STUDIES ON MEVALONATE KINASE, PHOSPHOMEVALONATE KINASE AND PYROPHOSPHOMEVALONATE DECARBOXYLASE IN DEVELOPING RAT BRAIN

Abstract— We have in the present study investigated the properties of mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase in the 105,000 g supernatant fractions from rat brain, and determined whether the activities of these enzymes change during brain development. All three enzymes in brain showed a specific requirement for ATP for optimal activity. The presence of Mg²⁺ as divalent cation was also required for optimal activity of mevalonate kinase and phosphomevalonate kinase. Both Mg²⁺ and Mn²⁺ were equally effective divalent metal ions for pyrophosphomevalonate decarboxylase in brain. Mevalonate kinase as well as phosphomevalonate kinase were active in a broad pH range of 6.5–8 while the pH curve for pyrophosphomevalonate decarboxylase showed a peak activity at approx 6. No age-dependent change occurred in the activities of mevalonate kinase and phosphomevalonate kinase in developing brain, whereas pyrophosphomevalonate decarboxylase activity in brain increased during the 1st week after birth, reached a peak value at about the 8th day of age and declined slowly thereafter. The K_m for brain mevalonate kinase in 2, 13 and 52 day old rats were 312, 400 and 434 μM, respectively. The V _max for the kinase in 2, 13 and 52 day old rats were in the range of 45–52 nmol/h/mg protein, respectively. This suggests that, like in liver (R amachandran & S hah , 1976), pyrophosphomevalonate decarboxylase in brain may also be one regulatory step for cholesterol synthesis.     

EFFECT OF PYRIDOXINE DEFICIENCY ON AROMATIC l-AMINO ACID DECARBOXYLASE IN THE DEVELOPING RAT LIVER AND BRAIN

—Maternal pyridoxine deficiency begun 2 weeks before mating and continued throughout pregnancy and the nursing period resulted in diminished wt. gains in the brain, the liver and the body in the first 16 days of life, as well as lowered levels of the aromatic l -amino acid decarboxylase in both brain and liver tissue. The fetus was protected from the effect of vitamin B₆ deficiency during pregnancy, since at birth the body wt., organ weights, and decarboxylase levels in these tissues were comparable to those of control litters. The brain was affected less than the liver, both in rate of wt. increase and decarboxylase activity. The cerebellum normally developed measurable decarboxylase activity only during the second week of life. The cortex normally slowly increased its low decarboxylase activity during the first week postnatally, with a more rapid increase during the second week. This rapid increase was primarily in the holoenzyme moiety. The rest of the brain, which had well developed levels of decarboxylase activity at birth, normally showed a sharp increase during the second week of life which was also largely in the holoenzyme portion. When the increasing weights of these tissues were considered, it became obvious that the total amount of apoenzyme as well as the amount of holoenzyme were increasing in the normally developing rat, although the greatest amount of the change was in the holoenzyme form. The liver normally showed a much more rapid increase in decarboxylase activity than did the brain, and showed the increase much earlier. The holoenzyme normally increased rapidly after the first 4 days, whereas the apoenzyme concentration levelled off at this time. The effect of the pyridoxine deficiency on decarboxylase activity was almost entirely on the holoenzyme form of the decarboxylase, since the apoenzyme form generally remained the same in the control and the deficient pups during development. There appeared to be no decarboxylase inhibitor present in pyridoxine deficient tissues, nor any evidence in control tissues for an enzyme required for the activation of the decarboxylase by cofactor.     

EFFECT OF FATTY ACIDS ON RAT BRAIN PARTICULATE HEXOKINASE

Abstract— The effect of free fatty acids on rat brain particulate hexokinase was studied in vitro. Hexokinase bound with brain mitochondrial fraction was found to be sensitive to the action of free fatty acids, resulting in the solubilization of at least part of bound enzyme activity into the supernatant. The decrease of total enzyme activity observed at the highest free fatty acid concentration was probably due to the inhibition of hexokinase. The physiological consequence of hexokinase solubilization by low concentrations of free fatty acids, similar to that observed in vivo , is discussed in relation to activity changes of soluble and particulate enzyme forms demonstrated previously under hypoxic conditions.     

INHIBITION OF THE BIOSYNTHESIS OF ISOPRENOID COMPOUNDS BY PHENOLIC ACIDS IN THE RAT BRAIN

—The incorporation of [2-¹⁴C]mevalonate into nonsaponifiable lipids by rat brain homogenates is inhibited by phenolic acids derived from tyrosine. The phenyl acids derived from phenylalanine are inhibitory only at very high concentrations compared with phenolic acids. The brain is more sensitive to inhibition by the phenolic acids than the liver. These studies indicate a possible role for phenolic acids in the impairment of cerebral sterol metabolism in phenylketonuria.     

KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INTERACTIONS WITH GLUTAMATE,PYRIDOXAL 5-PHOSPHATE AND GLUTAMATE-PYRIDOXAL 5-PHOSPHATE SCHIFF BASE1

Abstract— The kinetic behavior of glutamate decarboxylase from mouse brain was analyzed in a wide range of glutamate and pyridoxal 5′-phosphate concentrations, approaching three limit conditions: (I) in the absence of glutamate-pyridoxal phosphate Schiff base; (II) when all glutamate is trapped in the form of Schiff base; (III) when all pyridoxal phosphate is trapped in the form of Schiff base. The experimental results in limit condition (I) are consistent with the existence of two different enzyme activities, one dependent and the other independent of free pyridoxal phosphate. The results obtained in limit conditions (II) and (III) give further support to this postulation. These data show that the free pyridoxal phosphate-dependent activity can be abolished when either all substrate or all cofactor are in the form of Schiff base. The free pyridoxal phosphate-independent activity is also abolished when all substrate is trapped as Schiff base, but it is not affected by the conversion of free pyridoxal phosphate into the Schiff base. A kinetic and mechanistic model for brain glutamate decarboxylase activity, which accounts for these observations as well as for the results of previous dead end-inhibition studies, is postulated. Computer simulations of this model, using the experimentally obtained kinetic constants, reproduced all the observed features of the enzyme behavior. The possible implications of the kinetic model for the regulation of the enzyme activity are discussed.     

THE EFFECT OF DEVELOPMENT ON THE GANGLIOSIDES OF RAT AND PIG BRAIN

Abstract— The ganglioside content of the forebrain, brain stem and cerebellum have been studied, in the rat at various ages from 1 day to 27 months, and in the pig at various ages from 93 days gestation to 30 months. Each part of the brain was analysed for total ganglioside NANA and for four major gangliosides (G_Ml, G_D1a, G_Dlb and G_T1 in the nomenclature of S vennerholm , 1963). In the rat forebrain, the concentration of ganglioside NANA rose rapidly between 1 and 21 days after birth, fell to 3 months and subsequently rose to a mature value at 6 months. In the rat cerebellum, the peak concentration was reached at 2 months and the lower adult value at 9 months, whilst in the brain stern, the concentration rose more slowly and had a broad peak from 15 days to 2 months. Values are also given for the changes in the total amounts in each brain part. The changes in the concentrations and total amounts of ganglioside NANA, in the three parts of the pig brain were, on the whole, similar to those in rat brain except that the percentage distribution of the major gangliosides had almost attained the mature pattern at birth. In the forebrain of both species, the disialoganglioside, G_D1a, accounted for the highest percentage of the total gangliosides. The results are discussed with respect to their possible structural significance.     

THE EFFECT OF HIGH CONCENTRATIONS OF HISTIDINE ON THE LEVEL OF OTHER AMINO ACIDS IN PLASMA AND BRAIN OF THE MATURE RAT

—Changes in plasma and brain amino acids have been observed in adult rats 1 h after intraperitoneal injections of histidine and in others maintained on high histidine diets for 8 days. In the injection studies the compounds most consistently affected were the aromatic and branched chain amino acids and methionine. Reductions in their concentrations in the brain were explained by a competition with histidine for uptake into the tissue. There was little change in plasma amino acid levels. In the animals fed the highest concentration of histidine there was a generalized increase in brain, and a reduction in plasma, amino acid concentrations. A decrease in protein synthesis is postulated to explain this effect in brain.     

GLYCEROL KINASE AND DIHYDROXYACETONE KINASE IN RAT BRAIN

—The enzymatic phosphorylation of glycerol and dihydroxyacetone by ATP to sn-glycerol-3-phosphate and dihydroxyacetone phosphate respectively in various subcellular fractions of rat brain was studied. A sensitive radiochemical assay was used where the labelled phosphorylated products were separated from the radioactive substrates by high voltage paper electrophoresis and the radioactivity in these compounds determined. Using this assay the glycerol kinase (EC 2.7.1.30) activity was found to be associated with the mitochondrial fraction of the brain. Under optimum conditions 2.45 nmol of glycerol was phosphorylated/min per mg of protein. The K_m for glycerol was 70 μm at pH 7. This mitochondrial enzyme, like other glycerol kinases from different sources, also phosphorylated dihydroxyacetone. Under optimum conditions 1.7 nmol of dihydroxyacetone phosphate was formed/min per mg of mitochondrial protein. The K_m for dihydroxyacetone was 0.6 mm . Glycerol kinase activity was also present in the cytoplasm of brain. However, the specific activity of this enzyme in cytosol is about 15% of the mitochondrial glycerol kinase. Compared to glycerol, dihydroxyacetone was phosphorylated by ATP in cytoplasm at a much higher rate. The pH optimum for this soluble dihydroxyacetone kinase was much lower (pH 6.5) than that of the soluble or mitochondrial glycerol kinase (pH 10.0). Using ammonium sulfate, brain cytoplasm was fractionated to yield a fraction in which the dihydroxyacetone kinase was enriched 2–3 fold with no glycerol kinase activity. Under optimum conditions 1.0 nmol of dihydroxyacetone was phosphorylated/min per mg protein. The K_m for dihydroxyacetone was 60 μm . This cytosol fraction was also found to phosphorylate d -glyceraldehyde and l -glyceraldehyde at a rate of 30–40% to that of the dihydroxyacetone phosphorylation. The properties and the possible metabolic role of these enzymes in brain are discussed.     

PROPERTIES OF RAT BRAIN HISTIDINE DECARBOXYLASE

Abstract– The properties of histidine decarboxylase ( l -histidine carboxylyase EC 4.1,1.22) have been studied in a whole rat brain homogenate. Optimum pH depended upon substrate concentration; the variations of K _m and V _max were determined as a function of pH. pH values lower than 6.0 caused a loss of enzymic activity; activity was stable at pH values higher than 6.0. Enzyme activity was proportional to temperature in the range 30-45°C; temperature characteristic (μ) and Q₁₀ were determined and thermal inactivation was studied. Addition of pyridoxal 5'-phosphate increased enzyme activity. Dialysis of homogenates against phosphate buffer caused a partial loss of enzyme activity which could be restored by addition of the coenzyme to the incubation mixture. Enzyme activity was inhibited by α-methylhistidine and benzene and was unaffected by α-methyl DOPA. The properties correspond to those of a 'specific' histidine decarboxylase. However, the brain enzyme differs from the corresponding enzyme in peripheral tissues in the inability to achieve a total inhibition of activity by dialysis.     

EFFECT OF INCREASED RAT BRAIN TRYPTOPHAN ON 5-HYDROXYTRYPTAMINE AND 5-HYDROXYINDOLYL ACETIC ACID IN THE HYPOTHALAMUS AND OTHER BRAIN REGIONS

—Tryptophan was found at higher concentration in the rat hypothalamus than in other brain regions. This difference was explicable neither by regional differences in blood content nor by differences in tryptophan recovery from different weights of tissue. It was not due to interference by other known brain indoles. After food deprivation or tryptophan injection the tryptophan concentration rose in all regions. Total 5-hydroxyindole increases showed regional differences but relative changes were similar after both procedures. Increases in 5-hydroxytryptamine were clearest in midbrain + hippocampus. In general, 5-hydroxyindolylacetic acid increased more markedly than 5-hydroxytryptamine. The hypothalamus appeared refractory with negligible increases of both 5-hydroxyindoles upon either food deprivation or tryptophan administration even though hypothalamic tryptophan concentration rose considerably. Results are discussed in relation to other evidence suggesting special characteristics of 5-HT regulation in the hypothalamus.     

THE EFFECT OF BROMOCRIPTINE ON RAT STRIATAL ADENYLATE CYCLASE AND RAT BRAIN MONOAMINE METABOLISM

In slices and homogenate from rat brain striatum bromocriptine in marked contrast to DA. NA and apomorphine. had no stimulatory effect on adenylate cyclase activity, but antagonised the stimulatory effects of both NA and DA. Bromocriptine (10 mg/kg s.c.) decreased the turnover of DA in striatum and limbic structures 3h after drug administration. However, an increase in the turnover of NA in the brain stem and that of 5-HT in the cortex was observed 4h following treatment with bromocriptine. Possible modes of action of bromocriptine are discussed.     

THE EFFECT OF ISCHAEMIA AND RECIRCULATION ON PROTEIN SYNTHESIS IN THE RAT BRAIN

Abstract— Rats were subjected to cerebral compression ischaemia for 15min and were subsequently recirculated with blood for periods up to 3 h. In vivo incorporation of intravenously administered L-[1–¹⁴C]valine into total brain proteins was found to be severely inhibited (about 20% of controls) after 45 min of recirculation. After 3 h, protein synthesis had increased, the specific radioactivity of proteins then being about 40% of controls. The post-ischaemic inhibition of protein synthesis was accompanied by a breakdown in polyribosomes and a concomitant increase in ribosomal subunits. In vitro incorporation of L-[1–¹⁴C]phenylalanine by a postmitochondrial supernatant system derived from animals subjected to 15 min ischaemia and 15 min recirculation was also severely reduced and showed, in contrast to control animals, no response to the addition of a specific inhibitor of polypeptide chain initiation (Poly(I)). Together with the in vivo accumulation of ribosomal subunits this indicates a block in peptide chain initiation during the early stages of recirculation.
Polyribosomes from animals subjected to 15 min ischaemia without recirculation showed a normal rate of in vitro protein synthesis which was inhibited by Poly(I) to a similar extent as polyribosomes from control animals. These results suggest that the post-ischaemic inhibition in chain initiation develops during the early stages of recirculation rather than during the ischaemic period itself.     

PROPERTIES AND REGIONAL DISTRIBUTION OF HISTIDINE DECARBOXYLASE IN RAT BRAIN

—Properties of the histamine-forming enzyme in rat brain were studied, utilizing a sensitive fluorometric assay. The optimum pH was related to substrate concentration and found to be6·4 at 10^?2m -histidine; the apparent Km was about 4·10^?4m ; enzyme activity was inhibited by α-hydrazino -histidine and brocresine but was not affected by α-methyl DOPA or benzene. These different data suggest that the 'specific’histidine decarboxylase (EC 4.1.1.22)—and not the aromatic l -aminoacid decarboxylase—is involved. Determination of enzyme activity and histamine level in different areas of the rat brain revealed important regional differences, the two values being roughly parallel.     

CHANGES OF THYMIDINE KINASE IN THE DEVELOPING RAT BRAIN

Abstract— Thymidine kinase (ATP: thymidine-5'-phosphotransferase EC 2.7.1.21) of the supernatant fraction from 6-day-old rat brain possessed a pH optimum of 8.0 and required the presence of 5mM-ATP and 2.5 mM-MgCl₂ for maximum activity. The activity was completely inhibited by addition of 1.8 mM-TTP. The enzyme activity was lost if the same supernatant fraction was refrozen and thawed. K_m was 2.8 × 10⁻⁶ M for [6-³H]thymidine.
Following subcellular fractionation of rat brain, the greatest proportion and highest specific activity of thymidine kinase was found in the supernatant fraction. Thymidine kinase activities reached a maximum at 6 days of age and then dropped sharply during maturation. Comparative studies of thymidine kinase activities of cerebrum, cerebellum and the remainder of the brain during growth indicated that the activity in the cerebellum was usually higher than those in the cerebrum and the remainder, and the biggest differences obtained at 6 days after birth corresponded with the peak in cerebellar activity.     

EFFECTS OF UNDER NUTRITION AND PROTEIN DEFICIENCY ON GLUTAMATE DEHYDROGENASE AND DECARBOXYLASE IN RAT BRAIN

Abstract— Studies were made on the effects of undernutrition at different ages during the neonatal period and of the comparative effects of postweaning protein and calorie deficiencies in neonatally undernourished or normally reared animals. Neonatal undernutrition resulted in deficits in body wt, brain wt and the activities of brain glutamate dehydrogenase and glutamate decarboxylase. Percentage deficits in brain wt were maximum in the first week of life but those in brain enzymes were greater in the second week. Rehabilitation of neonatally undernourished animals reversed the deficits in brain wt and brain enzymes. Post-weaning protein deficiency produced similar deficits in brain enzymes in both neonatally undernourished and normally reared animals. With post-weaning undernutrition, however, these deficits were found only in animals subjected to neonatal undernutrition as well.