ONTOGENETIC DEVELOPMENT OF A PHOSPHODIESTERASE ACTIVATOR AND THE MULTIPLE FORMS OF CYCLIC AMP PHOSPHODIESTERASE OF RAT BRAIN

Abstract— The activity of cyclic AMP phosphodiesterase of rat cerebral homogenates increased several-fold between 1 and 60 days of age. Enzyme activity in the cerebellum, on the other hand, did not increase during this period. A kinetic analysis of the phosphodiesterase activity revealed evidence for multiple forms of the enzyme and indicated that the postnatal increase in phosphodiesterase activity of rat cerebrum was due almost exclusively to the high K_m enzyme. In cerebellum, the ratio of the high and low K_m enzyme remained fairly constant during ontogenetic development. Physical separation of the phosphodiesterases contained in 100,000 g soluble supernatant fractions of sonicated brain homogenates by polyacrylamide disc gel electrophoresis confirmed the presence of multiple enzyme forms. In adult rats we found six distinct peaks of phosphodiesterase activity (designated I to VI according to the order in which they were eluted from the column) in cerebellum and 4 forms of the enzyme (Peaks I through IV) in cerebrum. Brains of newborn rats had a different pattern and ratio of phosphodiesterase activities. For example, Peak I phosphodiesterase was undetectable in cerebrum or cerebellum of newborn rats. Moreover, in the cerebellum of newborn rats Peak II was the dominant peak whereas in the cerebellum of adult rats Peak III was the largest peak. A comparison of the multiple forms of phosphodiesterase from the cerebrum of newborn and adult animals suggested that the postnatal increase in phosphodiesterase activity previously seen in crude homogenates was due largely to an increase in a high K, Peak II phosphodiesterase. The ratios of activities of the other peaks and their sensitivities to an activator of phosphodiesterase were similar in newborn and adult rats. An endogenous heat-stable activator of phosphodiesterase was found in cerebrum, cerebellum and brain stem. In newborn rats, the cerebellum contained several-fold less activity of this activator than did cerebrum or brain stem. However, the activity of this activator increased with age in the cerebellum and would appear to have decreased postnatally in cerebrum and brain stem. These results suggest that some multiple forms of phosphodiesterase can develop independently and that changes in activities of these phosphodiesterases may occur by increases in the quantity of enzyme or by changes in the quantity of an endogenous activator of phosphodiesterase.     

ENZYMES OF PHOSPHOINOSITIDE METABOLISM DURING RAT BRAIN DEVELOPMENT

—The activities of four enzymes concerned with inositol lipid metabolism have been determined in homogenates of rat brains of different ages. The enzymes are CDP-diglyceride inositol phosphatidate transferase, phosphatidylinositol kinase, diphosphoinositide kinase and triphosphoinositide phosphomonoesterase. The activities of all the enzymes increased with age. Phosphatidylinositol kinase activity rose most sharply well before myelination, reaching a maximum at about 6 days of age. Diphosphoinositide kinase and triphosphoinositide phosphomonoesterase activities increased most rapidly during myelination. The increase in CDP-diglyceride inositol phosphatidate transferase showed no definite association with any period of development. It is concluded that triphosphoinositide metabolism is associated with myelin or a closely related structure.     

PHOSPHOINOSITIDE KINASES IN CHICK BRAIN AND SCIATIC NERVE, A DEVELOPMENTAL STUDY

Phosphatidylinositol kinase and diphosphoinositide kinase activities were measured in homogenates of brain and sciatic nerve of developing chick embryos and chicks. Characteristics of the chick nervous system enzymes were similar to those reported for rat brain. Diphosphoinositide kinase was inhibited by high concentrations of ATP and by low concentrations of triphosphoinositide. Both activities were greatly enhanced by the non-ionic detergent, Cutscum, and the ratio of detergent to protein in the reaction mixture was important. Optimum phosphatidylinositol kinase activity required a ratio of 7 : 1 for both tissues. The optimum ratio for diphosphoinositide kinase was 3:1 for nerve homogenates and 0.6:1 for brain. Cutscum increased the concentration of diphosphoinositide that is required for maximum diphosphoinositide kinase activity. Developmental changes were the same for both kinase activities, which were low in unmyelinated brain and sciatic nerve. The activities correlated with the concentration of polyphosphoinositides in chick brain where they increased 4-5 fold during the period of active myelination and remained high in the mature brain. The kinase activities correlated with the rate of triphosphoinositide deposition in sciatic nerve. Following a 2-3 fold increase during the initial phase of myelination the activities declined to values as low as those of embryonic nerve.     

The subcellular distribution of triphosphoinositide phosphomonoesterase in guinea-pig brain

1. Some properties of the triphosphoinositide phosphomonoesterase from the homogenates of guinea-pig brain were studied. The enzyme has an optimum pH range 6.7-7.3, is stimulated with KCl at a concentration of 0.1m, and under these conditions has K(m)1.43x10(-4)m. 2. A factor from the ;pH5 supernatant' of guinea-pig brain stimulates the enzyme activity over and above the stimulation produced by KCl. Subcellular fractions of guinea-pig brain varied in their response to the ;pH5 supernatant'. Maximum stimulation was observed with the P(1) fraction, containing myelin and nuclei. 3. An assay system for the enzyme was developed that contained optimum concentrations of both KCl and the ;pH5 supernatant'. Acid phosphatases were inhibited by NaF, but, in contrast with previous work, no EDTA was added to the assay system to inhibit the alkaline phosphatases. This reagent inhibited the triphosphoinositide phosphomonoesterase. It was estimated that the remaining fraction of non-specific phosphatases can account for only 14% of the observed triphosphoinositide phosphomonoesterase activity. 4. Subcellular fractions of guinea-pig brain were characterized by electron microscopy and subcellular markers. The triphosphoinositide phosphomonoesterase exhibited a distribution between the fractions similar to that of 5'-nucleotidase, but different from that of alkaline phosphatase.     

PROPERTIES AND REGIONAL DISTRIBUTION OF TRYPTOPHAN HYDROXYLASE IN THE CHICKEN BRAIN

Tryptophan hydroxylase in young chicken brain had a pH optimum of 7.5–8, depending on the buffer used. It had apparent K_m values for tryptophan and tetrahydrobiopterin of 49 μM and 32 μM respectively. The enzyme in chicken brain, but not rat brain, was cold-shock labile but was stable for up to 4 days at — 20°C. Lability was observed both in tissues and homogenates of these tissues subjected to cold shock, but the extent of loss of activity varied between brain regions. Supernatant fractions did not lose activity after cold shock. The highest level of tryptophan hydroxylase was found in the rostral region of the chicken brainstem. High levels were also found in the caudal region of the brainstem, the midbrain, thalamus, caudate and cerebral cortex. The cerebellum and optic chiasma contained only traces of activity.     

ACTIVITIES OF 3,4-DIHYDROXY-l-PHENYLALANINE AND 5-HYDROXY-l-TRYPTOPHAN DECARBOXYLASES IN RAT BRAIN: ASSAY CHARACTERISTICS AND DISTRIBUTION

Abstract— Optimal assay conditions for decarboxylation of 3,4-dihydroxy- l -phenylalanine (DOPA) and 5-hydroxy- l -tryptophan (5-HTP) were determined in homogenates of rat brain by use of a sensitive, precise microradiometric technique. The two activities exhibited widely different optima for pH, temperature and substrate concentrations. The activity of 5-HTP decarboxylase was stimulated 2-fold by added pyridoxal-5-phosphate and was relatively resistant to antagonists of pyridoxal-P. By contrast, the activity of DOPA decarboxylase was stimulated 20-fold by added coenzyme and could be completely inhibited by carboxyl trapping agents. DOPA decarboxylase activity in subcellular fractions of brain was associated predominately with the soluble fractions and its distribution in the various fractions closely paralleled that of lactic acid dehydrogenase. 5-HTP decarboxylase activity in brain was distributed almost equally between soluble and particulate fractions, and its distribution within the particulate fractions differed from that of succinic acid dehydrogenase. The two decarboxylases in brain exhibited a 7-fold divergence in relative specific activity when their respective distributions in subcellular fractions were compared. Similarly, the regional distributions of the two decarboxylases in rat brain did not parallel one another; e.g. there was a 4-fold difference between the ratio of the two activities in cerebellum and that found in the corpus striatum.     

ONTOGENETIC DEVELOPMENT OF ADENYL CYCLASE AND PHOSPHODIESTERASE IN RAT BRAIN

Abstract— The activities of adenyl cyclase and phosphodiesterase were determined in homogenates of cerebrum, cerebellum and brain stem of rats of 1 day to 9 weeks of postnatal age. The activity of cerebral and brain stem adenyl cyclase measured either in the absence or presence of sodium fluoride increased rapidly for 2 weeks, reached at 20 days a maximum about three times (brain stem) or six times (cerebrum) that seen on day 1 and then declined slightly during the next several weeks. In contrast, activity of cerebrellar adenyl cyclase increased more slowly and reached a maximum at about 32 days. Activity of phosphodiesterase in cerebrum and brain stem increased several-fold during brain maturation; however, enzymic activity in the cerebellum decreased during the entire 9 week period.
In the pineal gland, adenyl cyclase activity measured in the absence of norepinephrine or sodium fluoride did not change significantly with age. However, enzymic activity measured in the presence of these agents increased with the age of the rat. Bilateral removal of the superior cervical ganglia at 1 day of age is known to arrest the sympathetic innervation of the pineal gland but did not prevent the development of this adenyl cyclase system activated by catecholamines or fluoride.     

ON THE MECHANISM OF STIMULATION OF AMP-AMINOHYDROLASE ACTIVITY BY HEXOKINASE IN BRAIN TISSUE

—A hexokinase has been isolated from brain tissue on Sephadex G-100 and DEAE cellulose which is similar to yeast enzyme in stimulating the AMP-aminohydrolase activity of rat brain soluble fractions. This effect of hexokinase is influenced neither by N-acetyl-glucosamine nor noradrenaline. An isoenzyme of hexokinase isolated from brain tissue on DEAE cellulose, having properties similar to that of the muscle enzyme, has no effect on AMP-aminohydrolase activity. The activating effect of yeast hexokinase is not due to its oligomeric structure. Enzyme subunits obtained by the treatment of native yeast enzyme by urea also activate AMP-aminohydrolase of rat brain soluble fractions.     

THE DISTRIBUTION OF CALCIUM-DEPENDENT PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHODIESTERASE IN RAT BRAIN

Abstract— The properties of Ca2⁺-dependent phosphatidylinositol-phosphodiesterase in membrane fractions and supernatants prepared from rat brain have been examined with the aim of providing firm evidence for the existence of a membrane-bound activity distinct from the soluble enzyme found in the cytosol (EC 3.1.4.10). The soluble enzyme is either stimulated or inhibited at pH 7.0 by deoxycholate depending on the ratio of detergent to substrate. The effects of deoxycholate are pH dependent and result in a shift of the enzyme optimum to a higher pH if the enzyme is assayed in the presence of deoxycholate. The soluble enzyme cannot hydrolgse membrane-bound phosphatidylinositol (in ₃₂P-labelled rat liver microsomes) unless deoxycholate is present. The pH optimum is 6.7 for this detergent-dependent hydrolysis and this is probably dependent on the ionization of deoxycholic acid. The lactate dehydrogenase (EC 1.1.1.27) content of rat brain membrane fractions has been measured to estimate the contamination of these fractions by supernatant phosphatidylinositol-phosphodiesterase. No evidence has been found for phosphatidylinositol-phosphodiesterase activities that cannot be explained by such contamination. It is concluded that all the properties of calcium-dependent phospha-tidylinositol-phosphodicsterase in rat brain can be explained by the existence of only the solublc cyto-plasmic enzyme: no evidence confirming a distinct membrane-bound activity has been obtained.     

DEVELOPMENT OF MITOCHONDRIAL PYRUVATE METABOLISM IN RAT BRAIN

The activities of a number of mitochondrial enzymes involved in the metabolism of pyruvate during development of the rat brain were investigated. The rates of decarboxylation of [1-¹⁴C]pyruvate to ¹⁴CO₂ via pyruvate dehydrogenase and the fixation of H¹⁴CO₃^? in the presence of pyruvate via pyruvate carboxylase by brain homogenates were very low in newborn rats. These rates increased markedly by about four-fold and 15-fold respectively during 10–35 postnatal days. The rates of the fixation of H¹⁴CO₃^? by cerebral homogenates were supported by the development of the activity of pyruvate carboxylase in rat brain. The activities of citrate synthase, aconitase, NAD-malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and phosphoenol-pyruvate carboxykinase were very low in the particulate fraction of the newborn rat brain. The activities of all these enzymes increased makedly by about three- to 10-fold during 10–35 days after birth. The activity of mitochondrial phosphoenolpyruvate carboxykinase from rat brain was not precipitated by an antibody prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase suggesting that cerebral mitochondrial enzyme is immunologically different from that of the cytosolic form in hepatocytes. The significance of the development of the cerebral mitochondrial metabolism is discussed in relation to biochemical maturation of the brain.     

TWO FORMS OF ALANINE AMINOTRANSFERASE IN RAT BRAIN DURING ONTOGENY

Abstract— Alanine aminotransferase activity in subcellular fractions of rat brains was investigated during ontogenic development. The activity rose from the prenatal period until adulthood, the highest increase being observed during the period of morphological metabolic and functional maturation of the brain. The rise of the total activity was due predominantly to a rise in the activity of the cytosblic enzyme; the activity of the mitochondrial enzyme did not change markedly during ontogeny. CI-ions and elevated temperature (55°C) inhibited the activity only of the mitochondrial enzyme. Raised temperature stimulated the activity of the cytosolic enzyme while CI-ions did not influence its activity. Our results indicate that 2 alanine aminotransferase isoenzlmes are already present in the rat brain in the prenatal period. It is assumed that the cytosolic enzyme is involved in the regulation of tissue glycol)sis and alanine formation, while the mitochondrial enzyme plays a role in the amino nitrogen transport between mitochondria and cytosol.     

INTRACELLULAR LOCALIZATION OF ENZYMES IN SPLEEN : I. REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE CYTOCHROMEc REDUCTASE, CYTOCHROMEc OXIDASE, AND SUCCINIC DEHYDROGENASE IN THE RAT AND GUINEA PIG

1. The intracellular distribution of nitrogen, DPNH cytochrome c reductase, succinic dehydrogenase, and cytochrome c oxidase has been studied in fractions derived by differential centrifugation from rat and guinea pig spleen homogenates. 2. In the spleens of each species, the nuclear fraction accounted for 40 to 50 per cent of the total nitrogen content of the homogenate, and the mitochondrial, microsome, and supernatant fractions contained about 8, 12, and 30 per cent of the total nitrogen, respectively. 3. Per mg. of nitrogen, DPNH cytochrome c reductase was concentrated in the mitochondria and microsomes of both rat and guinea pig spleens. Seventy per cent of the total DPNH cytochrome c reductase activity was recovered in these two fractions. The reductase activity associated with the nuclear fraction was lowered markedly by isolating nuclei from rat spleens with the sucrose-CaCl₂ layering technique. The lowered activity was accompanied by the recovery of about 90 per cent of the homogenate DNA in the isolated nuclei, indicating that little, if any, of the reductase is present in spleen cell nuclei. 4. Per mg. of nitrogen, succinic dehydrogenase was concentrated about 10-fold in the mitochondria of rat spleen, and 65 per cent of the total activity was recovered in this fraction. 5. Cytochrome c oxidase was concentrated, per mg. of nitrogen, in the mitochondria of both rat and guinea pig spleens. The activity associated with the nuclear fraction was greatly diminished when this fraction was isolated from rat spleens by the sucrose-CaCl₂ layering technique. Only 50 to 70 per cent of the total cytochrome c oxidase activity of the original homogenates was recovered among the four fractions from both rat and guinea pig spleens, while the specific activities of reconstructed homogenates were only 55 to 75 per cent of those of the original whole homogenates. This was in contrast to the results with DPNH cytochrome c reductase and succinic dehydrogenase where the recovery of total enzyme activity approached 100 per cent, and the specific activities of reconstructed homogenates equalled those of the original homogenates. The recovery of cytochrome c oxidase was greatly improved when only the nuclei were separated from rat spleen homogenates. 6. Data were presented comparing the concentrations (ratio of activity per mg. of nitrogen of the fraction to activity per mg. of nitrogen of the homogenate) of DPNH cytochrome c reductase in mitochondria and microsomes derived from different organs of different animals. 7. Data were presented comparing the activities per mg. of nitrogen of DPNH cytochrome c reductase in homogenates from several organs of various animals.     

FATTY ACIDS OF LECITHIN IN SUBCELLULAR FRACTIONS DURING MATURATION OF BRAIN

Abstract— A study has been made of the fatty acyl profiles of lecithin in subcellular fractions of the brain in rat, guinea pig and rabbit. It was found that cerebral lecithins consisted of at least two groups with dissimilar fatty acyl profiles. The group obtained from myelin showed little variation with age of the rats or among two other species examined. The changes in lecithin fatty acyl composition of brain homogenates were in agreement with a progressively greater contribution of myelin lecithins to brain homogenate lecithins with increasing age.     

The hydrolysis of triphosphoinositide by a phosphodiesterase in rat kidney cortex

The hydrolysis of triphosphoinositide by a phosphodiesterase has been demonstrated in rat kidney cortex. Subcellular fractionation studies revealed that the enzyme activity was predominantly found in the supernatant fraction. After acid precipitation and ammonium sulfate fractionation, the soluble enzyme was free from triphosphoinositide phosphomonoesterase activity.Although the partially purified enzyme did not require added divalent cations for activity, it was strongly inhibited by EDTA (0.1 mm). In the absence of EDTA, added MgCl₂ or CaCl₂ depressed the enzyme activity. The enzyme preparation was specific to polyphosphoinositides; it did not attack phosphatidylinositol and other phospholipids. It hydrolyzed both diphosphoinositide and triphosphoinositide with the formation of 1,2-diglyceride and organic phosphate.     

THE EFFECT OF NEONATAL THYROIDECTOMY ON THE DEVELOPMENT OF THE ADENOSINE 3'',5''-MONOPHOSPHATE SYSTEM IN THE RAT BRAIN

Abstract— The effect of neonatal thyroidectomy on the cyclic AMP system in the developing rat brain was examined. Administration of ¹³¹I at birth led to a 16 per cent reduction in brain weight and a 70 per cent reduction in body weight by 40 days of age. The level of cyclic AMP in the brain increased 5-fold between birth and 40 days of age and this increase was partially reduced by early thyroidectomy. A similar increase in the activity of adenyl cyclase and phosphodiesterase was observed during development, but thyroidectomy produced no detectable changes in the activity of either enzyme. The activity of the cyclic AMP-dependent protein kinase was already maximal at birth and also was unaffected by thyroidectomy.
Norepinephrine increased levels of cyclic AMP 4- to 5-fold in brain slices prepared from adult rats, but was without effect on slices prepared from newborn or 3-day-old rats. The response to norepinephrine in thyroidectomized rats did not differ from that in control rats at any of the ages examined. Our findings indicate that neonatal hypothyroidism does not deleteriously affect the development of the cyclic AMP system in the rat brain.     

THE REGIONAL AND SUBCELLULAR DISTRIBUTION OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Catechol-O-methyl transferase (COMT) activities determined in different regions of rat brain showed small variations. Highest activities were found in the hypothalamus and corpora quadrigemina, and lowest activities in the hippocampus and corpus striatum. The regional distribution of COMT was thus at variance with the distribution of DOPA decar- boxylase in this study and with the distribution of catecholamines and tyrosine hydroxylase reported in the literature. Determinations of the subcellular distribution of COMT in rat forebrain showed that 50 per cent of the activity was recovered in the high speed supernatant fluid and about 33 per cent in the crude mitochondrial fraction. Further separation of the latter by discontinuous sucrose gradients showed that the particulate COMT was found in the synaptosomal fraction in an occluded form. Full enzyme activity was only obtained after treatment with a detergent or after resuspension in water. After hypo-osmotic rupture of the crude mitochondrial fraction, COMT was recovered in the cytoplasmic fraction. The subcellular distribution of COMT was very similar to the ones of lactate dehydrogenase and DOPA decarboxylase. The proportions of soluble COMT obtained from homogenates of various regions of the brain differed from that of choline acetyl transferase and DOPA decarboxylase but were similar to that of lactate dehydrogenase. In conclusion, COMT is a cytoplasmic enzyme almost evenly distributed in the CNS. Its distribution does not resemble the distributions of the catecholamines or of the enzymes participating in the synthesis of catecholamines.     

THE POSTNATAL DEVELOPMENT OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Abstract— The postnatal development of three enzymes in the rat forebrain was studied. When expressed per tissue weight the catechol- O -methyl transferase (COMT) increased 2-fold from birth to adult age, the lactate dehydrogenase (LDH) 4-fold and the monoamine oxidase (MAO) 12-fold. Expressed per mg protein the increase in the enzyme activities in the subcellular fractions which contained the main part of the different enzymes was still 2–4-fold for COMT and LDH while for MAO it was 4-fold.
There was a relative increase in the COMT activity in the P₂ fraction (synaptosomes and mitochondria). This increase was identical with a corresponding increase in LDH activity and protein and was probably due to growth of nerve terminals. The COMT in the cytoplasm of the synaptosomes showed the same increase relative to the proteins as did the 'free' cytoplasmic enzyme.
The conclusion is drawn that the enzymes in the rat brain show a certain degree of development during brain growth. An additional increase of some enzymes is due to the development of specialized structures such as mitochondria and nerve terminals with synapses. COMT is not related to any such specialized structure.     

BIOCHEMICAL CHANGES IN RAT BRAIN ASSOCIATED WITH THE DEVELOPMENT OF THE BLOOD-BRAIN BARRIER

—Subcellular fractions from brains of 5, 10, 13, 16, 21, 30 day-old and adult rats were prepared. Protein content and various enzyme activities were assayed in all fractions and brain homogenates. γ-Glutamyl transpeptidase activity and 5′-nucleotidase were very low at 5 days of life but steadily increased, reaching adult concentrations at about 30 days after birth. Alkaline phosphatase, instead slowly decreased with maturation, while monoamine oxidase after an initial decrease, increased rapidly to adult levels. The relation between the appearance of enzymatic activity in brain and the blood-brain barrier function is discussed.     

DIFFERENCES IN THE REGULATION OF TYROSINE AMINOTRANSFERASE IN BRAIN AND LIVER

Abstract— l -Tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5) activity in rat brain is not regulated in the same way as in rat liver. No diurnal rhythm in the activity of the cerebral enzyme was found in rats fed ad lib. although there was a marked diurnal variation in the activity of the hepatic enzyme. In adrenalectomized rats, hydrocortisone and glucagon induced the enzyme in liver but had no effect on the enzyme in brain. In normal rats, treatment with reserpine or exposure to cold elevated the activity of the hepatic enzyme without affecting the enzyme in brain. Thus, the tyrosine aminotransferase of brain differed from the enzyme in liver since it did not exhibit diurnal variations of activity and was not affected by hormones, drugs, or stress.     

EXISTENCE OF ASCORBIC ACID AS AN ACTIVATOR OF THIAMINE DlPHOSPHATASE IN RAT BRAIN

The effect of the supernatant fraction (105,000 g for 60 min) of rat brain on the microsomal thiamine diphosphatase activity was examined. The thiamine diphosphatase activity was increased by addition of the supernatant fraction. The factor activating the enzyme was a heat-stable and dialyzable substance. It caused lipid peroxidation in the microsomes and the increase of the enzyme activity was mediated through lipid peroxidation of the preparation. When the supernatant fraction was chromatographed on columns of Sephadex G-25 and Dowex 1 × 2, the activator was eluted in fractions containing ascorbic acid. The inhibitory factor of ATPase present in the supernatant fraction was also eluted with the activator. The u.v.-spectrum of the active fraction obtained by these chromatographies was the same as that of ascorbic acid. These findings indicate the existence of ascorbic acid as an activator of thiamine diphosphatase in rat brain and confirm the previous finding that the soluble factor inhibiting ATPase activity is ascorbic acid.