more than into fatty acids relative to the incorporations of ³H from [3-³H]glucose. Assuming that the ³H from [4-³H]glucose and from [2-³H]sorbitol were incorporated via the conversion, catalyzed by malic enzyme, of NADH to NADPH, this indicates the Compartmentation of the NADPH formed via malic enzyme catalysis from that formed via the pentose cycle. Alternatively, NADH provides reductive hydrogens for cholesterol synthesis in greater measure than in fatty acid formation or the stereochemistry of the synthetic processes are such that [A-³H]NADPH has greater excess than [B-³H]NADPH to cholesterol synthesis relative to fatty acid synthesis.     

Biosynthesis of serum albumin in rat liver. Isolation and probable structure of ''proalbumin'' from rat liver.

1. Two methods are described for the preparation of 'proalbumin' in good yields from rat liver. 2. One of the methods does not depend on the use of specific antisera. 3. The product from both methods is identical as judged by electrophoresis on polyacrylamide gel, isoelectric focusing on pH gradients, ion-exchange chromatography and quantitative immunoelectrophoresis. The protein also appears to be radiochemically pure by these criteria. 4. The protein is free from serum albumin as judged by isoelectric focusing and co-chromatography on ion-exchange columns. It is judged to be free from other proteins by these same criteria and by specific precipitation with antibody. 5. It is converted into serum albumin by limited tryptic hydrolysis. The albumin so produced has the same N-terminal (glutamic acid) and C-terminal (alanine) amino acids as reported for rat serum albumin. 6. A hexapeptide is liberated from the N-terminal end of 'proalbumin' simultaneously. It contains three arginine, one phenylalanine, one valine and one glycine residues.     

Conformational changes in rat liver chromatin after liver regeneration.

N-Pyrenemaleimide, a fluorescent probe that specifically labels histone H3 of rat liver chromatin in situ, was used to monitor the accessibility of histone H3 in chromatin isolated from rat liver at different times during degeneration. At times of maximum DNA synthesis (18--24 h after hepatectomy), the accessibility of the probe was found to be markedly (40--50%) increased. This increase is abolished, however, by treatment of the chromatin fibres with high salt (2 M-NaCl) or detergent. Tryptophan fluorescence was also enhanced at points of maximum DNA synthesis, suggesting that some non-histone tryptophan-containing protein was being synthesized. The polarization of the labelled histone H3 is not markedly altered, suggesting that fibre aggregation or dissociation does not occur. Mononucleosomes extracted from sham-operated and hepatectomized animals did not exhibit any difference in binding to the probe. Also, analysis of the chromatin protein by electrophoresis on detergent- and acid/urea/ Triton-X-100-containing polyacrylamide gels showed no detectable difference in histone H3 : 1, H3 : 2 or H3 : 3 subclasses.     

CDP-diacylglycerol synthesis in rat liver mitochondria.

CDP-diacylglycerol for polyglycerophosphatide biogenesis can be synthesized within rat liver mitochondria. This membrane-associated enzyme was predominantly located in the inner mitochondrial membrane. GTP had a significant effect in activating the microsomal CDP-diacylglycerol synthase, especially if the microsomes were preincubated with GTP in the presence of phosphatidic acid. This stimulatory effect of GTP on the microsomal enzyme was not detected in the mitochondrial fractions. The enzymes could be solubilized from the membrane fractions using CHAPS, and the detergent-soluble activity partially restored by addition of phospholipids. Mitochondrial and microsomal CDP-diacylglycerol synthase activity could be completely separated by anion-exchange column chromatography. The mitochondrial and microsomal CDP-diacylglycerol synthases appear to be two distinct enzymes with different localization and regulatory characteristics.     

Futile cycles in isolated perfused rat liver and in isolated rat liver parenchymal cells.

Isolated livers from fed rats were perfused with a medium containing glucose labeled uniformly with ¹⁴C and specifically with ³H. There was considerable formation of glucose from endogenous sources but simultaneously uptake of about half of the ¹⁴C in glucose. After 2 hours the ${}^3\text{H}{}^{14}\text{C}$ ratios in perfusate glucose decreased by 55–60% with (2-³H, U-¹⁴C), 40–50% with (5-³H, U-¹⁴C), 25–30% with (3-³H or 4-³H, U-¹⁴C) and by 10–15% with (6-³H, U-¹⁴C) glucose. Qualitatively comparable patterns were obtained with rat hepatocytes. These results demonstrate recycling of carbon between glucose and pyruvate. Superimposed upon this there is an extensive futile cycle between glucose and glucose 6-P. There is also futile cycling between fructose 6-P and fructose 1,6 P₂ and to a small extent between phosphoenol pyruvate and pyruvate.     

Gluconeogenesis from L-serine in rat liver.

Livers of fed and fasted rats were perfused $\text{in situ}$ in the presence and absence of 4.8 mM quinolinate, an $\text{in vivo}$ inhibitor of phosphoenolpyruvate carboxykinase. An assay of the hepatic activities of serine dehydratase and serine pyruvate transaminase and a comparison of the $\text{in vivo}$ incorporation of radioactivity from serine 3-¹⁴C and serine U-¹⁴C into blood glucose were also carried out in the above nutritional states. Our results demonstrate that gluconeogenesis from L-serine proceeds through two pathways. One, involving the reversal of the biosynthetic route of serine, bypasses conversion to pyruvate phosphoenolpyruvate and oxaloacetate and is not inhibited by quinolinate. This pathway appears to be the only one active in the fed state but produces a very insignificant amount of glucose. The other involves serine dehydratase mediated conversion of serine to pyruvate, is inhibited by quinolinate and becomes predominant during starvation.     

Iron-induced changes in rat liver isoferritins.

The effects of single and of multiple iron injection on the distribution of isoferritins was studied in rat liver with the aid of 14C-labelling either after or before iron treatment. Several effects of iron can be seen. Analysis of protein and labelling patterns show that it not only produces a disproportionate increase in the more-basic isoferritins, but may, in sufficient dose, actually lead to a decrease in the more-acidic isoferritins. Use of iron injection after radioactivity shows that it must give rise to post-assembly changes causing acidic isoferritins to become more basic. With a moderate iron dose this change is relatively slow, taking several hours, and seems to occur in addition to a differential stimulation of the synthesis of the more-basic isoferritins. With higher iron dosage the post-assembly changes may be so rapid that it is difficult to distinguish them from a switch in the pattern of synthesis.     

Glucosamine metabolism in regenerating rat liver.

1. Glycoprotein synthesis was investigated with [1-14C]glucosamine in vivo. [14C]Glucosamine was administered intravenously 24h after hepatectomy to rats. 2. Incorporation into the acid-soluble fraction was maximum at 15 min after injection both in sham-operated and hepatectomized rats. 3. Enhancement of incorporation into UDP-N-acetylhexosamine in regenerating liver was observed. However, its specific activity was lower, because of a greater enhancement of synthesis de novo of the amino sugar. 4. In the liver acid-insoluble fraction, maximum incorporation of [14C]glucosamine was at 30 min in sham-operated rats and 2 h in hepatectomized rats respectively. 5. In sham-operated rats, incorporation into the plasma acid-insoluble fraction followed that of the liver acid-insoluble fraction, but hepatectomy resulted in a rapid enchancement of incorporation into plasma. 6. It is concluded that synthesis of liver glycoproteins is stimulated after partial hepatectomy and that glycoproteins synthesized are released rapidly into the plasma.     

Proline transport in rat liver mitochondria.

Proline transport across the inner membrane of rat liver mitochondria shows the following properties: (a) It is stereospecific; the penetration of l-proline is two times faster than the penetration of dl-proline. (b) Proline is accumulated against a concentration gradient, (c) The transport of proline is enhanced in the presence of respiratory substrates such as succinate or tetramethylphenylenediamine + ascorbate; it is inhibited by uncouplers of oxidative phosphorylation. (d) Proline transport is inhibited by mersalyl and p-chloromercuribenzoate, but not by hydrophobic thiol blocking reagents; thus, proline transport involves thiol groups located in a very hydrophilic environment. The penetration of several other neutral amino acids (alanine, glycine, serine) is almost insensitive to mersalyl. These results suggest that proline does not travel across the mitochondrial membrane by free diffusion, but that its transport is mediated by a specific carrier. The rate of proline transport has been compared with the rates of the first two steps of proline oxidation: All of these rates are very similar, indicating that proline transport is not a limiting factor of proline metabolism in rat liver mitochondria.