The effect of linoleic acid and benzyl alcohol on the activity of glycosyltransferases of rat liver golgi membranes and some soluble glycosyltransferases

The effects of the membrane perturbing reagents linoleic acid and benzyl alcohol on the activities of four rat liver Golgi membrane enzymes, N-acetylglucosaminyl-, N-acetylgalactosaminyl-, galactosyl-, and sialytransferases and several soluble glycosyltransferases, bovine milk galactosyl- and N-acetylglucosaminyltransferases and porcine submaxillary N-acetylgalactosaminyltransferases have been studied. In rat liver Golgi membranes, linoleic acid inhibited the activities of N-acetylgalactosaminyl- and galactosyltransferases by 50% or greater, sialyltransferase by 10–15%, and N-acetylglucosaminyltransferase not at all. The isolated bovine milk N-acetylglucosaminyltransferase and porcine submaxillary N-acetylgalactosylaminyltranferase were not inhibited but bovine milk galactosyltransferase was inhibited by 95% or greater. The inhibition by linoleic acid on Golgi membrane galactosyltransferase appears to be a direct effect of the reagent on the enzyme. Incorporation of bovine milk galactosyltransferase into liposomes formed from saturated phospholipids, DMPC, DPPC, and DSPC (dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine) prevented inhibition of the enzyme activity suggesting that the lipid formed a barrier which did not allow linoleic acid access to the enzyme. The water soluble benzyl alcohol was more effective in inhibiting enzymes of the isolated rat liver Golgi complex. All four glycosyltransferases were inhibited, the N-acetylglucosaminyl- and N-acetylgalactosaminyltransferases by more than 95%. A higher concentration of benzyl alcohol was necessary to inhibit the galactosyltransferases than was required for the other Golgi enzymes. Benzyl alcohol also inhibited the isolated bovine milk N-acetylglucosaminyl- and galactosyltransferases 90% to 95%, respectively, but did not affect the isolated porcine submaxillary gland N-acetylgalactosaminyltransferase. Benzyl alcohol did not inhibit the milk galactosyltransferase incorporated into DMPC or DPPC liposomes but showed a complex effect on the activity of the enzyme incorporated into DSPC vesicles, a stimulation of activity at low concentrations followed by an inhibition. A lipid environment consisting of saturated lipids appears to present a barrier to inhibiting substances such as linoleic acid and benzyl alcohol, or lipid may stabilize the active conformation of the enzyme. The different effects of these reagents on four transferases of the Golgi complex suggest that the lipid environment around these enzymes may be different for each transferase.     

Insulin degradation V. Unmasking of glutathione-insulin transhydrogenase in rat liver microsomal membrane

Glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) inactivates insulin by cleaving its disulfide bonds. The distribution of GSH-insulin transhydrogenase in subcellular fractions of rat liver homogenates has been studied. From the distribution of insulin-degrading activity and marker enzymes (glucose-6-phosphatase and succinate-INT reductase) (INT, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) after cell fractionation by differential centrifugation, the immunological analysis of the isolated subcellular fractions with antibody to purified rat liver GSH-insulin transhydrogenase, and chromatographic analysis (on a column of Sephadex G-75 in 50% acetic acid) of the products formed from ¹²⁵I-labelled insulin after incubation with the isolated subcellular fractions, it is concluded that GSH-insulin transhydrogenase is located primarily in the microsomal fraction of rat liver homogenate. An enzyme(s) that further degrades insulin by proteolysis is located mainly in the soluble fraction; a significant amount of the protease(s) activity is also present in the mitochondrial fraction. The possibility has been discussed that the protease(s) acts upon the intermediate product of insulin degradation, A and B chains of insulin, rather than upon the intact insulin molecule itself.The GSH-insulin transhydrogenase in intact microsomes occurs in a latent state; it is readily released from the microsomal membrane and its activity is greatly increased by treatments which affect the lipoprotein membrane structure of microsomal vesicles. There include homogenization with a Polytron homogenizer, sonication, freezing and thawing, alkaline pH, the nonionic detergent Triton X-100, and phospholipases A and C.     

A liver factor that interconverts multiple forms of tyrosine aminotransferase.

Three activity peaks of rat liver soluble tyrosine aminotransferase have been resolved using hydroxyl-apatite chromatography. These peaks interconvert during storage of the soluble enzyme preparation in ice for 20 h. A component of a particulate fraction of liver which will interconvert the forms of tyrosine aminotransferase in vitro with no alteration of total enzyme activity has been detected. This factor is present in a 31, 000 gh pellet of liver and is solubilized by sonication. When the factor is subjected to dialysis or incubation at 25°C for 30 min. its effect on tyrosine aminotransferase is greatly diminished.     

Localization of an Amikacin 3′-Phosphotransferase in Escherichia coli

A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of Escherichia coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and β-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H₃³²PO₄ were shown to utilize internally generated adenosine 5′-triphosphate in the phosphorylation of amikacin. The absence of ³²P-phosphorylated amikacin after incubation of [γ-³²P]adenosine 5′-triphosphate with spheroplasts confirmed that exogenous adenosine 5′-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.     

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.     

Evidence for the presence of diacylglycerol kinase in rat brain myelin

The previous demonstration that incubation of brain slices with [³²P]phosphate brings about rapid tabeling of phosphatidic acid in myelin suggests that the enzyme involved should be present in this specialized membrane. DAG kinase (ATP:1,2-diacyglycerol 3-phosphotransferase, E.C. 2.7.1.107) is present in rat brain homogenate at a specific activity of 2.5 nmol phosphatidic acid formed/min/mg protein, while highly purified myelin had a much lower specific activity (0.29 nmol/min/mg protein). Nevertheless, the enzyme appears to be intrinsic to this membrane since it can not be removed by washing with a variety of detergents or chelating agents, and it could not be accounted for as contamination by another subcellular fraction. Production of endogenous, membrane-associated, diacylglycerol (DAG) by PLC (phospholipase C) treatment brought about translocation from soluble to particulate fractions, including myelin. Another level of control of activity involves inactivation by phosphorylation; a 10 min incubation of brain homogenate with ATP resulted in a large decrease in DAG kinase activity in soluble, particulate and myelin fractions.     

Evidence for the formation of a steroid S-glutathione conjugate from an epoxysteroid precursor.

Biotransformation of [4-¹⁴C]cholesterol α-epoxide (5α,6α-epoxycholestan-3β-ol) to the S-glutathione conjugate, 3β,5α-dihydroxycholestan-6β-yl-S-glutathione, by S-glutathione transferase of the rat liver soluble supernatant fraction, has been described. After the isolation from the biological incubation mixture by n-butanol extraction, followed by the Amberlite XAD-2 column treatment, the conjugate was chromatographically identified with a synthetic specimen which was prepared from cholesterol α-epoxide and glutathione in an ethanolic solution of sodium hydroxide. Further identification of the biologically formed conjugate with the synthetic one, including structural assignment, was established from the result that they yielded the same desulfrization product, 3β,5α-dihydroxycholestane, by the treatment with Raney nickel in an atmosphere of hydrogen.     

Drosophila polyribosomes. The characterization of two populations by cell fractionation and isotopic labeling with nucleic acid and protein precursors

Two populations of polyribosomes have been isolated from third instar larvae of D. melanogaster. One population appeared to be soluble while the second seemed membrane-bound. Short-term labeling of the two RNP fractions with radioactive nucleic acid and protein precursors was achieved by using a feeding stimulant. RNA was extracted from both polyribosomal fractions following 25, 40, and 60 min of in vivo uridine-³H incorporation. Soluble polyribosomes exhibited more rapid uptake of uridine into ribosomal and heterogeneous RNA fractions than did membrane-bound polyribosomes at comparable time periods. In vivo amino acid incorporation into the two polyribosomal populations was examined after 10, 20, 40, 60, and 80 min of incubation in leucine-³H. In this case, the membrane-bound polyribosomes reached a higher specific activity than did the soluble ones. These functional differences confirmed the observation, based on cellular fractionation studies, that the two classes of polyribosomes represented functionally distinct populations. These data have been compared with those from studies on other metazoan systems. In addition, dithiothreitol has been demonstrated to be a powerful ribonuclease inhibitor.     

Subcellular distribution of intraportally injected 125I-labeled insulin in rat liver

Time course studies revealed that at 30 s after intraportal injection of 200 μU of ¹²⁵I-labeled insulin per 100 g rat 47.9 ± 2.8% of the injected radioactivity was recovered from the liver homogenate by precipitation with trichloroacetic acid. Trichloroacetic acid precipitable radioactivity declined to very low levels during the next 30 min whereas trichloroacetic acid soluble radioactivity reached a peak value of 9.56 ± 1.9% at 5 min and declined gradually thereafter. At 30 s mean peak accumulations ±SE of 6.83 ± 0.42, 5.06 ± 0.27, 14.90 ± 1.85, and 3.58 ± 0.58% of injected radioactivity were recovered in trichloroacetic acid precipitates from the 700g (nuclei + debris), 10,000g (mitochondria + lysosome), 105,000g (microsomes), and supernatant (cytosol) subfractions, respectively. Mean peak values of 0.72 ± 0.08, 0.12 ± 0.02, and 1.11 ± 0.16% of injected radioactivity were recovered in the partially purified mitochondrial fraction, purified nuclei, and plasma membranes, respectively, as trichloroacetic acid precipitable material. Most of the trichloroacetic acid precipitable activities in the subfractions were immunoprecipitable. Trichloroacetic acid soluble radioactivity was found mainly in the cytosol and microsomal fractions. Peak specific activity (percentage of injected dose/mg protein × 10^?3) was highest in the microsomes, intermediate in the plasma membranes, and very low in the purified nuclei and partially purified mitochondrial fraction. The specific activity of the microsomes remained at or near peak levels for 5 min after ¹²⁵I-labeled insulin injection and then declined, whereas specific activity of the plasma membranes dropped precipitously to 25% of peak values at 5 min. Sephadex gel filtration of the radioactivity in the deoxycholate soluble fraction of microsomes at 5 min after ¹²⁵I-labeled insulin injection resulted in the elution of a major peak (Peak I) in the region of ¹²⁵I-labeled insulin and a minor peak (Peak II) in the region of the labeled A and B chains. Incubation of the fraction for 30 min at 37 °C with 3 mm reduced glutathione and 15 mm EDTA resulted in a reciprocal fall in Peak I and rise in Peak II. The data suggest that intraportally injected ¹²⁵I-labeled insulin is rapidly internalized and concentrated in the rat liver microsomes. The time courses of appearance and disappearance of trichloroacetic acid precipitable radioactivity in plasma membrane and microsomes further suggest, although do not prove, that insulin binds to plasma membranes before it is internalized. They also provide presumptive evidence suggesting that the sequential degradative pathway is operative in vivo.     

6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The V_max for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent K_m was 90 μM. Cytochrome b₅ was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r²=0.97) and testosterone 6β-hydroxylation (r²=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation (r²=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent K_m for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

The metabolism of dl-(1,6-14C)lipoic acid in the rat

dl-[1,6-¹⁴C]Lipoic acid was synthesized and administered to rats or incubated in vitro with rat liver systems. The urinary excretion of radioactivity after labeled lipoate was administered intraperitoneally at a level of 0.5 mg/100 g body weight was maximal at 3–6 hr, with 60% of the injected radioactivity recovered within 24 hr. Respiratory ¹⁴CO₂ from the same animals is maximal at 3 hr, after which it falls off markedly. Approximately 30% of the injected radioactivity was recovered as ¹⁴CO₂ within 24 hr. The excretion of radioactivity after lipoate was administered by stomach tube was similar to that after intraperitoneal injection. Localization of radioactivity in the body was greatest in liver, intestinal contents, and muscle in all cases. Ionexchange and paper chromatographies of 24-hr pooled urine revealed several watersoluble radioactive metabolites. Incubation of [¹⁴C]lipoate with homogenates or mitochondrial preparations in vitro resulted in the production of ¹⁴CO₂, which was decreased by incubation with unlabeled fatty acids and unaffected by the addition of carnitine or (+)-decanoylcarnitine. The rat, like certain bacteria, metabolizes lipoate via β-oxidation of the valeric acid side chain and by other metabolic reactions on the dithiolane ring, which render the molecule more water soluble.     

Studies on the binding of d-α-tocopherol to rat liver nuclei

The present study describes the nature and characteristics of the intranuclear binding sites of [³H]d-α-tocopherol in rat liver. When radioactively labeled d-α-tocopherol was intravenously administered to rats, approximately 55% of the nuclear radioactivity was associated with an intranuclear nucleoprotein complex. This complex, which was extractable by high concentrations of NaCl, was characterized by equilibrium density ultracentrifugation on a 30 to 60% linear sucrose gradient. About 50% of the high-salt-extracted radioactivity was coprecipitable with macromolecules by 10% ice-cold trichloroacetic acid (TCA). This TCA-precipitable radioactivity was completely ethanol soluble. Alkaline conditions favored the solubilization of the vitamin-receptor complex. Among various enzymes tested, only Pronase and trypsin were capable of dissociating the vitamin-receptor complex. Both ionic (sodium dodecyl sulfate) and nonionic (Triton X-100) detergents solubilized α-tocopherol from the nuclei and concomitantly released some of the associated macromolecules. In addition, treatment of nuclei with low concentrations of Triton X-100 showed that about 30% of the nuclear bound α-tocopherol is associated with inner core sites in the nucleoprotein complex with very high affinity for the vitamin. Dissociation of the nucleoprotein complex (chromatin) by high-salt solubilization and subsequent partial reassociation of the components by salting out procedures revealed the high affinity association of α-tocopherol with the reconstituted DNA-protein complex. Subfractionation of this complex further revealed that α-tocopherol is predominantly associated with the fraction containing phenol-soluble nonhistone proteins having a high affinity for DNA. In vitro binding studies also showed that there are specific saturable binding sites for d-α-tocopherol in rat liver nuclei.     

Preferential dephosphorylation of protein bound phosphorylthreonine and phosphorylserine residues by cytosol and mitochondrial "casein phosphatases".

The partial purification of a rat liver cytosol protein phosphatase is described, resulting in a preparation active on casein but not on phosvitin, cytosol phosphopeptides, ATP, ADP and p-nitrophenylphosphate, which, on the contrary, are still dephosphorylated by the protein phosphatase purified from rat liver mitochondria. Moreover the activity of the former enzyme on casein appears to involve only a limited amount of phosphoric sites which are also preferentially phosphorylated by soluble protein kinase. The isolation and evaluation of ³²P-serine and ³²P-threonine from protein-kinase-dependently labelled phosvitin and casein, before and after incubation with the two enzymes, led to the conclusion that mitochondrial protein phosphatase hydrolyzes more actively the phosphorylserine residues, while the cytosol “casein phosphatase” promotes a preferential breakdown of the ³²P-threonine residues.     

Mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase

Hepoxilins are lipid signaling molecules derived from arachidonic acid through the 12-lipoxygenase pathway. These trans-epoxy hydroxy eicosanoids play a role in a variety of physiological processes, including inflammation, neurotransmission, and formation of skin barrier function. Mammalian hepoxilin hydrolase, partly purified from rat liver, has earlier been reported to degrade hepoxilins to trioxilins. Here, we report that hepoxilin hydrolysis in liver is mainly catalyzed by soluble epoxide hydrolase (sEH): i) purified mammalian sEH hydrolyses hepoxilin A₃ and B₃ with a V_max of 0.4–2.5 μmol/mg/min; ii) the highly selective sEH inhibitors N-adamantyl-N’-cyclohexyl urea and 12-(3-adamantan-1-yl-ureido) dodecanoic acid greatly reduced hepoxilin hydrolysis in mouse liver preparations; iii) hepoxilin hydrolase activity was abolished in liver preparations from sEH^−/− mice; and iv) liver homogenates of sEH^−/− mice show elevated basal levels of hepoxilins but lowered levels of trioxilins compared with wild-type animals. We conclude that sEH is identical to previously reported hepoxilin hydrolase. This is of particular physiological relevance because sEH is emerging as a novel drug target due to its major role in the hydrolysis of important lipid signaling molecules such as epoxyeicosatrienoic acids. sEH inhibitors might have undesired side effects on hepoxilin signaling.     

Biosynthèse de l'udpglucose par les microsomes des hépatocytes de ratBiosynthesis of UDPglucose by rat liver microsomes

Evidence is presented to show that all enzymes and all intermediary metabolites of a UDPglucose biosynthesis pathway are present in the microsomal membranes of rat liver. Glucose 6-phosphate, glucose 1-phosphate and UDPglucose are characterized by chromatography.The properties of phosphoglucomutase and UTP: D-Glucose-1-phosphate uridyltransferase are studied. The K_m values of phosphoglucomutase at pH 7.2 and 42°C were 0.26 · 10^?3 mM for glucose 1,6-diphosphate and 80 · 10^?3 mM for glucose 1-phosphate. The K_m values of UTP: D-glucose-1-phosphate uridyltransferase at pH 8.5 and 37°C were 220 · 10^?3 mM for UTP and 166 · 10^?3 mM for glucose 1-phosphate. These values are compared to the given values for enzymes from different species, and to those found for soluble enzymes. The significance of this membranous pathway is discussed.     

Effects of perfluorocarboxylic acids on the activities of acyl-CoA elongations in vivo and in vitro

Effects of perfluorocarboxylic acids (PFCAs) on proportions of oleic acid and cis-vaccenic acid through acyl-CoA chain elongation systems have been studied in the liver of rats. Administration of PFCAs caused a significant increase in palmitoyl-CoA chain elongation activity while these chemicals did not affect palmitoleoyl-CoA chain elongation activity in vivo.Condensation for both palmitoyl-CoA and palmitoleoyl-CoA were inhibited by PFCAs in vitro at the concentrations, which were physiologically found in the liver of rats treated with the PFCAs. Δ⁹ Desaturase, which catalyzes both stearoyl-CoA desaturation and palmitoyl-CoA desaturation, was induced by the treatments of rats with the PFCAs. The administration of the PFCAs to rats caused a marked increase in proportion of oleic acid, while that of cis-vaccenic acid was not affected at all. These results strongly suggest that the induced palmitoyl-CoA chain elongation by PFCAs, which exist in the liver, effectively produces oleic acid in concert with the induced stearoyl-CoA desaturase, but the inhibitory effects of PFCAs on either palmitoyl-CoA chain elongation or palmitoleoyl-CoA chain elongation are not crucial for the formation of the elongated fatty acids in vivo.     

Elongation of fatty acids by microsomal fractions from the brain of the developing rat.

Elongation of fatty acids by microsomal fractions obtained from rat brain was measured by the incorporation of [2-14C]malonyl-CoA into fatty in the presence of palmitoyl-CoA or stearoyl-CoA. 2. Soluble and microsomal fractions were prepared from 21-day-old rats; density gradient centrifugation demonstrated that the stearoyl-CoA elongation system was localized in the microsomal fraction whereas fatty acid biosynthesis de novo from acetyl-CoA occurred in the soluble fraction. The residual activity de novo in the microsomal fraction was attributed to minor contamination by the soluble fraction. 3. The optimum concentration of [2-14C]malonyl-CoA for elongation of fatty acids was 25 mum for palmitoyl-CoA or stearoyl-CoA, and the corresponding optimum concentrations for the two primer acyl-CoA esters were 8.0 and 7.2 muM respectively. 4. Nadph was the preferred cofactor for fatty acid formation from palmitoyl-CoA or stearoyl-CoA, although NADH could partially replace it. 5. The stearoyl-CoA elongation system required a potassium phosphate buffer concentration of 0.075M for maximum activity; CoA (1 MUM) inhibited this elongation system by approx. 30%. 6. The fatty acids formed from malonyl-CoA and palmitoyl-CoA had a predominant chain length of C18 whereas stearoyl-CoA elongation resulted in an even distribution of fatty acids with chain lengths of C20, C22 and C24. 7. The products of stearoyl-CoA elongation were identified as primarily unesterified fatty acids. 8. The developmental pattern of fatty acid biosynthesis by rat brain microsomal preparations was studied and both the palmitoyl-CoA and stearoyl-CoA elongation systems showed large increases in activity between days 10 and 18 after birth.     

Enzymic studies of glycol and glycerol lipids containing O-alkyl bonds in liver and tumor tissues

The utilization of [1-¹⁴C]hexadecyl-[2-³H]ethyleneglycol and [1-¹⁴C]hexadecyl-[2-³H]glycerol as substrates for acyltransferase, phosphotransferase, phosphorylcholine, and phosphorylethanolamine transferase, and O-alkyl cleavage activities in cell-free preparations from normal rat liver and preputial gland tumors of mice was investigated. Our studies demonstrate that alkylethyleneglycols, like alkylglycerols, can serve as substrates for acyltransferases in both the liver and tumor microsomes; the product alkylacylethyleneglycerol can be readily deacylated by pancreatic lipase. A polar lipid was formed from the alkylethyleneglycol by the tumor homogenates in the presence of ATP and Mg²⁺; although the small quantities formed precluded absolute identification, its thin-layer Chromatographic behavior in acidic and basic solvent systems indicated that a free phosphate group was present. As expected, phosphorylbase transferases in these preparations did not utilize either the alkylethyleneglycol or alkylglycerol as substrates. The O-alkyl moiety of hexadecyl-ethyleneglycol was oxidized to hexadecanal by a tetrahydropteridine-dependent cleavage enzyme in rat liver microsomes, whereas in the tumor microsomes this activity was not present. We conclude that alkylethyleneglycols are metabolized in a manner similar to alkylglycerols and perhaps by identical enzymes.     

The influence of ammonia on purine and pyrimidine nucleotide biosynthesis in rat liver and brain in vitro

1. The effect of ammonia on purine and pyrimidine nucleotide biosynthesis was studied in rat liver and brain in vitro. The incorporation of NaH¹⁴CO₃ into acid-soluble uridine nucleotide (UMP) in liver homogenates and minces was increased 2.5–4-fold on incubation with 10mm-NH₄Cl plus N-acetyl-l-glutamate, but not with either compound alone. 2. The incorporation of NaH¹⁴CO₃ into orotic acid was increased 3–4-fold in liver homogenate with NH₄Cl plus acetylglutamate. 3. The 5-phosphoribosyl 1-pyrophosphate content of liver homogenate was decreased by 50% after incubation for 10min with 10mm-NH₄Cl plus acetylglutamate. 4. Concomitant with this decrease in free phosphoribosyl pyrophosphate was a 40–50% decrease in the rates of purine nucleotide synthesis, both de novo and from the preformed base. 5. Subcellular fractionation of liver indicated that the effects of NH₄Cl plus acetylglutamate on pyrimidine and purine biosynthesis required a mitochondrial fraction. This effect of NH₄Cl plus acetylglutamate could be duplicated in a mitochondria-free liver fraction with carbamoyl phosphate. 6. A similar series of experiments carried out with rat brain demonstrated a significant, though considerably smaller, effect on UMP synthesis de novo and purine base reutilization. 7. These data indicate that excessive amounts of ammonia may interfere with purine nucleotide biosynthesis by stimulating production of carbamoyl phosphate through the mitochondrial synthetase, with the excess carbamoyl phosphate in turn increasing pyrimidine nucleotide synthesis de novo and diminishing the phosphoribosyl pyrophosphate available for purine biosynthesis.     

The distribution of phosphatidylinositol in microsomal membranes from rat liver after biosynthesis de novo. Evidence for the existence of different pools of microsomal phosphatidylinositol by the use of phosphatidylinositol-exchange protein

1. The phosphatidylinositol-exchange protein from bovine brain was used to determine to what extent phosphatidylinositol in rat liver microsomal membranes is available for transfer. 2. The microsomal membranes used in the transfer reaction contained either phosphatidyl[2-³H]inositol or ³²P-labelled phospholipid. The ³²P-labelled microsomal membranes were isolated from rat liver after an intraperitoneal injection of [³²P]P_i. The ³H-labelled microsomal membranes and rough- and smooth-endoplasmic-reticulum membranes were prepared in vitro by the incorporation of myo-[2-³H]inositol into phosphatidylinositol by either exchange in the presence of Mn²⁺ or biosynthesis de novo in the presence of CTP and Mg²⁺. 3. Tryptic or chymotryptic treatment of the microsomes impaired the biosynthesis de novo of phosphatidylinositol. It was therefore concluded that the biosynthesis of phosphatidylinositol and/or its immediate precursor CDP-diacylglycerol takes place on the cytoplasmic surface of the microsomal membrane. 4. Under the conditions of incubation 42% of the microsomal phosphatidyl[2-³H]inositol was transferred with an estimated half-life of 5min; 38% was transferred with an estimated half-life of about 1h; the remaining 20% was not transferable. Identical results were obtained irrespective of the method of myo-[2-³H]inositol incorporation. 5. Both measurement of phosphatidylinositol phosphorus in the microsomes after transfer and the transfer of microsomal [³²P]phosphatidylinositol indicate that phosphatidyl[2-³H]-inositol formed by exchange or biosynthesis de novo was homogeneously distributed throughout the microsomal phosphatidylinositol. 6. We present evidence that the slowly transferable pool of phosphatidylinositol does not represent the luminal side of the microsomal membrane; hence we suggest that this phosphatidylinositol is bound to membrane proteins.