Sulphydryl Inhibitors Research Articles

Purification and Characterization of Carboxylesterase from the Seeds of Jatropha curcas

Carboxylesterases are hydrolases which catalyze the hydrolysis of various types of esters. Carboxylesterase from the seeds of Jatropha curcas has been purified to homogeneity using ammonium sulfate fractionation, CM-cellulose chromatography, Sephadex G-100 chromatography and preparative polyacrylamide gel electrophoresis (PAGE). The homogeneity of the purified enzyme was confirmed by PAGE, iso-electrofocusing and SDS-PAGE. The molecular weight of the purified enzyme was determined by both gel-permeation chromatography on Sephadex G-150 and SDS-PAGE. The molecular weight determined by Sephadex G-150 chromatography and SDS-PAGE both in the presence and absence of 2-mercaptoethanol was 31 kDa. The isoelectric point of the purified enzyme was found to be 8.9. JCSE-I (J. curcas seed esterase-I) was classified as carboxylesterase on the basis of substrate and inhibitor specificity. The K(m) of JCSE-I with 1-naphthyl acetate, 1-naphthyl propionate, 1-naphthyl butyrate and 2-naphthyl acetate as substrates were found to be 0.0,794, 0.0,658, 0.0,567 and 0.1 mM, respectively. The enzyme exhibited an optimum temperature of 45 °C and an optimum pH of 6.5. The enzyme was stable up to 15 min at 65 °C. The enzyme was resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB) and inhibited by organophosphates (dichlorvos, parathion and phosphamidon).

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization

Two carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CM-cellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS–PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS–PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K m of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K m was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45 °C (ME-III) and 37 °C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35 °C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB).

Preliminary evidence on existence of transplasma membrane electron transport in Entamoeba histolytica trophozoites: a key mechanism for maintaining optimal redox balance

Entamoeba histolytica, an amitochondriate parasitic protist, was demonstrated to be capable of reducing the oxidized form of alpha-lipoic acid, a non permeable electron acceptor outside the plasma membrane. This transmembrane reduction of non permeable electron acceptors with redox potentials ranging from -290 mV to +360 mV takes place at neutral pH. The transmembrane reduction of non permeable electron acceptors was not inhibited by mitochondrial electron transport inhibitors such as antimycin A, rotenone, cyanide and azide. However, a clear inhibition with complex III inhibitor, 2-(n-heptyl)-4-hydroxyquinoline-N-oxide; modifiers of sulphydryl groups and inhibitors of glycolysis was revealed. The iron-sulphur centre inhibitor thenoyltrifluoroacetone failed to inhibit the reduction of non permeable electron acceptors whereas capsaicin, an inhibitor of energy coupling NADH oxidase, showed substantial inhibition. p-trifluromethoxychlorophenylhydrazone, a protonophore uncoupler, resulted in the stimulation of alpha-lipoic acid reduction but inhibition in oxygen uptake. Mitochondrial electron transport inhibitors substantially inhibited the oxygen uptake in E. histolytica. Transmembrane reduction of alpha-lipoic acid was strongly stimulated by anaerobiosis and anaerobic stimulation was inhibited by 2-(n-heptyl)-4-hydroxyquinoline-N-oxide. Transmembrane redox system of E. histolytica was also found to be sensitive to UV irradiation. All these findings clearly demonstrate the existence of transplasma membrane electron transport system in E. histolytica and possible involvment of a naphthoquinone coenzyme in transmembrane redox of E. histolytica which is different from that of mammalian host and therefore can provide a novel target for future rational chemotherapeutic drug designing.

Pyrimidine 5' nucleotidase deficiency.

Pyrimidine 5' nucleotidase deficiency.

Use of electrospray ionization mass spectrometry and tandem mass spectrometry to study binding of F0 inhibitors to ceroid lipofuscinosis protein, a model system for subunit c of mitochondrial ATP synthase.

Ceroid lipofuscinosis protein (CLP), the major accumulating protein in several forms of ceroid lipofuscinosis, has an amino acid sequence that is identical to that of the F0 subunit c of normal bovine ATP synthase. Electrospray ionization mass spectrometry (ESI-MS) has shown that ovine CLP and normal bovine F0 subunit c are identical, including a 42 mass unit post-translational modification. Although the identity and the location of this modification have not been fully established in both species, CLP can be used as a convenient and a unique source of subunit c for studies of F0 inhibitor interactions by ESI-MS analysis. Analysis of mixtures of CLP incubated with several known F0 inhibitors showed that N, N'-dicyclohexylcarbodiimide and organotins bind covalently to CLP but interactions with oligomycin and venturicidin were not observed. The sulphydryl inhibitors, 2,3-dimethoxy-5-methyl-1,4,-benzoquinone (UQ0) and N-ethyl maleimide (NEM) were also shown to bind covalently to the protein. The binding stoichiometry and the relative rate of reaction were then determined for each inhibitor. Tandem mass spectrometry experiments performed on the [M+5H]5+ ion of the intact CLP and of the complexes UQ0-CLP and NEM-CLP allowed the identification of 80% of the CLP sequence and revealed that UQ0 and NEM are both bound to cysteine-64. This work shows the exceptional utility of ESI-MS in studies of the interaction of CLP with a range of inhibitors which are applicable to studies of the F0 component of ATP synthase.

Use of Electrospray Ionization Mass Spectrometry and Tandem Mass Spectrometry to Study Binding of F 0 Inhibitors to Ceroid Lipofuscinosis Protein, a Model System for Subunit c of Mitochondrial ATP Synthase

Ceroid lipofuscinosis protein (CLP), the major accumulating protein in several forms of ceroid lipofuscinosis, has an amino acid sequence that is identical to that of the F0 subunit c of normal bovine ATP synthase. Electrospray ionization mass spectrometry (ESI-MS) has shown that ovine CLP and normal bovine F0 subunit c are identical, including a 42 mass unit post-translational modification. Although the identity and the location of this modification have not been fully established in both species, CLP can be used as a convenient and a unique source of subunit c for studies of F0 inhibitor interactions by ESI-MS analysis. Analysis of mixtures of CLP incubated with several known F0 inhibitors showed that N, N′-dicyclohexylcarbodiimide and organotins bind covalently to CLP but interactions with oligomycin and venturicidin were not observed. The sulphydryl inhibitors, 2,3-dimethoxy-5-methyl-1,4,-benzoquinone (UQ0) and N-ethyl maleimide (NEM) were also shown to bind covalently to the protein. The binding stoichiometry and the relative rate of reaction were then determined for each inhibitor. Tandem mass spectrometry experiments performed on the [M+5H]5+ ion of the intact CLP and of the complexes UQ0–CLP and NEM–CLP allowed the identification of 80% of the CLP sequence and revealed that UQ0 and NEM are both bound to cysteine-64. This work shows the exceptional utility of ESI-MS in studies of the interaction of CLP with a range of inhibitors which are applicable to studies of the F0 component of ATP synthase.

Pyruvate dehydrogenase complex and acetyl-CoA carboxylase in pea root plastids: their characterization and role in modulating glycolytic carbon flow to fatty acid biosynthesis

The pyruvate dehydrogenase complex (PDC) and acetyl-CoA carboxylase (ACC, EC 6.4.1.2) have been characterized in pea root plastids. PDC activity was optimum in the presence of 1.0 mM pyruvate, 1.5 mM NAD+ 0.1 mM CoA, 0.1 mM TPP, 5 mM MgCl2, 3.0 mM cysteine-HCl, and 0.1 M Tricine (pH 8.0) and represents approximately 47% of the total cellular activity. ACC activity was greatest in the presence of 1.0 mM acetyl-CoA, 4 mM NaHCO3 mM ATP, 10 mM MgCl2, 2.5 mM dithiothreitol, and 100 mM Tricine (pH 8.0). Both enzymes were stimulated by reduced sulphydryl reagents and inhibited by sulphydryl inhibitors. ACC was also inhibited by malonyl-CoA while PDC was inhibited by both malonyl-CoA and NADH. Both enzymes were stimulated by DHAP and UDP-galactose while ACC was also stimulated by PEP and F1,6P. Palmitic acid and oleic acid both inhibited ACC, but had essentially no effect on PDC. Palmitoyl-CoA inhibited both enzymes while PA and Lyso-PA inhibited PDC, but stimulated ACC. The results presented support the hypothesis that PDC and ACC function in a co-ordinated fashion to promote glycolytic carbon flow to fatty acid biosynthesis in pea root plastids.

Adenosine triphosphate-lead histochemical reactions in ependymal epithelia of murine brains do not represent calcium transport adenosine triphosphatase.

The strong enzyme histochemical reactions for adenosine triphosphatase (ATPase) seen in ependymal tanycytes after incubation in calcium-containing media have previously been reported as calcium transport ATPase. Investigation of these reactions showed that: (1) any nucleoside triphosphate can serve as a substrate; (2) diphosphates and monophosphates cannot replace triphosphates; this includes p-nitrophenyl phosphate which is readily hydrolysed by plasma membrane transport ATPases; (3) strong localization occurs in the presence of millimolar concentrations of either calcium or magnesium ions; there is no absolute requirement for calcium ions; (4) they are not inhibited by sulphydryl inhibitors or calmodulin antagonists; (5) lead phosphate precipitates are localized almost entirely on the external face of tanycyte plasma membranes. In addition, the technique gives strong localization to vessels in the choroid plexus but not to the choroidal epithelium. Immunohistochemistry with a primary antibody raised against Ca2+, Mg2(+)-ATPase stains the choroidal epithelium but not the vessels or the ependymal tanycytes. These results are inconsistent with identification of the reaction as calcium transport ATPase but support characterization as an ecto-ATPase.

Differences in glucose transport between bloodstream and procyclic forms of Trypanosoma brucei rhodesiense

In African trypanosomes the requirements for glucose and its metabolism vary in different stages of the life cycle. Here we present evidence that cultured procyclic trypanosomes of Trypanosoma brucei rhodesiense uptake glucose against a concentration gradient in a time and dose-dependent manner. Moreover, glucose transport is completely inhibited by the sulphydryl inhibitor N-ethylmaleimide, suggesting the presence of a protein moiety as the carrier molecule. Comparison of glucose uptake in bloodstream and procyclic trypanosomes point to the possibility that different transporters may function in the 2 developmental stages. Glucose uptake by bloodstream trypanosomes requires Na + ions and is inhibited by phlorizin, an inhibitor of Na +-dependent glucose transporters in mammalian cells. Conversely, procyclic trypanosomes transport glucose in a Na +-independent manner, and transport is not affected by phlorizin. Finally, the putative procyclic glucose transporter has a higher affinity for glucose (apparent K m 23 μM) than the bloodstream carrier (apparent K m 237 μM).

Purification and characterization of alkaline endo-1,4- -glucanases from alkalophilic Bacillus sp. KSM-635

Summary: Two carboxymethylcellulases (CMCase, 1,4-1,4-β-d-glucan glucanohydrolase, EC 3.2.1.4), designated E-H and E-L, were purified to homogeneity from a culture filtrate of the alkalophilic Bacillus sp. KSM-635, by chromatography on DEAE-Toyopearl 650S and gel filtration on Bio-Gel A-0.5m. The purified CMCases both contained approximately 2–3% (w/w) glucosamine. Molecular masses deduced from SDS-PAGE were 130 kDa for E-H and 103 kDa for E-L. The pH optima of the enzymes were both about 9.5, and their optimum temperatures were around 40°C. Activities of both enzymes were inhibited by Hg2+, Cu2+, Fe2+ and Fe3+, but sulphydryl inhibitors, such as N-ethylmaleimide, monoiodoacetate and 4-chloromercuribenzoate, had either no effect or a slightly inhibitory effect. N-Bromosuccinimide was strongly inhibitory, suggesting that a tryptophan residue is essential for the activity of the CMCases from Bacillus. In addition, the activities of both E-H and E-L were stimulated by Co2+, and they required Mg2+, Ca2+, Mn2+ or Co2+ for stabilization. Both enzymes efficiently hydrolysed carboxymethylcellulose (β-1,4-linkage) and lichenan (β-1,3; 1,4-linkage), but crystalline cellulosic substrates, curdlan (β-1,3-linkage), laminarin (β-1,3; 1,6-linkage) and 4-nitrophenyl-β-d-glucopyranoside were hydrolysed very little, if at all. 4-Nitrophenyl-β-d-cellobioside was hydrolysed by both enzymes to liberate 4-nitrophenol, and their hydrolysis rates were higher at neutral pH than at alkaline pH.

Effects of the sulphydryl inhibitor N-ethyl-maleimide on the phrenic nerve and diaphragm muscle of the rat

N-Ethyl-maleimide (NEM, 2.5 × 10 −5M) inhibited the compound action potential of the phrenic nerve and increased the spontaneous release of transmitter from the nerve terminals, recorded as miniature endplate potentials. The first effect was the cause of a blockade of the phrenic nerve diaphragm preparation, during indirect stimulation. The left phrenic nerve was more susceptible to inhibition than the right. An increase of the threshold was observed during the progression of the inhibition. The inhibition was not use-dependent and there was no synergistic interaction with the local anaesthetic drug, tetracaine. The inhibition was partly antagonized by di-thio-threitol (3.0 × 10 −3 M). The increase of spontaneous release of transmitter was not accompanied by an increase of the stimulus-evoked release since the amplitude of the endplate potential was not increased and partial inhibition caused by d-tubocurarine or magnesium chloride was not antagonized. When the concentration of NEM was increased to 2.75 × 10 −4 M, the directly-elicited twitches were inhibited, and the baseline tension was increased. This increase of tension was slightly reduced in a preparation depolarized with potassium chloride; a small depolarization could partly explain this effect. It was not reduced by dantrolene or in a calcium-free solution. The inhibition of the twitch and the increased baseline tension (probably a rigor) might be caused by a reduced sensitivity of the contractile proteins for calcium ions and an inhibition of the myosin ATPase activity, respectively.

Some properties of a β‐galactosidase from an extremely thermophilic bacterium

An inducible beta-galactosidase from an extremely thermophilic organism, Thermus strain 4-1A, has been isolated and partially purified. There were significant dissimilarities to T. aquaticus beta-galactosidase. It had a pl of 4.5, was inhibited by sulphydryl inhibitors and a number of transition metals, and was activated by EDTA and SH-containing reagents. The beta-galactosidase showed strong product inhibition, and weaker inhibition by some other mono- and disaccharides. It was very stable up to 90 degrees C at pH 8. On immobilization by diazonium linkage to porous glass, the pH optimum (6.0), the K(M) with ONPG (5mM) and the product inhibition were not altered.

Association of acylglyceride and retinyl palmitate hydrolase activities with zinc and copper metalloproteins in a high molecular weight lipid-protein aggregate fraction from chick liver cytosol

A lipid-protein aggregate fraction of molecular weight approx. 1.8·10 6 was isolated by gel filtration from chick liver cytosol. This aggregate fraction had a hydrated density range of 1.06–1.13, was 45% lipid, contained zinc and copper and was associated with triolein, phosphatidylcholine and retinyl palmitate hydrolase activity. Hydrolytic activities were stimulated by albumin and cholate, but not by dihydroxy bile acids, and inhibited by serine esterase and sulphydryl inhibitors. Incubation of the aggregate with fatty acid-labelled acylglycerides resulted in protein-binding fatty acid fractions with molecular weights of 150000, 60000, 13000 and approx. 2000. Incubation of the aggregate with [ 3H]retinyl palmitate yielded retinol-containing fractions with molecular weights of 150 000, 60 000 and 15 000. The latter peak appears, on the basis of amino acid composition, to be similar to the cellular retinol-binding protein. In addition, on incubation of the aggregate fraction, zinc and copper peaks are found with molecular weights of 150000, 60000 and 12000–8000. The latter were further purified to yield a copper-rich metalloprotein similar to ‘copper-chelatin’ and a zinc-rich metalloprotein, possibly zinc-metallothionein. Both these metalloprotein fractions had acyl hydrolase activity which was depressed in zinc-depleted animals. This may provide a possible explanation for the documented nutritional interactions between zinc and retinol.

Response of blood glucose, hepatic glycogen and pancreatic islets to treatment of sulphydryl inhibitors inClarias batrachus L.

The correlative effects of the two sulphydryl inhibitors viz., the mono-and dithiol reagent, alloxan and cobalt chloride, were studied in the fresh water air breathing catfish,Clarias batrachus. Alloxan induced a typical mammalian-like triphasic response and necrobiotic changes in the islet Β-cells. Cobalt chloride elicited a discontinuous hyperglycemia with cytopathological changes in Β-cells following nearly the same sequence as those induced by alloxan. However, the α-cells of cobalt-treated fish showed, unlike those after alloxanization, specific degranulation, vacuolization and nuclear enlargement. The changes in the hepatic glycogen content of two experimental groups were observed to be equal. Both the drugs appeared to be detrimental to the normal islet function and were, thus, overtly or potentially diabetogenic agents to the fish.

Energy-dependent calcium sequestration activity in a Golgi apparatus fraction derived from lactating rat mammary glands

A fraction isolated from lactating rat mammary glands was shown by marker enzyme assays to be rich in Golgi apparatus vesicles. This Golgi apparatus-rich fraction was shown to accumulate calcium in the presence of ATP but not in its absence. Other nucleoside triphosphates were only partially effective in promoting calcium transport. Mg 2+ was required for the uptake which was also temperature and pH dependent. The uptake was sustained by the use of oxalate and phosphate as intravesicular trapping agents. In the presence of 10 mM oxalate the apparent K m for calcium uptake was 0.24 μM ionized calcium. The V was 4.45 nmol calcium/min per mg protein. Preloaded calcium could be rapidly released by the addition of the ionophore A23187 indicating an intravesicular location for the sequestered ion. Addition of ethylene glycol bis( β-aminoethyl ether)- N, N′-tetraacetic acid resulted in a slower release of preaccumulated calcium, indicating the existence of one or more efflux routes by which calcium leaves the vesicles in the presence of MgATP. Ruthenium red partially inhibited the uptake but lanthanum and particularly the sulphydryl inhibitor p-hydroxymercuribenzoate were much more effective. The properties of the calcium-sequestering system in the Golgi apparatus-rich fraction were similar to those reported for other non-muscular tissues and lend support to the hypothesis that calcium is secreted into milk via the Golgi apparatus of the mammary gland secretory cell.

Some characteristics of Ureaplasma urealyticum. Urease activity in a simple buffer: effect of metal ions and sulphydryl inhibitors.

Urealytic activity of the cytoplasmic fraction of Ureaplasma urealyticum prepared by digitonin lysis was assayed in a simple buffer system (HEPES plus EDTA) by measuring the release of 14CO2 from [14C]urea. The Km of this preparation agreed with our previous observations of the same activity measured in a more complex reaction mixture. The substrate concentration at which maximum velocity occurred was approximately 20 mM. The activity was sensitive to heavy metals and inhibitors which react with sulphydryl groups such as N-ethylmaleimide and p-chloromercuribenzoate. It was not inhibited by Ca2+ or Mg2+ or by the reaction products, ammonia and carbon dioxide.

The neonatal platelet: evaluation of platelet malonyl dialdehyde formation as an indicator of prostaglandin synthesis.

Summary. It is recognized that neonates exhibit a transient thrombocytopathy. Whether the cause of the platelet abnormality is due to a storage pool defect, or an‘aspirin‐like’abnormality with inhibition of platelet prostaglandin synthesis is unclear. In an attempt to assess neonatal platelet prostaglandin synthesis, platelet rich plasma (PRP) from 10 normal control maternal–neonatal pairs was obtained contemporaneously with PRP from donors who had ingested 600 mg aspirin within 12‐18 h prior to study. When platelet malonyl dialdehyde production in the presence of the sulphydryl inhibitor N‐ethyl maleimide (NEM), or thrombin, was measured as an indicator of platelet prostaglandin synthesis, a significant decrease was observed in neonatal platelets (2.46 ± 0.61 and 0.90 ± 0.19 nmol/109 platelets respectively) when compared to those of the paired maternal or adult controls (3. 23 ±0.31 and 1.30 ± 0.17 nmol/109 platelets). As expected, a marked decrease in platelet malonyl dialdehyde production in response to NEM or thrombin was also seen in the platelets from the donors who had ingested aspirin (0.25±0.14 and 0.10 ± 0.09 nmol/109 platelets). When compared to the normal maternal control PRP both the neonatal and aspirin donor PRPs demonstrated abnormalities in second phase platelet aggregation in response to ADP and epinephrine, and an abnormal response to collagen. However, mutual correction and secondary irreversible aggregation was obtained when both these abnormal PRPs were mixed in varying proportions. No mutually corrective effect was seen using neonatal PRP obtained from infants where maternal documentation of aspirin ingestion was present within 48 h prior to delivery, or when normal neonatal PRP was preincu‐bated with aspirin in vitro prior to mixing with PRP from donors who had recently ingested aspirin. These findings provide evidence that the neonatal platelet dysfunction cannot be attributed to an abnormality in prostaglandin synthesis or an‘aspirin‐like’defect. Although platelet malonyl dialdehyde formation in response to NEM and thrombin was decreased when compared to the adult the amount synthesized by the neonatal platelet in response to external stimuli appears to be

Membrane proteins of incubated erythrocytes: effect of sulphydryl inhibition.

Incubation of erythrocytes with various sulphydryl inhibitors for 24 h leads to an accumulation in isolated membranes of globin as well as an unidentified polypeptide of apparent molecular weight 24 000 on polyacrylamide gel electrophoresis. Accumulation of both these components does not appear to be directly related to erythrocyte adenosine triphosphate and reduced glutathione levels. The 24 000 molecular weight polypeptide is probably derived from the erythrocyte cytosol, as determined by polyacrylamide gel electrophoresis of the non-haemoglobin proteins of the cytosol. Together with globin it forms an integral part of the 'spectrin-actin lattice' of sulphydryl-inhibited erythrocytes, as both these components can be detected, together with spectrin and actin, after extraction of such membranes by Triton X-100. We postulate that this leads to inherent damage and distortion to the membranes of such erythrocytes.

Acid phosphatase and protease release by the insectivorous plant Drosera rotundifolia

The leaves of the insectivorous plant Drosera rotundifolia L. produced extracellular hydrolytic enzymes in response to feeding with gelatin. Enzyme release was first detected 1 to 2 days after feeding, reached a maximum on day 4, and then gradually declined. Optimal activity of both acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) and protease enzymes was in the acidic range of pH. The acid phosphatase attacked a range of phosphorylated compounds but its p-nitrophenylphosphatase (PNPPase) and ribonucleoside triphosphatase activities were highest. It displayed relatively low phosphomonoesterase (EC 3.1.3.1, 3.1.3.2) activity. Both acid phosphatase and protease enzymes were insensitive to the sulphydryl inhibitor N-ethylmaleimide. The acid phosphatase was strongly inhibited by fluoride and orthophosphate. The nature of the apparent induction of hydrolase enzyme activity is briefly discussed.

SUBCELLULAR DISTRIBUTION OF DOPAMINE‐β‐HYDROXYLASE AND INHIBITORS IN THE HIPPOCAMPUS AND CAUDATE NUCLEUS IN SHEEP BRAIN

Abstract—The enzyme dopamine‐β‐hydroxylase (EC 1.14.17.1) which converts dopamine to noradrenaline was found to be present in substantial amounts in sheep brain hypothalamus and caudate nucleus and was located to the synaptic vesicle fractions in these two brain regions by subcellular fractionation. This dopamine‐β‐hydroxylase was associated with paniculate matter in these two brain regions since it was resistant to solubilization with butan‐1‐ol and 0.1% Triton X‐100. As highly significant levels of dopamine‐β‐hydroxylase were present in the caudate nucleus, factors other than a simple lack of this enzyme must operate to maintain the low levels of noradrenaline and high levels of dopamine in the caudate nucleus. Purified adrenal dopamine‐β‐hydroxylase was substantially inhibited by two factors prepared from sheep brain hypothalamus and caudate nucleus. These were found to be cupric ions and a sulphydryl inhibitor. High levels of the sulphydryl inhibitor of dopamine‐β‐hydroxylase were found in synaptosomal fractions from sheep brain hypothalamus and caudate nucleus and the levels were comparable in both regions. Upon subfractionation of a synaptosome‐containing fraction from the hypothalamus, the inhibitor was located predominantly in the soluble fraction, although there were significant levels in the synaptic vesicle fraction. Therefore, the sulphydryl inhibitor must be considered as a possible regulator of dopamine‐β‐hydroxylase activity.Free cupric ion concentrations as low as 2·5 μM were found to inhibit purified adrenal dopamine‐β‐hydroxylase in vitro and the concentration of copper in the soluble tissue component of hypothalamus and caudate nucleus was well above this minimal copper concentration. The percentage content of soluble copper in the caudate nucleus was significantly higher than in the hypothalamus. The importance of the soluble to particulate‐bound ratio of copper in brain was shown in studies of the developing rat brain. A rapid increase in the level of copper in brain was found in the first 4 weeks but the level was constant by 2 months of age. The percentage of soluble copper, however, was maximal soon after birth and had declined to a constant figure by 2 months of age. A scheme for the regulation of dopamine‐β‐hydroxylase activity involving these factors is proposed.