Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Effects of pyridoxine deficiency and dl-p-chlorophenylalanine administration to rats on 5-hydroxytryptamine and noradrenaline concentrations in brain and 5-hydroxytryptamine concentration in blood

Pyridoxine deficiency in post-weanling rats caused a marked decrease in body weight and a small but significant decrease in brain weight. Although the concentration of circulating 5-hydroxytryptamine was markedly decreased, the concentrations of 5-hydroxytryptamine and noradrenaline in the brain were not affected. p-Chlorophenylalanine, an inhibitor of 5-hydroxytryptamine synthesis, decreased the 5-hydroxytryptamine content of brain to very low values in both the deficient and control animals, whereas the noradrenaline contents were not appreciably affected. The concentration of 5-hydroxytryptamine in blood, the origin of which is primarily gastrointestinal, was decreased only in the controls but not in the deficient animals after p-chlorophenylalanine treatment. These results suggest that whereas l-tryptophan hydroxylase (EC 1.14.3.2) is rate-limiting in the brain as has been reported by others, the pyridoxal 5'-phosphate-dependent enzyme 5-hydroxytryptophan decarboxylase (EC 4.1.1.28) may be more important in the gastrointestinal tract in the regulation of 5-hydroxytryptamine synthesis.     

Ovine placental lactogen stimulation of ornithine decarboxylase activity in brain and liver of neonatal rats

Ovine placental lactogen (oPL), growth hormone (oGH), prolactin (oPRL) and human placental lactogen (hPL) were administered intracisternally (ic) or intraperitoneally (ip) to 17 day old rats and brain and liver ODC activities determined four hours later. When given ic, oPL, oGH and oPRL caused significant increases in brain ODC activity, while hPL had no significant effect. After ip administration, oPL and oGH also caused a significant increase in brain as well as liver ODC activity but oPRL and hPL were without significant effect. The stimulation of polyamine metabolism by oPL together with earlier reports of its potent somatotropic effects and its high concentration in the fetus supports the hypothesis that oPL may be important in the regulation of fetal growth.     

Changes of gastric lipase activity after ethanol and indomethacin administration: influence of pretreatment with allopurinol, pentoxifylline and L-DOPA

Gastric lipase (GL) plays an important role in emulsification and digestion of food fat. Lipids are components of the hydrophobic mucus and mucosa barrier. Damage of the gastric mucosa may therefore be related to changes in the lipid content and GL activity. In the present paper, we studied the effect of administration of a single dose of 96 % ethanol (E) and indomethacin 20 mg x kg(-1) (IND) on the activity of GL and on the concentrations of nonesterified fatty acids (NEFA) and triacylglycerols (TG) in the gastric mucosa of rats. Furthermore, we studied how these changes are affected by allopurinol (ALO), pentoxifylline (PX) and L-DOPA pretreatment 30 min before administration of E or IND. The effect of sialoadenectomy (SA) on these parameters was also evaluated. We found: 1) significant (p < 0.01) inhibition of GL activity after administration of E and IND and also ALO, as well as after pretreatment with ALO before E and PX before IND. L-DOPA administered alone stimulated GL activity, but its administration before IND significantly (p < 0.01) inhibited this enzymatic activity. GL activity was decreased to the threshold values in SA rats and after administration of E to SA animals. 2) NEFA concentrations were decreased after E and increased significantly (p < 0.01) after IND administration. A marked significant (p < 0.01) decrease in NEFA was found after PX and L-DOPA administration. The administration of ALO also lowered the concentration of NEFA. Pretreatment by drugs before E and IND resulted in a significant increase of NEFA in comparison with the drugs given alone (p < 0.05 for ALO + E; p < 0.01 for PX + IND). 3) TG were also decreased in all experimental groups in comparison with the control group, i.e. after E and IND, after ALO and SA and also after pretreatment by ALO before E. The concentration of TG decreased after PX, significantly (p < 0.05) after L-DOPA and after pretreatment by PX before IND. Pretreatment by ALO before E and L-DOPA before IND resulted in the increase of TG in comparison with drugs alone. Thus, these results suggest certain protective effect of pretreatment with ALO, PX and L-DOPA against the E- and IND-induced decrease in NEFA and TG during injury of the gastric mucosa. On the other hand, inhibition of GL activity was also apparent after administration of these drugs before E and IND, which suggest presence of a persisting impairment of lipid digestion in the stomach.     

A sensitive radiometric assay for cysteic acid decarboxylase activity in crude enzyme preparations of rat liver and brain.

1. A method is described for the synthesis of L-[U-14C]cysteic acid from L-[U-14C] cysteine hydrochloride and for its subsequent utilisation as a substrate for cysteic acid decarboxylase activity in liver and brain. 2. The enzyme determination relies on the entrapment of radio-labelled carbon dioxide in Hyamine hydroxide. 3. The assay is sensitive, reliable and convenient and is particularly suitable for measuring the activity of the decarboxylase in crude enzyme preparations.     

Activation and inactivation of rabbit liver pyridoxamine (pyridoxine) 5'-phosphate oxidase activity by urea and other solutes.

Urea, guanidine hydrochloride, and neutral salts both activate and denature pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) from rabbit liver. Activation occurs at lower concentrations (e.g. 2-2.5 m for urea) of these compounds and is rapid and reversible. Greater structural changes leading to inactivation occur slowly under “activating conditions” but rapidly at higher concentrations of urea. Both reversibly and irreversibly inactivated species are formed. Activation by urea does not involve either dissociation of the enzyme to subunits or aggregation to multimers, and there is little disruption of protein secondary structure. The V and K_m for substrates, K_i for product, and the rate of release of product from the enzyme are increased by urea, and substrate inhibition is decreased; urea has little effect on the reactivity of reduced enzyme with oxygen. Both flavin and tryptophanyl fluorescence increase in the presence of urea; at lower concentrations of urea (≤2 m), there is a rapid increase followed by slower, sigmoidal increases. The polarization of flavin fluorescence of the oxidase is increased upon the addition of 2 m urea, which corresponds to the initial enhancement of protein and flavin fluorescence intensities, and then decreases. The near-ultraviolet-visible absorption spectra of native enzyme and that treated with 2 m urea are only slightly different; however, a considerable change at the flavin-binding site is reflected by the circular dichroism spectra. Hence, it appears that urea yields a rapidly formed, “activated” species of the oxidase that is changed primarily at the active site in a manner that allows increased dissociation of substrate and product.     

Biochemical properties and kinetic parameters of dihydroxyphenylalanine--5-hydroxytryptophan decarboxylase in brain, liver, and adrenals of cat.

Biochemical properties and kinetic parameters of nonpurified dihydroxyphenylalanine-5-hydroxytryptophan decarboxylase extracted from brain and two peripheral organs, liver and adrenals, were studied in the cat. This study shows that decarboxylase activity in brain is lower than in peripheral organs and that 5-hydroxytryptophan can be decarboxylated without exogenous addition of pyridoxal-5'-phosphate (PLP). However, the addition of PLP substantially increases the enzyme activity. Excess of coenzyme (greater than 60 muM) induces inhibition in adrenals and liver but not in the central nervous system (CNS). The observed inhibition might be related to the presence of a tetrahydroisoquinoline derivative formed in the medium. Differentiation between mechanisms of action of decarboxylase in the CNS and peripheral organs is suggested.     

Purification of uroporphyrinogen decarboxylase from human erythrocytes. Immunochemical evidence for a single protein with decarboxylase activity in human erythrocytes and liver.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified 4419-fold to a specific activity of 58.3 nmol of coproporphyrinogen III formed/min per mg of protein (with pentacarboxyporphyrinogen III as substrate) from human erythrocytes by adsorption to DEAE-cellulose, (NH4)2SO4 fractionation, gel filtration, phenyl-Sepharose chromatography and polyacrylamide-gel electrophoresis. Progressive loss of activity towards uroporphyrinogens I and III occurred during purification. Experiments employing immunoprecipitation, immunoelectrophoresis and titration with solid-phase antibody indicated that all the uroporphyrinogen decarboxylase activity of human erythrocytes resides in one protein, and that the substrate specificity of this protein had changed during purification. The purified enzyme had a minimum mol.wt. of 39 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gel filtration gave a mol.wt. of 58 000 for the native enzyme. Isoelectric focusing showed a single band with a pI of 4.60. Reaction with N-ethylmaleimide abolished both catalytic activity and immunoreactivity. Incubation with substrates or porphyrins prevented inactivation by N-ethylmaleimide. An antiserum raised against purified erythrocyte enzyme precipitated more than 90% of the uroporphyrinogen decarboxylase activity from human liver. Quantitative immunoprecipitation and crossed immunoelectrophoresis showed that the erythrocyte and liver enzymes are very similar but not identical. The differences observed may reflect secondary modification of enzyme structure by proteolysis or oxidation of thiol groups, rather than a difference in primary structure.     

Changes in chick liver and brain mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase during development

Phosphorylation and decarboxylation of mevalonate in chick liver and brain was investigated during early post hatching stages of development. In chick liver, both mevalonate kinase and mevalonate-5-phosphate kinase increased their activity from day 5 of age while pyrophosphate decarboxylase activity remained low during the first days after hatching, increased sharply up to day 9 of age, and remained practically unchanged thereafter. The developmental pattern obtained in brain shows a slight decrease in the phosphorylation and decarboxylation of mevalonate after the first week of postnatal development. Further studies were performed using the specific substrate of mevalonate-5-pyrophosphate decarboxylase, corroborating the results obtained using mevalonate as substrate. Changes in hepatic decarboxylase were more pronounced than those observed in mevalonate-phosphorylating enzymes, thus suggesting an important role for decarboxylase in the control of cholesterogenesis during postnatal development.     

Delta 5-desaturase activity in liver and brain microsomes during development of the pig.

Microsomes isolated from liver and brain tissue were assayed to examine transitions in metabolic capability to synthesize tetraenes and pentaenes by chain elongation-desaturation of C20:3(8,11,14) during the perinatal development of the pig. Rates of synthesis of tetraenes and pentaenes by chain elongation-desaturation of C20:3(8,11,14) were greatest in liver. During the latter half of gestation, the capability to synthesize tetraenes increased 7- or 23-fold on a per mg of microsomal protein basis for brain and liver respectively. Increase in the capacity to synthesize tetraenes from C20:3(8,11,14) suggests a significant transition in the activity of the delta 5-desaturase during the last half of gestation. These observations indicate that in liver and brain the capability to chain elongate-desaturate C18:2(9,12) to longer chain homologues increases significantly during early development as a function of transitions in the activity of the delta 5-desaturase.