PHOSPHOPROTEIN PHOSPHATASE OF PINEAL GLAND: SOME PROPERTIES OF THE ENZYME AND THE IDENTIFICATION OF AN ENDOGENOUS ACTIVATOR

Abstract— Both soluble and insoluble fractions of rat pineal glands catalyze the dephosphorylation of phosphohistone. The phosphoprotein phosphatase in cytosol as well as in insoluble fraction is inhibited by ZnCl₂ and NaF. Guanosine triphosphate, ATP and MnCl₂ activate the soluble enzyme but not the enzyme in the insoluble fraction suggesting that with solubilization from membranes some unfunctional changes of the enzyme may occur. Fractionation of the soluble enzyme preparation revealed the existence of two forms of enzyme differing in molecular weight. These two forms can be further differentiated by their sensitivities to MnCl₂ and deoxycholate. A thermostable factor which activates the soluble but not the insoluble enzyme was demonstrated in both beef and rat pineal glands. The thermostable factor is protein in nature because it is nondialyzable and trypsin labile. Whether in vivo the endogenous activator mediates the regulation of the phosphoprotein phosphatase in pineal remains to be investigated.     

Properties and Ontogenic Development of Membrane-Bound Histidine Decarboxylase from Rat Brain

Abstract: Histidine decarboxylase (HD) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates. Twenty-eight percent of the HD activity was associated with membranes, and the remaining was soluble. Several properties of the soluble and membrane-bound HD were compared. No significant differences in the values of K _m for histidine and pyridoxal 5'-phosphate were observed. The solubilization of membrane-bound HD with Triton X-100 resulted in an increase of 60% over the nonsolubilized activity with no changes in the K _m for substrate and cofactor. The proportion of free pyridoxal 5'-phosphate-independent activity was identical in both fractions. The soluble and membrane-bound forms of the enzyme differ slightly in their pH-activity profiles, although both enzymes showed an optimum pH near 6.5. The HD activities present in soluble and membrane fractions were determined at different postnatal ages. The soluble activity increased until day 90, whereas the membrane-bound activity became stabilized from day 20.     

SUBCELLULAR FRACTIONS OF NORMAL HUMAN SUBSTANTIA NIGRA AND CAUDATE NUCLEUS; A STUDY OF THEIR MORPHOLOGY AND SOME ENZYMES INCLUDING GLUTAMATE DECARBOXYLASE AND CHOLINE ACETYLTRANSFERASE

Abstract— Subcellular fractions have been prepared from normal human caudate nucleus and substantia nigra by a standard fractionation technique and the fractions assayed for the following enzymes, which were studied because of their relevance to neurotransmission and pathological change: glutamate decarboxylase (EC 4.1.1.15), choline acetyltransferase (EC 2.3.1.6), acetylcholinesterase (EC 3.1.1.7), acid phosphatase (EC 3.1.3.2) and succinate dehydrogenase (EC 1.3.99.1). The distribution of these enzymes was assessed in relation to the morphology of the fractions as observed by electron microscopy. As with preparations from animal cerebral cortex, acetylcholinesterase and acid phosphatase were found mainly in fractions known to contain plasma membranes, synaptosomal membranes and microsomes. The levels of choline acetyltransferase in fractions from the substantia nigra were too low to measure but, in the caudate nucleus, the enzyme was concentrated in the crude mitochondrial fraction (P₂), especially in the P₂B and P₂C subfractions. A high proportion of the glutamate decarboxylase activity was present in the P₂ fractions of the substantia nigra and caudate nucleus and, although the synaptosomal (P₂B) fraction contained the enzyme, significant amounts were found in the mitochondrial (P₂C) fraction. This may have been due to some contamination of the mitochondria with small synaptosomes. Succinate dehydrogenase showed a conventional bimodal distribution between synaptosomes and mitochondria with a concentration in the latter.     

Soluble and membrane-bound forms of lipoprotein lipase in mouse lung tissue

Homogenates of mouse lungs were separated by differential centrifugation into two fractions containing lipoprotein lipase, namely, a soluble and a membrane-bound fraction. Lipoprotein lipase was specifically identified by its inhibition by both protamine sulfate (3 mg/ml) and sodium chloride (0.9 mol/l). The enzymatic activity of each fraction was enhanced when serum was preincubated with the enzyme. Both enzyme fractions showed optimum activity at alkaline pH, but the membrane-bound enzyme showed a higher pH optimum. In addition, the apparent Km of the soluble enzyme was lower than that of the membrane-bound enzyme. It is concluded that there are two different forms of lipoprotein lipase in mouse lung tissue that differ in a number of aspects.     

Thiol-protein disulphide oxidoreductases. Assay of microsomal membrane-bound glutathione-insulin transhydrogenase and comparison with protein disulphide-isomerase.

1. Inhibition of endogenous microsomal NADPH oxidase by CO enables membrane-bound glutathione-insulin transhydrogenase (EC 1.8.4.2) to be assayed conveniently by a linked assay involving NADPH and glutathione reductase (EC 1.6.4.2). 2. The specific activity of the enzyme in rat liver microsomal preparations is of the order of 1 nmol of oxidized glutathione formed/min per mg of membrane protein. 3. The specific activity of the enzyme is comparable in rough and smooth microsomal fractions, and the activity is not affected by treatment with EDTA and the removal of ribosomes from rough microsomal fractions. 4. Membrane-bound glutathione-insulin transhydrogenase is not affected by concentrations of deoxycholate up to 0.5%, whereas protein disulphide-isomerase (EC 5.3.4.1) is drastically inhibited. 5. On these grounds it is concluded that, in rat liver microsomal fractions, glutathione-insulin transhydrogenase and protein disulphide-isomerase activities are not both catalysed by a single enzyme species.     

PURIFICATION OF PROTEIN CARBOXYMETHYLASE FROM OX BRAIN

Abstract— The enzyme protein carboxymethylase from the soluble fraction of ox brain was purified to electrophoretic homogeneity. Brain protein carboxymethylase activity was also detected in a membrane-bound form which could only be solubilized by treatment with detergent. The solubilized membrane-bound form differed from the 'native' soluble form in that the former irreversibly lost activity on removal of the detergent. The two forms, however, have several similarities, having a molecular weight of 35,000, a K _m of 2.7 × 10⁻⁶ M for S -adenosyl-L-methionine, and a pH optimum of 6.2 when ovalbumin was used as the methyl acceptor.     

Some properties of acetylcholinesterase from rat retina.

Acetylcholinesterase (AChE, EC 3.1.1.7) of rat retina was studied with respect to its kinetic and other properties, and a comparison was made with the enzyme from brain. The subcellular distribution of the retinal AChE showed that the enzyme was concentrated in the synaptosomal-mitochondrial fraction although in the brain the AChE was distributed more evenly between the fractions studied. The enzyme from both retina and brain was easily solubilised and exhibited a Km of the order of 10(-4) M. The pH optimum was 8.3-8.6 for the AChE from both tissues for both the soluble and particulate enzyme.     

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.     

Is Na + K ATPase a Myelin-Associated Enzyme?

The Na + K ATPase activity associated with purified myelin has been investigated. On the basis of marker enzyme studies, the Na + K ATPase activity of myelin was higher than could be accounted for by microsomal contamination. Fractions prepared from white matter-enriched areas of rat brain showed a threefold enrichment in Na + K ATPase activity in myelin as compared with the white matter homogenate. The ATPase activity in myelin was stimulated fourfold by treatment with sodium deoxycholate, but the activity in the whole brain homogenate and the microsomal fraction was only doubled. This discontinuity temperature for Na + K ATPase activity was significantly higher for the myelin fraction (29 degrees C) than for the microsomal fraction (21 degrees C), but the energies of activation, both above and below the discontinuity temperature, were the same for both fractions, Myelin Na + K ATPase had a lower affinity for strophanthidin than the microsomal enzyme, but both fractions were inhibited to the same extent by 10-3 M-strophanthidin. The evidence thus indicated that much of the ATPase activity of myelin is not the result of microsomal contamination. Although the possibility of axolemmal contamination cannot be ruled out conclusively, indirect evidence suggest that this is not a significant factor and that Na + K ATPase may be a myelin-associated enzyme.     

BIOSYNTHESIS OF PHOSPHATIDIC ACID IN RAT BRAIN VIA ACYL DIHYDROXYACETONE PHOSPHATE

Abstract— The enzymes for the biosynthesis of phosphatidic acid from acyl dihydroxyacetone phosphate were shown to be present in rat brain. These enzymes were mainly localized in the microsomal fraction of 12–14 day old rat brains. The brain microsomal acyl CoA: dihydroxyacetone phosphate acyl transferase (EC 2.3.1.42), exhibited a broad pH optimum between pH 5 and 9 with maximum activity at pH 5.4. K _m for DHAP at pH 5.4 was 0.1 m m and V _max was 0.86nmol/min/mg of microsomal protein. The corresponding microsomal enzyme for the glycerophosphate pathway (acyl CoA: sn -glycerol-3-phosphate acyl transferase EC 2.3.1.15) was shown to have a different pH optimum (pH 7.6). On the basis of the differences in pH optima, differential effects of sodium cholate in the enzymes and a common substrate competition study, these acyl transferases were postulated to be two different microsomal enzymes.
Acyl DHAP:NADPH oxidoreductase (EC 1.1.1.101) in brain microsomes was found to be quite specific for NADPH as cofactor, being able to utilize NADH only at very high concentrations. This enzyme exhibited a K _m of 8.6 μ m with NADPH and V _mx of 0.81 nmol/min/mg protein. The presence of these two enzymes and the known presence of l-acyl- sn -glycerol-3-phosphate: acyl CoA acyl transferase in brain (F leming & H ajra , 1977) demonstrated the biosynthesis of phosphatidic acid in brain via acyl dihydroxyacetone phosphate. Phosphatidic acid was shown to form when dihydroxyacetone phosphate, acyl CoA, NADPH and other cofactors were incubated together with brain microsomes. Further properties of the enzymes and the probable importance of the presence of this pathway in brain were discussed.     

Purification and Characterization of Bovine Brain 5'-Nucleotidase

Abstract: The 5'-nucleotidase located in the cytoplasmic fraction of bovine brain cortex was purified to electrophoretic homogeneity. The molecular weight was 134,000 daltons in the presence of sodium deoxycholate, whereas the enzyme formed high molecular weight aggregates in the absence of detergent. The purified enzyme showed the same kinetic and electrophoretic behaviour as the enzyme present in the original cytoplasmic fraction, and the presence of surfactants did not change the K_m and V_m, values. The nucleotidase from this source was a phosphohydrolase of 5'-mononucleotides acting on the deoxyribonucleotides and ribonucleotides of purines and pyrimidines. 5'-IMP was the preferred substrate; the optimum pH was 7.5. The study of the influence of the temperature on the initial reaction rates allowed calculation of the δE_a, and δH^o values. The variation of V_m and K_m with a change in pH suggests the existence of a sulfhydryl group and an imidazole group in the enzymesubstrate complex.     

Ca2+ and Phospholipid-Dependent Protein Kinase Activity and Phosphorylation of Endogenous Proteins in Bovine Adrenal Medulla

Abstract: Soluble and membrane fractions of bovine adrenal medulla contain several substrates for the Ca²⁺/ phospholipid-dependent and cyclic AMP-dependent protein kinases. The phosphorylation of soluble proteins (36 and 17.7 kilodaltons) and a membrane protein (22.5 kilo-daltons) showed an absolute requirement for the presence of both Ca²⁺ and phosphatidylserine; other substrates showed less stringent phosphorylation requirements and many of these proteins were specific for each of the protein kinases. The Ca²⁺/phospholipid-dependent phosphorylation was rapid, with effects seen as early as at 30 s of incubation. Measurement of enzyme activities with histone HI as an exogenous substrate demonstrated that the Ca²⁺/phospholipid-dependent protein kinase was equally distributed between the soluble and membrane fractions whereas the cyclic AMP-dependent enzyme was predominantly membrane-bound in adrenal medulla and chromaffin cells. The activity of the soluble Ca²⁺/phos-pholipid-dependent protein kinase of adrenal medulla was found to be about 50% of the enzyme level present in rat brain, a tissue previously shown to contain a very high enzyme activity. These results suggest a prominent role for the Ca²⁺/phospholipid-dependent protein kinase in chromaffin cell function.     

4-Aminobutyraldehyde Dehydrogenase Activity in Rat Brain

Abstract: An enzyme with NAD⁺-dependent 4-aminobutyraldehyde dehydrogenase activity was purified about 360-fold from rat brain extract. AMP-Sepharose chromatography was effective in separating the enzyme from other NAD⁺-dependent aldehyde dehydrogenases included in the extract. The K _ms for the substrates NAD⁺ and 4-aminobutyraldehyde were 4.8 × 10⁻⁴ and 8.3 × 10⁻⁵ M , respectively. The pH optimum for the enzyme was about 8.0. The ratio of activities toward 4-aminobutyraldehyde, propionaldehyde, succinate semialdehyde, and benzaldehyde was 1.00:0.17:0.24:0.09:0.03 when the activity toward 4-aminobutyraldehyde was set equal to 1.00. The enzyme activity in subcellular fractions of rat brain was localized in cytosol.     

SUBCELLULAR LOCALIZATION AND PARTIAL PURIFICATION OF A CHLORIDE DEPENDENT ANGIOTENSIN-I CONVERTING ENZYME FROM RAT BRAIN

Abstract— Angiotensin converting enzyme (peptidyl dipeptide hydrolase EC 3.4.15.1) was extracted from particulates of rat brain using the nonionic detergent Triton X-100. Enzyme activity in subcellular fractions was associated with purified synaptosomes and present in the microsomal fraction, but absent in purified mitochondria and water-shocked myelin. Partial purification was achieved by chromatography on DEAE-cellulose and hydroxylapatite columns. The enzyme had a pH optimum of pH 7–8 and an apparent K_m of 2.2 m m using hippuryl-histidyl-leucine as substrate; it was chloride dependent, inhibited by (Sar¹-Ala⁸)-angiotensin-II (saralasin), and, at lower concentrations, by the specific nonapeptide inhibitor SQ 20881. Associated with the purified enzyme was an aminopeptidase, cleaving N-terminal Asp from the native substrate, which could be involved in the production of the active heptapeptide, angiotensin III (des-Asp-angiotensin-II). Also present was a carboxypeptidase-like enzyme removing C-terminal Phe following the liberation of His-Leu by converting enzyme, which may be involved in the inactivation of angiotensin II or III.     

Characterization of Membrane-Bound and Soluble Catechol-O-Methyltransferase from Human Frontal Cortex

Abstract: Catechol- O -methyltransferase (COMT; E.C. 2.1.1.6) from human frontal cortex occurs in both a soluble and membrane-bound form. Attempts to solubilize the membrane-bound transferase by repeated washing or by extraction into solutions of high ionic strength were unsuccessful. The finding that Triton X-100 was capable of solubilizing membrane-bound COMT suggested that the membrane-bound transferase is an integral membrane protein. The membrane-bound and soluble enzymes did not differ in their requirements for magnesium ions or in their pH-activity profiles; both enzymes showed an optimum near pH 8.0 when assayed in phosphate buffer. In addition, the two enzymes did not differ in the degree of inhibition caused by CaCl₂, both enzymes displaying 65% inhibition at 2.5 m M CaCl₂. The competitive inhibitors tropolone and nordihydroguaiaretic acid displayed K _i values for the membrane-bound transferase five- to 10-fold lower than those observed for the soluble transferase. Solubilization of membrane-bound COMT in Triton X-100 resulted in an increase in the apparent K _m value of the membrane-bound transferase for dopamine. The increase in K _m appeared to be due to apparent competitive inhibition by Triton X-100 and reached a limiting value of approximately 80 μM. These results confirm that membrane-bound COMT is an integral membrane protein that may be structurally distinct from soluble COMT.     

l-Myo-inositol-1-phosphate synthase from lower plant groups: Partial purification and properties of the enzyme from Euglena gracilis

A survey for the enzyme L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been conducted among various members of the lower plant groups, mainly algac, bryophytes and fungi; some properties of the partially purified enzyme from Euglena gracilis Z . are presented. The enzyme was detected in Chloropycean algae, Marchantiales and the Basidiomycetous fungi. The enzyme from Euglena had a pH optimum at 7.5. The Km for glucose-6-P was 2.1 m M and for NAD⁺ 80 μ M . When assayed in the absence of added NAD⁺, the enzyme showed a basal activity suggesting the presence of bund NAD⁺ in the system. NH₄Cl increased the enzyme activity two-fold, altough the enzyme was inactivated by (NH₄)₂SO₄.     

Some characteristics of sn-glycero-3-phosphocholine diesterases from rat brain.

1. sn-Glycero-3-phosphocholine diesterase activities, glycerophosphohydrolase (EC 3.1.4.2) and choline phosphohydrolase (EC 3.1.4.38) from rat brain have been partially purified and characterized using sn-glycere-3-[32P]phosphocholine as substrate and separating the reaction products by anion-exchange chromatography and ionophoresis. 2. Rat brain contained particulate (75%) and soluble (25%) activity from both diesterases. No difference in pH optimum or metal ion requirement for the particulate compared to the soluble enzymes was observed. 3. Glycerophosphohydrolase (EC 3.1.4.2) was purified 60-fold, choline phosphohydrolase (EC E.1.4.38) 120-fold from rat brain supernatant fraction by DEAE-cellulose ion-exchange chromatography and sucrose density gradient centrifugation. The density gradient results in conjunction with dodecyl sulphate-polyacrylamide gel disc electrophoresis yielded molecular weight estimates of 230 000 (monomer 62 000) for choline phosphohydrolase and 120 000 (monomer 70 000) for glycerophosphohydrolase (EC 3.1.4.2). 4. Glycerophosphohydrolase (EC 3.1.4.2) has a pH optimum of 8.9 and a Km for sn-glycero-3-phosphocholine of 0.6 mM. The enzyme is inhibited by EDTA and reactivated by Ca2+. Choline phosphohydrolase (EC 3.1.4.38) has pH optimum 10.5, a Km of 2 mM and is unaffected by EDTA. Both enzymes require Ca2+ for maximum activity.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

myo-Inositol dehydrogenase(s) from Acetomonas oxydans. Optimization of conditions for solubilization of membrane-bound enzyme

Methods are described for the first reported successful isolation of the soluble form of the membrane-bound myo-inositol dehydrogenase(s) from Acetomonas oxydans. Conditions for optimum yields of active enzyme in a crude membrane-free protein extract were established. The relevant conditions are (a) cell rupture by solid-shearing, (b) solubilization of the complete 60000g pellet with sodium deoxycholate at pH7.2, (c) rapid separation of the released protein from sodium deoxycholate by gel chromatography.     

Substrate specificity of human alpha-L-fucosidase.

Human α-l-fucosidase is a soluble lysosomal enzyme which hydrolyzes α-l-fucose residues linked to the 2 position of galactose or the 3, 4, or 6 position ofN-acetylglucosamine. Demonstration of activity towards natural oligosaccharide or glycosphingolipid substrates was achieved by measuring liberated l-fucose by coupling to fucose dehydrogenase and NAD and measuring NADH production spectrophotometrically. Activity of purified human spleen, brain, and cultured skin fibroblast or crude cell extracts towards 4-methylumbelliferyl-α-l-fucoside had a pH optimum of 4.5 to 5.5 and was unaffected by the presence of neutral detergents such as Triton X-100. However, the addition of sodium taurocholate or other bile salts to the incubation mixture caused a marked inhibition at pH 5 and a shift in pH optimum to the pH 6–7 region. Sodium taurocholate effected a threefold reduction in the apparent K_m for α-l-fucosidase at pH 6.0, but studies on fucosidosis tissue (α-fucosidase deficiency) or subcellular fractions derived from rat liver failed to indicate the existence of a membrane-bound α-l-fucosidase. The response of other lysosomal hydrolases to the presence of bile salts was investigated and was found to be variable, perhaps depending upon the hydrophilic or hydrophobic nature of the natural substrate and/or the state of association of the active enzyme.