Diverse function of aromatase and the N-terminal sequence deleted form

The diverse function of human placental aromatase including estradiol 6-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6,7-³H₂,4-¹⁴C]estradiol, 7-ethoxycoumarin, and [N-methyl-³H₃]cocaine. 6-Hydroxy[7-³H,4-¹⁴C]estradiol was isolated as the metabolite of estradiol and the ³H-water release method based on the 6-³H label was established. The initial rate kinetics of the 6-hydroxylation gave K_m of 4.3 μM, V_max of 4.02 nmol min⁻¹mg⁻¹, and turnover rate of 0.27 min⁻¹. Testosterone competed dose-dependently with the 6-hydroxylation and showed the K_i of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed K_m of 200 μM, V_max of 12.5 nmol min⁻¹mg⁻¹ and turnover rate of 1.06 min⁻¹. The N-demethylation of cocaine was analysed by the ³H-release method, giving K_m of 670 μM, V_max of 4.76 nmol min⁻¹mg⁻¹, and turnover rate of 0.49 min⁻¹. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min⁻¹mg⁻¹ (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min⁻¹.     

Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.     

Regulation of pathways of glucose metabolism in kidney: Specific linking of pentose phosphate pathway activity with kidney growth in experimental diabetes and unilateral nephrectomy

The pentose phosphate pathway operates at an elevated level in rat kidney following induction of diabetes and in the compensatory hypertrophy following unilateral nephrectomy in control and alloxan-diabetic rats, as shown by the yields of ¹⁴Co₂ from [1-¹⁴C]glucose, [6-¹⁴C]glucose and ³H₂O yields from [2-³H]glucose. The elevated flux through the pentose phosphate pathway is correlated with the increased RNA content and weight of the kidney. The direct utilization of NADPH for reductive synthetic reactions and the potential for indirect utilization via the sorbitol route and the linked transhydrogenase reactions of the glucuronate-xylulose pathway, for NADH and ATP generation, are also discussed.     

Oxidation of [1-C]linoleic acid in isolated microsomes from pea leaves

The oxidation of [1-¹⁴C]linoleate in isolated microsomes from pea leaves was found to be stimulated by NADPH addition. The formation of one of the main metabolites, 12-hydroxy-9(Z)-dodecenoic acid is particularly NADPH-dependent. The predominant products in the absence of NADPH were hydroperoxides and in the presence of NADPH, 12-hydroxy-9(Z)-dodecenoic acid. Exogenous [1-¹⁴C]-13-hydroperoxy-9(Z), 11(E)-octadecadieoic acid and [1-¹⁴C]-12-oxo-9(Z)-dodecenoic acidwere the efficient precursors of 12-hydroxy9(Z)-dodecenoic acid. It was concluded that 12-hydroxy-9(Z)-dodecenoic acid is formed by NADPH-dependent enzymatic reduction of 12oxo-9(Z)-dodecenoic acid. The observed inhibition of linoleate oxidation in isolated microsomes by CO and metryapone suggests the involvement of cytochrome P-450 in the reaction. The relative contribution of lipoxygenase and monooxygenase activity to linoleate oxidation in microsomes is discussed.     

Modulation of protein kinase C activity by NaF in bone marrow derived macrophages

Stimulation of murine bone marrow derived macrophages with NaF, prelabeled with [1-¹⁴C]oleate and [³H]inositol, increased the production of inositol phosphates and the release of 1,2-[¹⁴C]diacylglycerol (DAG). Moreover, NaF also induced activation of protein kinase C. These results indicate that bone marrow derived macrophages exhibit a phosphatidyl-4,5-bisphosphate phospholipase C activity, sensitive to NaF, which might be modulated by G-proteins. Activation of protein kinase C could have been mediated by NaF-induced release of DAG.     

On the mechanism of biosynthesis of leukotrienes and related compounds

[10D-³H; 3-¹⁴C]- and [10L-³H; 3-¹⁴C]arachidonic acids were incubated with human polymorphonuclear leukocytes and with human platelets. Leukotriene B₄ and 5(S),12(S)-dihydroxy-6trans,8cis,10trans,14-cis-eicosatetraenoic acid (5,12-DHETE) were isolated and the ³H/¹⁴C ratios determined. It could be concluded that the 10D (pro-R)-hydrogen is eliminated in the conversion of 5(S)-hydroperoxy-6trans,8cis,11cis,14cis-eicosatetraenoic acid into leukotriene A₄ whereas in the conversion of arachidonic acid into 5,12-DHETE the 10L (pro-S)-hydrogen is lost. Incubation of the doubly labeled arachidonic acids with human platelets confirmed and extended previous data on the stereochemistry of the hydrogen removal from C-10 during the conversion into 12(S)-hydroperoxy-5cis,8cis,10trans,14cis-eicosatetraenoic acid, i.e., the 10L (pro-S)-hydrogen is eliminated and the 10D (pro-R)-hydrogen retained.     

Determination of δC values of valine in protein hydrolysate by gas chromatography–combustion isotope ratio mass spectrometry

A gas chromatographic–combustion isotope ratio mass spectrometric (GC–C-IRMS) method for the determination of [1-¹³C]valine enrichments in protein hydrolysates is described. Using a quick derivatization method, δ¹³C values of the N-methoxycarbonyl methyl ester of valine can be determined from baseline separated GC peaks. Evaluation studies with respect to precision, accuracy, linearity, reduction capacity of the CuO combustion furnace and isotope dilution as a result of derivatization, showed that our GC–C-IRMS system allows robust measurement of enrichments of [1-¹³C]valine in the range 0 to 1.5 MPE (S.D.±0.01 MPE, n=3). Therefore this method is suited to determine fractional synthetic rates (FSRs) of proteins as low as one-tenth of the FSR of human albumin, in studies using a primed, continuous (6 h) infusion with [1-¹³C]valine plasma enrichments of approximately 15 MPE and an hourly sampling schedule.     

Increased biological potency of hexafluorinated analogs of 1,25-dihydroxyvitamin D3 on bovine parathyroid cells

1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is known to be involved in regulating the proliferation of parathyroid cells and PTH synthesis through reactions involving its nuclear receptor. We evaluated the effects of 1,25-(OH)₂D₃ and its hexafluorinated analog, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D₃ (26,27-F₆-1,25-(OH)₂D₃), on parathyroid cells. The 1,25-(OH)₂D₃ and 26,27-F₆-1,25-(OH)₂D₃ each inhibited [³H]thymidine incorporation and ornithine decarboxylase (ODC) activity, which is important in cell proliferation, in primary cultured bovine parathyroid cells. The inhibitory effect of 26,27-F₆-1,25-(OH)₂D₃ on PTH secretion from parathyroid cells was significantly more potent than that of 1,25-(OH)₂D ₃ between 10⁻¹¹ M and 10⁻⁸ M. Study of 26,27-F₆-1,25-(OH)₂D₃ metabolism in parathyroid cells in vitro elucidated its slower degradation than that of 1,25-(OH)₂D₃. After 48 h of incubation with [1β-³H]26,27-F₆-1,25-(OH)₂D₃, two HPLC peaks, one for [1β-³H]26,27-F₆-1,25-(OH)₂D₃, and a second larger peak for [1β-³H]26,27-F₆-1,23(S),25-(OH)₃D₃, were detected. No metabolites were detected after the same period of incubation with 1,25-(OH)₂[26,27-³H]D₃. We observed that 26,27-F₆-1,23(S),25-(OH)₃D₃ was as potent as 1,25-(OH)₂D₃ in inhibiting the proliferation of parathyroid cells.

Data suggest that the greater biological activity of 26,27-F₆-1,25-(OH)₂D₃ is explained by its slower metabolisms and by the retention of the biological potency of 26,27-F₆-1,25-(OH)₂D₃ even after 23(S)-hydroxylation.     

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnovers in neuroblastoma × glioma hybrid cells (NG 108-15)

In neuroblastoma × glioma hybrid cells (NG 108-15) labelled with [³²P]-trisodium phosphate, [³H]-inositol and [¹⁴C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) while it had no effect on the release of [¹⁴C]-arachidonic acid (AA). The effect on PIP, was time- and dose-dependent with a maximal effect on [³H]-inositol- and [³²P]-labelled cells after 10–30 s of stimulation with 10⁻⁶ M bradykinin. However, the hydrolysis of [¹⁴C]-AA labelled PIP₂ was delayed compared to the effect on [³H]- and [¹⁴C]-PIP₂ and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [³H]-inositol phosphates (IP) and [³²P]- and [¹⁴P]- and [¹⁴C]-phosphatidic acid (PA) but the time course for PA formation did not allow the time-course for PIP₂ hydrolysis. A reduced labelling of [²³P]- and [¹⁴C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP₂. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A₂, in NG 108-15 cells.     

Biogenesis of nonacosan-15-one in brassica oleracea

The incorporation of [4-¹⁴C]4-oxostearic acid, [4-¹⁴C]stearic acid, and [1-¹⁴C]eicosanoic acid, respectively, into the nonacosan-15-one of Brassica oleracea has been investigated. While the oxo-acid was found to be a poor precursor of nonacosan-15-one, the two n-fatty acids were efficiently incorporated as intact units into n-nonacosane and the related 15-ketone. In accordance with expectation, the label in the 4-position of stearic acid appeared in the carbonyl carbon of the ketone. These results suggest that the C₂₉ hydrocarbon is first formed and subsequently oxidized to nonacosan-15-one.     

THE RELEASE OF AMINO ACIDS FROM THE HEMISECTED SPINAL CORD DURING STIMULATION

Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate. l -[¹⁴C]aspartate, l -[¹⁴C]serine, l [¹⁴C]threonine or l -[³H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate and l -[¹⁴C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[¹⁴C]serine, l -[¹⁴C]threonine, l -[¹⁴H]leucine, [¹⁴C]mannitol or [¹⁴C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.     

Glutamine and Glucose as Precursors of Transmitter Amino Acids: Ex Vivo Studies

Abstract: Radiolabelled glutamine and glucose were infused into lateral ventricles of rats in order to label transmitter amino acid pools in vivo . Brain regions close to the lateral ventricle (hippocampus, corpus striatum, hypothalamus) were labelled more effectively than more distant structures such as cerebral cortex or cerebellum. All regions were labelled to much the same extent over 30-150 min by [U-¹⁴C]glucose, [U-¹⁴C]glutamine, or [³H]glutamine administered alone or together in doublelabel experiments when allowance was made for any differences in precursor specific radioactivities. Slices of cerebral cortex or hippocampus from brains labelled in vivo were incubated and stimulated in vitro with veratrine (75 μ M ); tetrodotoxin (1 μ M ) was present in the control medium. Single-label experiments showed that [U-¹⁴C]- glutamine was more effective than [U-¹⁴C]glucose for labelling releasable glutamate and GABA. Double-label experiments showed that [³H]glutamine and [U-¹⁴C]- glucose given together in vivo labelled glutamate and GABA releasable in vitro to a similar extent. Both types of experiment empbasise the large contribution made by glutamine in vivo to pools of transmitter glutamate and GABA.     

Oligodendroglial Glycerophospholipid Synthesis: Incorporation of Radioactive Precursors into Ethanolamine Glycerophospholipids by Calf Oligodendroglia Prepared by a Percoll Procedure and Maintained in Suspension Culture

Abstract: Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyr-rolidone-coated silica gel) density gradient centrifugation survived in culture on polylysine-coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short-term suspension culture by dual label time course and substrate concentration dependence experiments with [2-³H]glycerol and either [1,2-¹⁴C]ethanolamine or L-[U-¹⁴C]serine. Rates of incorporation of ³H from the glycerol and of ¹⁴C from the ethanolamine into EGP were constant for 14 h. In medium containing 3 mM-[1,2-¹⁴C]ethanolamine and 4.8 mM-[2-³H]glycerol, rates of incorporation of ¹⁴C and ³H into diacyl glycerophosphoethanolamine (diacyl GPE) were similar. Under the same conditions, ³H specific activities of alkylacyl GPE and alkenylacyl GPE were much lower than ¹⁴C specific activities, likely as a result of the loss of tritium during synthesis of these forms of EGP via dihydroxyacetone phosphate. L-[U-¹⁴C]serine was incorporated into serine glycerophospholipid (SGP) by base exchange rather than de novo synthesis. ¹⁴C from L-[U-¹⁴C]serine also appeared in EGP after an initial lag period of several hours. Methylation of oligodendroglial EGP to choline glycerophospholipid (CGP) was not detected.     

The sequence of initiation of RNA, DNA and protein synthesis in the wheat grains during germination

The syntheses of main macromolecular substances, in a whole wheat grain allowed to germinate, are triggered in the following order: RNA, protein, DNA. The RNA synthesis, as judged by [2-¹⁴C]uridine incorporation, is initiated almost immediately after the seeds are exposed to the optimal germination conditions, whereas [1-¹⁴C]leucine and [2-¹⁴C]thymidine incorporation begins to occur only 3 and 4 hr later, respectively. The initiation of protein synthesis is accompanied by an apparent cessation of uridine incorporation.     

Concerning the pathway from 19-oxoandrost-4-ene-3,17-dione to estrone

The conversion of a molecule of 19-oxoandrost-4-ene-3,17-dione [1a] to estrone [2a] by human placental aromatase requires a molecule of oxygen and of NADPH. An atom of this molecule of oxygen is incorporated into the extruded formic acid derived from C-19 of [1a]. It was proposed that the 0₂ is utilized for the enzymatic 2β-hydroxylation of [1a] and the released intermediate 2β-hydroxy-19-oxoandrost-4-ene-3, 17-dione [5a]aromatizes nonenzymatically. Should [5a] be an obligatory intermediate of estrogen biosynthesis, then all the oxygen of its 2β-hydroxyl must be incorporated into the extruded formic acid. We have previously synthesized [2β-¹⁸0;19-³H][5c] and proved that none of its 2β-¹⁸0 was incorporated in the formic acid extruded in the aromatization. On this basis we concluded that [5a] can not be an obligatory precursor of estrogen biosynthesis.

The trapping of radioactive androst-4-ene-2β,3β,17β,19-tetrol in a reductively terminated incubation of a mixture of radioactive androst-4-ene-3, 17-dione and [5a] with crude placental aromatase was interpreted as evidence in support of the intermediacy of [5a]. We confirmed that the tetrol can indeed be trapped in the reductively terminated incubations. However, considering that the crude placental enzyme preparation very likely contains numerous activated oxygen species capable of a variety of oxidation reactions, most of which may not be related to estrogen elaboration, and in view of our results quoted above, the origin and the eventual biosynthetic role of the parent compound of the tetrol remains to be determined.     

The synthesis of [26,27-11C]dihydroxyvitamin D(3), a tracer for positron emission tomography (PET).

1,25-Dihydroxyvitamin D₃, an endogenous ligand with the highest affinity for the vitamin D receptor (VDR), was labeled with ¹¹C for use in biological experiments. The radionuclide was incorporated via the reaction of [¹¹C]methyllithium on a methyl ketone precursor in tetrahydrofuran at −10 °C. Deprotection of the labeled intermediate yielded 2.5–3 GBq [26,27-¹¹C]1,25-dihydroxyvitamin D₃ [¹¹C-1,25(OH)₂ D₃] with specific radioactivity averaging 100 GBq/μmol at the end of synthesis and HPLC purification. The entire process took 48 min from the end of radionuclide production. In vitro binding experiments in rachitic chick purified VDR demonstrated the high affinity binding of this novel tracer. Thus; ¹¹C-1,25(OH)₂ D₃ is available for in vivo distribution studies and may be suitable for the positron emission tomography (PET) determination of VDR levels and occupancy in animals and humans.     

SYNTHESIS OF GLYCOPROTEINS AND GANGLIO-SIDES IN DEVELOPING RAT BRAIN

Abstract— Intracerebral injections of radioactive fucose into developing rats resulted in specific labelling of the brain glycoproteins in their fucose moieties. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the radioactive glycoproteins were very heterogeneous with regard to molecular weight. A procedure utilizing [³H]fucose and [¹⁴C]fucose together with double-label counting techniques was developed for comparing the electrophoretic patterns of newly synthesized glycoproteins from different samples of tissue. By the use of this procedure we showed that the incorporation of radioactive fucose into the glycoproteins of high mol. wt. was relatively greater in the brains of 5-day-old rats than in those of 25-day-old rats. Intracerebral injection of N -[ Ac -³H]acetyl- d -mannosamine resulted in a high degree of specificity for the labelling of sialic acid moieties in glycoproteins and gangliosides. The ratio of the d.p.m. of N -[³H]acetylmannosamine incorporated into glycoproteins to the d.p.m. incorporated into gangliosides was higher in 5-day-old rats than in 15- or 25-day-old rats. Experiments in which 15-day-old rats were injected with a mixture of [¹⁴C]fucose and N -[³H]acetylmannosamine showed that there were differences in the relative degrees of incorporation of the two radioactive precursors into the various glycoproteins. The greatest incorporation of [¹⁴C]fucose relative to that of N- [³H]acetylmannosamine occurred in some of the glycoproteins of smaller mol. wt.     

[2-3H]GLYCEROL AS A PRECURSOR OF PHOSPHOLIPIDS IN RAT BRAIN: EVIDENCE FOR LACK OF RECYCLING

Abstract— When [2-³H]glycerol was injected intracranially into young rats, it was presented as a pulse label, leaving the brain rapidly and giving up much of its labelled hydrogen to water. [2-³H]glycerol was efficiently incorporated into brain lipids, especially into choline and ethanolamine phospholipids. Following injection of a mixture of [³H]- and [¹⁴C]-labelled glycerol, the ratio of ³H to ¹⁴C in the phospholipids of both whole brain and the microsomal fraction decreased as a function of time after injection. This finding indicated less recycling of the tritium label. This lack of recycling was further indicated by the finding that 94 per cent of the tritium label of phosphatidyl choline was in the glycerol portion of the molecule rather than in the fatty acids. At 2 weeks following injection with [³H]glycerol, 93 per cent of the total radioactivity in brain appeared in the lipid fraction. In contrast, following injection with [¹⁴C]glycerol, only 57 per cent of the radioactivity appeared in lipid, with about 20 per cent in protein.     

NEUROTRANSMITTER UPTAKE INTO SLICES OF RAT CEREBRAL CORTEX IN VITRO: EFFECT OF SLICE SIZE

Abstract— The uptake of [¹⁴C]GABA, [¹⁴C]taurine, [³H] β -alanine and [¹⁴C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [¹⁴C]Taurine and [³H] β -alanine uptake increased whereas [¹⁴C]GABA uptake decreased with increasing slice size. [¹⁴C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [³H] β -alanine and [¹⁴C]GABA uptake by l -2,4-diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.     

THE INCORPORATION OF DOUBLE-LABELLED ACETATE INTO GLUTAMATE AND RELATED AMINO ACIDS FROM ADULT MOUSE BRAIN: COMPARTMENTATION OF AMINO ACID METABOLISM IN BRAIN

Abstract– We have determined the incorporation of [³H]-, [1-¹⁴C]- and [2-¹⁴C]acetate into glutamate, glutamine and aspartate of the adult mouse brain. All these three acetates were incorporated more extensively into glutamine than into glutamate. This has been reported by several authors for each of these labelled acetates in separate experiments. It was shown that [³H, 2-¹⁴C]acetate can be used to obtain an acetate labelling ratio analogous to the previously used [2-¹⁴C]acetate/[1-¹⁴C]acetate labelling ratio. From these acetate labelling ratios of glutamine and glutamate conclusions can be deduced about the dynamic relationship of these amino acids with each other and with the tricarboxylic acid cycle.
A fairly large isotope effect between acetate and glutamate was observed. As this isotope effect is very likely caused by the citrate synthase reaction, it can be argued that citrate synthase involved in the conversion of labelled acetate into glutamate is far out of equilibrium in vivo. Comparing our data with literature data, the possibility can be suggested that citrate synthase in the acetate metabolizing compartment is in situ kinetically distinct from citrate synthase in other compartments of the brain.