High-performance liquid chromatography of proteins: Purification of the acidic isozyme of adenylosuccinate synthetase from rat liver

High-performance ion-exchange liquid chromatography was utilized for the purification of the acidic isozyme of adenylosuccinate synthetase from rat liver. Initial steps in the purification included ammonium sulfate fractionation and DEAE-cellulose and agarose-GTP affinity columns. The final steps were done on a SynChropak AX-300 anion-exchange support. The enzyme was purified 3000-fold with an overall yield of 10%. The enzyme preparation exhibited only one protein band on gel electrophoresis.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

Purification and crystallization of rat liver fatty acid synthetase

A purification procedure for rat liver fatty acid synthetase has been developed using polyethylene glycol. This procedure results in high yields of the enzyme which is essentially free of endogenous proteolytic nicking and also free of any contaminating proteases. The fatty acid synthetase obtained has a specific activity range of 1.8–2.1 measured at 25 °C and is stable at 4 °C for a few weeks and indefinitely when frozen. Approximately 1 mg of enzyme can be obtained per gram of induced rat liver. The enzyme is pure as determined by sodium dodecyl sulfate-gel electrophoresis, sedimentation velocity, and immunoelectrophoresis. The first crystallization of rat liver fatty acid synthetase is also reported.     

Structural studies of a mitochondria-free plasma membrane fraction from rat liver

Liver plasma membranes virtually free of contaminating mitochondria have been prepared. Sodium dodecylsulfate-polyacrylamide gel electrophoresis reveals a membrane protein resistant to papain digestion in the intact membranes but readily hydrolyzed in membranes disrupted by detergent or sonication.Electron microscopy of mechanically deformed membranes reveals fibrils within the membrane which appear to be protein in nature but which also persist in papain digested membranes.     

DNA Replication by a membrane-DNA complex from rat liver mitochondria

The results presented here indicate that mitochondrial DNA (mtDNA) synthesis occurs on the inner mitochondrial membrane and that a membrane-DNA complex, enriched in newly synthesized DNA, can be isolated. The complex is able to synthesize DNA in vitro. Enrichment studies demonstrated that mtDNA synthesis occurs on an intact membrane-DNA complex in vitro and that pulse-labeled mtDNA could be chased from the membrane-DNA complex to the top fraction of the discontinuous sucrose gradient. The membrane-DNA complex was also shown to carry out replicative synthesis of mtDNA in vitro. Replication was shown to be asynchronous with heavy-strand synthesis preceding light-strand synthesis. The progression of mtDNA replication by the membrane-DNA complex was shown to be from small fragments (<13 S) to larger fragments (14–24 S) liberated from closed circular molecules, to a heat-stable 27 S molecule, and finally to a 38 S heat-stable molecule. The time estimated to progress from small fragments to the 38 S molecule is 120 min.     

Haem-binding proteins of the rat liver cytosol

Gel filtration of soluble supernatant fraction obtained from livers of rats 10 min after an injection of the haem precursor 5-amino [³h] laevulinic acid shows the presence of a major radioactive fraction which upon gel filtration is similar in elution volume to ligandin. 20 min after administration of the precursor four previously minor components also come into prominence. This pattern is a characteristic of in vivo binding since a different elution pattern is obtained if soluble supernatant fraction from rat liver is labelled in vitro by incubation either with [³H] haem-labelled mitochondria, [³H] haem-labelled microsomes or with [³H] haemin.These results are discussed with particular reference to ligandin.     

Effect of the oral hypoglycemic agent 2-tetradecylglycidic acid on fatty acid oxidation in suckling rats in vivo and in perfused liver

The hypoglycemic agent, 2-tetradecylglycidic acid (TDGA), administered in vivo lowered the concentration of plasma glucose and ketone bodies but raised the concentration of liver and plasma triglycerides in 10-day-old suckling rats. Phospholipid and cholesterol content of the plasma and liver were unaffected by drug treatment. TDGA inhibited the in vivo oxidation of [1-¹⁴C]palmitate but not that of [1-¹⁴C]decanoate. In suckling rat liver perfusion, TDGA totally inhibited ketone body formation from palmitate and depressed ketone body production from decanoate by 20%. Liver ATP and ADP content in the presence of TDGA decreased although this was probably a reflection of the increased triglyceride content of the liver since the $\text{ATP}\text{ADP}$ was the same as control livers. The results are discussed in relation to the diet and to the inhibition of carnitine acyl transferase in suckling rats.     

Glucagon and fasting do not activate peroxisomal fatty acid beta-oxidation in rat liver

In a study of the endocrine control of peroxisomes, the effects of acute glucagon treatment and fasting on hepatic peroxisomal beta-oxidation in rats have been investigated. The activity of the rate-limiting peroxisomal beta-oxidation enzyme, fatty acyl-CoA oxidase, was measured to determine whether activation of peroxisomal beta-oxidation could account for the increase in total hepatic fatty acid oxidation following acute glucagon exposure. Catalase, a peroxisomal enzyme not directly involved in beta-oxidation, was also measured as a control for total peroxisomal activity. No changes with acute glucagon treatment of intact animals were observed with either activity as measured in liver homogenates or partially purified peroxisomal fractions. These observations indicate the lack of acute control by glucagon of peroxisomal function at the level of total enzyme activity. Previous work on the effects of fasting on hepatic fatty acid beta-oxidation [H. Ishii, S. Horie, and T. Suga (1980) J. Biochem. 87, 1855-1858] suggested an enhanced role for the peroxisomal beta-oxidation pathway during starvation. It was found that the peroxisomal beta-oxidation system, as measured by fatty acyl-CoA oxidase activity, does increase with duration of fast when expressed on a per gram wet weight liver basis. However, when this activity is expressed as total liver capacity, a decline in activity with increasing duration of fast is observed. Furthermore, this decline in peroxisomal capacity parallels the decline in total liver capacity for citrate synthase, a mitochondrial matrix enzyme, and total liver protein. These data indicate that peroxisomal beta-oxidation activity is neither stimulated nor even preferentially spared from proteolysis during fasting.     

Isolation and characterization of a tryptic fragment containing the thioesterase segment of fatty acid synthetase from the uropygial gland of goose.

Trypsin treatment of purified fatty acid synthetase from the uropygial gland of goose released a 33,000 molecular weight peptide from the 270,000 molecular weight synthease. A combination of ammonium sulfate precipitation, Sephadex G-100 gel filtration, anion-exchange chromatography with QAE-Sephadex, and cation-exchange chromatography with cellulose phosphate gave rise to the first homogeneous preparation of the 33,000 molecular weight fragment containing fatty acyl-CoA thioesterase activity. Amino acid composition of this peptide was quite similar to that of the intact fatty acid synthetase except for a lower valine content; a partial specific volume of 0.734 was calculated for the thioesterase fragment. The pH optimum for the thioesterase was near 7.5 and the enzyme showed a high degree of preference for CoA esters of fatty acids with 16 or more carbon atoms. Palmitoyl-CoA inhibited the enzyme and therefore the rate of hydrolysis was not proportional to the amount of protein at low concentrations. Inclusion of bovine serum albumin in the reaction mixture prevented this inhibition. Disregarding the substrate inhibition, an apparent K_m of 5 × 10^?5m and a V of 340 nmol/min/mg were calculated. The thioesterase was inhibited by active serine-directed reagents such as phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate as well as by SH-directed reagents as p-chloromercuribenzoate and N-ethylmaleimide. The isolated thioesterase fragment generated antibodies in rabbits and the antithioesterase inhibited the enzymatic activity of fatty acid synthetase. The antithioesterase showed immunoprecipitant lines with fatty acid synthetase from the uropygial gland and the synthetase from the liver of goose. Anti-fatty acid synthetase prepared against the enzyme from the gland cross-reacted with the thioesterase segment. Even though the synthetase from the uropygial gland synthesizes multimethyl-branched fatty acids in vivo, the thioesterase segment of this synthetase appears to be quite similar to that isolated from the rat.     

Synthesis of multimethyl-branched fatty acids by avian and mammalian fatty acid synthetase and its regulation by malonyl-CoA decarboxylase in the uropygial gland

Fatty acid synthetase, partially purified by gel filtration with Sepharose 4B from goose liver, showed the same relative rate of incorporation of methylmalonyl-CoA (compared to malonyl-CoA) as that observed with the purified fatty acid synthetase from the uropygial gland. In the presence of acetyl-CoA, methylmalonyl-CoA was incorporated mainly into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8,10-pentamethyl-dodecanoic acid by the enzyme from both sources. Methylmalonyl-CoA was a competitive inhibitor with respect to malonyl-CoA for the enzyme from the gland just as previously observed for fatty acid synthetase from other animals. Furthermore, rabbit antiserum prepared against the gland enzyme cross-reacted with the liver enzyme, and Ouchterlony double-diffusion analyses showed complete fusion of the immunoprecipitant lines. The antiserum inhibited both the synthesis of n-fatty acids and branched fatty acids catalyzed by the synthetase from both liver and the uropygial gland. These results suggest that the synthetases from the two tissues are identical and that branched and n-fatty acids are synthesized by the same enzyme. Immunological examination of the 105,000g supernatant prepared from a variety of organs from the goose showed that only the uropygial gland contained a protein which cross-reacted with the antiserum prepared against malonyl-CoA decarboxylase purified from the gland. Thus, it is concluded that the reason for the synthesis of multimethyl-branched fatty acids by the fatty acid synthetase in the gland is that in this organ the tissue-specific and substrate-specific decarboxylase makes only methylmalonyl-CoA available to the synthetase. Fatty acid synthetase, partially purified from the mammary gland and the liver of rats, also catalyzed incorporation of [methyl-¹⁴C]methylmalonyl-CoA into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8-tetramethylundecanoic acid with acetyl-CoA and propionyl-CoA, respectively, as the primers. Evidence is also presented that fatty acids containing straight and branched regions can be generated by the fatty acid synthetase from the rat and goose, from methylmalonyl-CoA in the presence of malonyl-CoA or other precursors of n-fatty acids. These results provide support for the hypothesis that, under the pathological conditions which result in accumulation of methylmalonyl-CoA, abnormal branched acids can be generated by the fatty acid synthetase.     

Induction of experimental phenylketonuria-like conditions in chick embryo. Effect on amino acid concentration in brain,liver and plasma

Experimental hyperphenylalaninemia has been induced in chick embryos between 11–20 days of incubation by daily injection of α-methylphenylalanine and phenylalanine. Brain and liver weight decreased after 8 days of treatment. An increase of nearly 14-fold in the brain phenylalanine/tyrosine ratio was observed after 9 days of treatment. Similar results were obtained in liver, although the increase found in this case was smaller than in brain. Chronic hyperphenylalaninemia induced a clear rise in the levels of plasma and liver valine, leucine and isoleucine, while in brain these levels did not change significantly. Plasma and brain glycine content was also enhanced by this treatment. Brain tyrosine concentration was clearly decreased in these conditions, in contrast to the enhancement reported after this and other treatments in various animal species. Thus, the higher value of the brain phenylalanine/tyrosine ratio obtained by α-methylphenylalanine plus phenylalanine administration was due to both an increase in the phenylalanine and a decrease in the tyrosine levels, conditions that have been also found in human phenylketonurics. Therefore, the treatment here reported was an excellent method for imitating the conditions of phenylketonuria during the period of rapid myelination in the chick, one of the most dramatic in nervous system development.     

Measurement of a folate binding protein from rat liver cytosol by radioimmunoassay

A radioimmunoassay has been developed for the folate binding protein from rat liver cytosol with a molecular weight of 150,000 which was recently purified to homogeneity (Suzuki, N., and Wagner, C., 1980, Arch. Biochem. Biophys.199, 236–248). This method has indicated that the binding protein (FBP-CII) is found primarily in the liver. A significant amount of FBP-CII was also found in the kidney and much reduced levels in spleen, serum, brain, lung, and heart. No FBP-CII could be detected in small intestine, skeletal muscle, or testes. Small amounts of cross-reacting material were found in the livers of mouse, dog, chick, and humans. Levels of FBP-CII were not decreased in the livers of folate-deficient rats. Assays of rat fetal liver and kidney 2 days prior to birth showed much lower levels which increased rapidly at birth. These data are consistent with the FBP-CII fulfilling a role as a folate storage protein in rat liver.     

Resolution and reconstitution of soluble components of rat liver microsomal vitamin D3-25-hydroxylase

Solubilized components of the vitamin D₃-25-hydroxylase, isolated from intact rat liver microsomes known to catalyze the C-25 oxidation of vitamin D₃in vitro, have been separated into two submicrosomal fractions enriched in detergent-solubilized NADPH-cytochrome c reductase and cytochrome P-450 or P-448. The P-450 hemoprotein-containing fraction was obtained by solubilization with cholic acid followed by treatment with the nonionic detergent, Emulgen 911, yielding a final preparation with a specific content of 7.25 nmol/mg microsomal protein. The reduced triphosphopyridine nucleotide-dependent cytochrome P-450 reductase activity, as detected by its ability to reduce the artificial electron acceptor, cytochrome c, was isolated free of cytochromes b₅ or P-450 by solubilization with deoxycholate and chromatography on DEAE-cellulose. The reductase component was found to exhibit kinetic properties with Michaelis constants: K_m(NADPH) = 3.14 μM, K_m(NADH) = 31.25 μM, and K_{m(cyt c)} = 12.34 μM. The NADPH-cytochrome c reductase activity was sensitive to NADPH-reversible inhibition by NADP, but not rotenone or cyanide. When the isolated components were incubated in the presence of an NADPH-generating system and carbon monoxide under anaerobic conditions, enzymatic reduction of the P-450 hemoprotein was measured by the appearance of characteristic absorbances at 420 and 450 nm of the reduced carbon monoxide vs. reduced difference spectrum. Furthermore, when the soluble submicrosomal components were reconstituted with excess reduced triphosphopyridine nucleotide, ³H-labeled vitamin D₃, and soluble cytosolic supernatant, full vitamin D₃-25-hydroxylase activity was restored at rates of up to 7.68 pmol/h/mg protein, with an apparent turnover number of cytochrome P-450 of 1.16 to 1.20 under conditions where the concentrations of the hemoprotein were rate limiting for net product formation. These results strongly support the hypothesis that the rat liver microsomal mixed-function oxidase, vitamin D₃-25-hydroxylase, consists of at least two membrane-bound protein components, NADPH-cytochrome c reductase and a cytochrome P-450 terminal oxidase, for the catalytic conversion of vitamin D₃ to 25-hydroxyvitamin D₃.