Effect of dicarboxylic acids on the peroxidase-IAA oxidase isozymes of soybean callus

Multiple forms of peroxidase with indole-3-acetic acid (IAA) oxidase activity were detected in callus cultures from soybean seeds [ Glycinc max (L.) Merrill, cv. Acme] using ion-exchange chromatography and polyacrylamide gel electrophoresis. The properties of the IAA oxidase were studied with a partially purified fraction eluted from a DEAE cellulose column. At pH 5.7. p-coumaric acid and MnCl₂ were required as cofactors and H₂O₂ was not able to replace them, but H₂O₂ eliminated the usual lag period of the reaction. Activation effects obtained with some dicarboxylic acids acting only on IAA oxidase are shown. These effects were studied at different pH values and oxalic acid was found to be the most efficient activator, particularly at pH 4.6. Activation by oxalic acid occurred even in the absence of MnCl₂, but the presence of this salt produced a synergistic effect. IAA oxidase showed a sigmoidal kinetic behaviour at pH 5.7 changing to hyperbolic at pH 4.6     

STUDIES ON SOLUBLE PROTEINS RELEASED FROM THE SYNAPTIC VESICLES OF RAT BRAIN CORTEX

Abstract— The soluble proteins released from the synaptic vesicles of rat cerebral cortex were studied. One fraction (D₄) of these proteins was released in parallel with release of acetylcholine when synaptic vesicles were incubated at 37°C for 10 min in isotonic medium. Another fraction (Dj) was liberated from synaptic vesicles when their membranes were ruptured by mild treatment under hyposmotic conditions and freeze-thawing after release of D₁ fraction. Fractions D₁ and D₂ contained 12 and 9 per cent, respectively, of the total protein in the synaptic vesicles. Some properties of these fractions were investigated by zone electrophoresis and ultracentrifugation, and by measuring their binding capacities for [¹⁴C]acetylcholine and various enzyme activities related to acetylcholine metabolism.     

STUDIES ON THE MONOAMINE OXIDASE OF MONKEY BRAIN

Abstract— A study of the enzyme monoamine oxidase (MAO) was carried out in the monkey brain. From the monkey brain mitochondrial fraction a lysolecithin-soluble form of the enzyme (MAO_s and an insoluble form (MAO_p) were isolated. The latter required freezing, thawing and sonication for solubilization. Both these forms of MAO had identical electrophoretic mobilities, a pH optimum of 7 and comparable thermal stabilities. The enzyme which could not be solubilized and which remained membrane-bound also gave the same pH optimum of 7 and a similar thermal stability profile. Both MAO_s and MAO_p had comparable K _m values of 2.2 × 10⁻⁵ m and 5.0 ×^105- m respectively when using tyramine as substrate and 7.4 ×⁻⁵ M and 7.7 × 10⁻⁵ m respectively with benzylamine as substrate. The K _m values of the membrane-bound enzyme were 1.0 × 10"⁵m with tyramine as substrate 2.5 × m with benzylarnine as substrate. The MAO inhibitors, tranylcypromine, isocarboxazid and iproniazid inhibited both MAO_s and MAO_p to approximately the same extent. The extent of inhibition of the membrane-bound enzyme however, was relatively different with all three inhibitors. Immunodiffu-sion techniques using anti-MAO_p indicated the immunological identity among MAO_p, MAO_s and the mitochondrial fraction. Substrate specificity and substrate competition experiments as well as the use of the selective inhibitor pargyline indicated the presence of both the 'A' and 'B' type of activity in the MAO isolated from monkey brain.     

Properties of β-galactosidase of Lactobacillus kefiranofaciens K-1 isolated from kefir grains

β-Galactosidase from Lactobacillus kefiranofaciens K-1 was isolated and characterized. Optimal temperature and pH for the enzyme reaction were 50^°C and pH 6.5, respectively. Molecular weight was estimated to be approximately 311000. Glucose and galactose inhibited the activity, but the inhibition by galactose was rather weaker than observed in other β-galactosidases. MnCl₂ and MgCl₂ had no effect on the activity. FeSO₄, AgNO₃ and HgCl₂ acted as the inhibitor. β-Mercaptoethanol and L-cysteine activated the enzyme, while iodoacetamide inhibited the activity. The K _m values were 4.92 mmol/1 for ONPG and 1.27 mmol/1 for lactose.     

Characterization of a Neutral Aminoacyl-Peptide Hydrolase from Naegleria fowleri1

An intracellular alpha-aminoacyl-peptide hydrolase (EC 3.4.11.-) from Naegleria fowteri nN68 (ATCC 30894) has been characterized. The enzyme preparation hydrolyzed phenylalanyl-, tyrosyl-, leucyl-, arginyl-, alanyl-, tryptophanyl-, histidyl-, methionyl-, and lysyl-naphthylamide but not benzoylleucyl-, leucylglycyl-, glycylprolylleucyl-, glycyl-, threonyl-, aspartyl-, or glutamyl-naphthylamide. The aminopeptidase activity was inhibited by the cysteine-protease inhibitors—hydroxymercuribenzoate, chloromercurisulfate, and iodoacetate- by the aminopeptidase inhibitors-bestatin and trans-epoxysuccinyl-leucyl-agmatine- by an inhibitor of soluble alanyl aminopeptidase EC 3.4.11.14, puromycin, and by the metalloprotease inhibitor, o-phenanthroline. The exopeptidase activity was not inhibited by the chelator, ethylenediaminetetraacetate, or the serine-protease inhibitor, phenylmethylsulfonylfluoride. The pH optimum of the exopeptidase was between 7.0 and 8.0. Enzyme activity was stable at 55°C for 30 min, but all activity was lost after 15 min at 80°C. Enzyme activity was inhibited by 100 μM HgCI₂ and CdCl₂ but not by 1 mM CoCl₂, CuCl₂, MnCl₂, NiCl₂, FeCl₂, or ZnCl₂. Enzyme activity was inhibited by 0.1% sodium dodecyl sulfate but not by 0.2% Brij 35, Tween 20, Tween 80, or Triton X-100.     

LIMITATIONS OF PHOTOSYNTHESIS IN DIFFERENT REGIONS OF THE ZEA MAYS LEAF

The progressive development of the photosynthetic apparatus occurring along the length of the Zea mays leaf offers a convenient system with which to examine the limitations to photosynthetic CO₂ assimilation during biogenesis of a C₄ leaf. Changes in light-induced O₂ evolution and CO₂ assimilation, chlorophyll content, activity of PEP-carboxylase, NADP-malic enzyme and the 'R5P system' (consisting of d -ribose-5-phosphate-keto isomerase, ATP- d -ribulose-5 phosphate 1-phosphotransferase and d -ribulose-1,5-bisphosphate carboxylase) and fluorescence emission characteristics were examined along the length of the second leaf of 7-day-old plants grown under a diurnal light regime. The results suggest that the major limitation to CO₂ assimilation in the leaf sheath lies within the chlorenchyma and is either energy supply for carboxylation or the capacity of key photosynthetic enzymes. In the leaf blade stomatal resistance to CO₂ diffusion constitutes a major fraction of the total leaf resistance to CO₂ assimilation implicating the stoma as the major limiting factor to photosynthetic CO₂ assimilation.     

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 .     

NOREPINEPHRINE INCREASES THE [32P]LABELLING OF A SPECIFIC PHOSPHOLIPID FRACTION OF POST-SYNAPTIC PINEAL MEMBRANES

Abstract— Cultured pineal glands incorporated ³²P into membrane phospholipids. Treatment of cultured glands with norepinephrine, which is known to stimulate membrane- bound pineal adenyl cyclase and to increase the production and secretion of melatonin, stimulated the incorporation of ³²P into a phospholipid fraction of membranes and particulates containing phosphatidyl serine and phosphatidyl inositol. The labelling of other phospholipid fractions and the total ³²P in the gland were not changed by norepinephrine treatment. Experiments with chronically-denervated pineal glands indicated that the effect of norepinephrine on the [³²P]labelling of phospholipids occurred at a postsynaptic site. When norepinephrine-stimulated secretion of melatonin was partially inhibited by p -chlorophenylalanine (a compound which blocks the synthesis of melatonin precursors), the norepinephrine-stimulated labelling of phospholipids was still observed. Conversely, when melatonin secretion was stimulated in the absence of norepinephrine by treatment with the immediate precursor of melatonin, N -acetylserotonin, a stimulation of ³²P- labelling of phospholipids did not occur. These observations suggest that the increased [³²P]- labelling of a phospholipid fraction caused by the norepinephrine treatment is not related to the secretion of melatonin. This effect on phospholipids may be associated with the interaction of norepinephrine with a membrane-bound postsynaptic receptor. Stimulation by norepinephrine of [³²P]-incorporation into phospholipids has not been previously reported to occur in a tissue in which cholinergic fibres are absent.     

ACCELERATION OF CHOLINE UPTAKE AFTER DEPOLARIZATION-INDUCED ACETYLCHOLINE RELEASE IN RAT CORTICAL SYNAPTOSOMES

Abstract— Activation of nerve elements in vivo and in vitro is associated with an increased rate of choline uptake by a Na⁺-dependent high affinity transport system. Following the methodology of B arker (1976), rat cortical synaptosomes were depolarized (37°C, 10min) by 25mM-KCl in the presence of CaCl₂ (1 mM) or other divalent cations. After reisolation by centrifugation, the rate of ³H-choline uptake (1.25μM) was measured by Millipore filtration. KCl treatment alone failed to accelerate the rate of uptake in the reisolated synaptosomes. CaCl₂, BaC1₂ or SrCl₂ (but not MgCl₂ or MnCl₂) were necessary (1 mM) to observe the KCl induced acceleration. Moreover, RbCl, but not LiCl or CsCl, also produced the calcium-dependent rate enhancement in the reisolated synaptosomes. The conditions mediating the enhanced rate of choline uptake correlated strongly with those associated with neurotransmitter release. To test this possibility, synaptosomal acetylcholine content was measured in response to the various salt treatments. Treatment with KCI (25 mM) and CaCl₂ (1 mM), but not KCl alone, reduced the synaptosomal acetylcholine content from 154 to 113pmol/mg protein. The respective rates of choline uptake increased about 60%. The increased rate was reversed by incubation with 50 μM-choline followed by synaptosome reisolation. This procedure also normalized the acetylcholine content. In summary, the rate of choline uptake by the high affinity choline uptake system is inversely related to the synaptosomal acetylcholine content.     

THE SUBUNIT STRUCTURE OF MAMMALIAN ACETYLCHOLINESTERASE: CATALYTIC SUBUNITS, DISSOCIATING EFFECT OF PROTEOLYSIS AND DISULPHIDE REDUCTION ON THE POLYMERIC FORMS

Abstract— An analysis of the [³H]DFP-labelled catalytic subunits of mammalian (bovine SCG) acetylcholinesterase (AChE, EC 3.1.1.7.) indicates a monomer molecular weight of 75,000. This is equivalent to the mass previously determined for the smallest active form and demonstrates that the globular, or G forms, are respectively monomeric (G₁ form, 4S), dimeric (G₂ form, 6.5S) and tetrameric (G₄ form, 10S). In the tetrameric G₄ form the catalytic chains are associated in dimers, by disulphide bonds.
The effect of reduction and proteolysis has shown that the dimeric form (G₂ form, 6.5S) is readily reduced into G₁, while the tetramer G₄ is very stable, being only dissociated by a combination of reduction and proteolysis by high concentration of trypsin. The asymmetric forms A₁₂ (16S), A₈ (13S) and A₄ (9S) are not sensitive to reduction, but are readily dissociated by low concentrations of trypsin, into each other, progressively liberating isolated tetramers. We obtained essentially identical results with AChE preparations from rat brain or superior cervical ganglion. These observations support a general model for the quaternary structure of acetylcholinesterase molecular forms.     

Paramecium Has Two Regulatory Subunits of Cyclic AMP-Dependent Protein Kinase, One Unique to Cilia

ABSTRACT. The subunit composition and intracellular location of the two forms of cAMP-dependent protein kinase of Paramecium cilia were determined using antibodies against the 40-kDa catalytic (C) and 44-kDa regulatory (R₄₄) subunits of the 70-kDa cAMP-dependent protein kinase purified from deciliated cell bodies. Both C and R₄₄ were present in soluble and particulate fractions of cilia and deciliated cells. Crude cilia and a soluble ciliary extract contained a 48-kDa protein (R₄₈) weakly recognized by one of several monoclonal antibodies against R₄₄, but not recognized by an anti-R₄₄ polyclonal serum. Gel-filtration chromatography of a soluble ciliary extract resolved a 220-kDa form containing C and R₄₈ and a 70-kDa form containing C and R₄₄. In the large enzyme, R₄₈ was the only protein to be autophosphorylated under conditions that allow autophosphorylation of R₄₄ The subunits of the large enzyme subsequently were purified to homogeneity by cAMP-agarose chromatography. Both C and R₄₈ were retained by the column and eluted with 1 M NaCl; no other proteins were purified in this step. These results confirm that the ciliary cAMP-dependent protein kinases have indistinguishable C subunits, but different R subunits. The small ciliary enzyme, like the cell-body enzyme, contains R₄₄, whereas R₄₈ is the R subunit of the large enzyme.     

LOW CONCENTRATIONS OF LITHIUM INHIBIT THE SYNTHESIS OF CYCLIC AMP AND CYCLIC GMP IN THE RAT PINEAL GLAND

Abstract— Lithium chloride (2 m m ) significantly inhibited the increases in cyclic AMP and in cyclic GMP caused by norepinephrine or high concentrations of potassium in intact rat pineal glands. Adenylyl cyclase activity in homogenates and its stimulation by isoproterenol, a β-adrenergic agonist, were also inhibited. Lithium reduced the apparent V _max of isoproterenol-stimulated adenylyl cyclase activity without significantly affecting the apparent affinity for isoproterenol. There was no effect on the binding of the antagonist [³H]dihydroalprenolol to the β-adrenergic receptors, nor on the competition for binding sites by isoproterenol. Inhibition of adenylyl cyclase activity by lithium was inversely related to the magnesium concentration in the reaction mixture. There was no differential effect of lithium on adenylyl cyclase activity from supersensitive vs subsensitive glands. Lithium may inhibit cyclic nucleotide synthesis by interfering with the role of divalent cations.     

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.     

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.     

Effects of Phospholipases on the Kinetic Properties of Rat Striatal Membrane-Bound Tyrosine Hydroxylase

Abstract: Rat striatal tyrosine hydroxylase can be isolated in both a soluble and a synaptic membrane-bound form. The membrane-bound enzyme, which exhibits lower K _ms for both tyrosine (7 μ M ) and reduced pterin cofactor (110 μ M ) relative to the soluble enzyme (47 μ M and 940 μ M , respectively), can be released from the membrane fraction with mild detergent, and concomitantly its kinetic properties revert to those of the soluble enzyme. Treatment of membrane-bound tyrosine hydroxylase with C. perfringens phospholipase C increased the K _m of the enzyme for tyrosine to 27 μ M and the V _max by 60% without changing the K _m for cofactor. In contrast, treatment of membrane-bound tyrosine hydroxylase with V. russelli phospholipase A₂ increased the K _m for tyrosine to 48 μ M increased the V _max and increased the K _m for cofactor to 560 μ M . The enzyme remained bound to the membrane fraction following both phospholipase treatments. Addition of phospholipids to treated enzyme could partially reverse the effects of phospholipase A₂ treatment, but not the effects of phospholipase C treatment. The kinetic properties of phospholipase-treated, detergent-solubilized tyrosine hydroxylase were identical to those of the control solubilized enzyme. Tyrosine hydroxylase appears to interact with synaptic membrane components to produce at least two separately determined consequences for the kinetic properties of the enzyme.     

Studies on a Capillary-Rich Fraction Isolated from Brain: Histaminic Components and Characterization of the Histamine Receptors Linked to Adenylate Cyclase

Abstract: A fraction enriched in capillaries has been prepared from the guinea pig cerebral cortex. The purity of this fraction was checked by light- and electron-microscopic examination and by its high enrichment in alkaline phosphatase and γ-glutamyl transpeptidase. In the capillary-rich fraction, the endogenous level of histamine was 1.9%'of that measured in the initial hornogenate. The histamine-synthesizing enzyme, I-histidine decarboxylase, and the metabolizing enzyme, histamine-N-methyltransferase, were barely detectable. In addition, histamine elicits a twofold stimulation in the accumulation of cyclic AMP in this capillary fraction with an EC₅₀ of 5 γM. Agonists and antagonists of the two types of histamine receptors (H₁ and H₂) were used for the characterization of the receptors mediating this action: H₂-receptor agonists were able to activate the adenylate cyclase with "relative potencies" similar to that found on typical H₂-receptors, and cimetidine, a specific H₂-receptor antagonist, competitively inhibited the response to histamine with a K₁ value reflecting its interaction with a single population of H₂-receptors. On the contrary, data obtained with H₁-receptor agonists and antagonists reflect their interaction with H₂-receptors rather than H₁-receptors. Thus H₂-receptors are involved in the activation of adenylate cyclase of the capillary fraction.     

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.     

Induction of a proteinase by heat-shock in yeast

Abstract In Saccharomyces cerevisiae heat-shock induces an increase in proteinase activity. The induction is probably due to newly synthesized enzyme molecules, since the increase in proteinase activity can be inhibited by cycloheximide. Degradation of endogenous proteins is enhanced by EDTA, while the azocasein assay is not affected by MnCl₂, MgCl₂, or EDTA. The proteinase has a pH optimum of 8, and phenylmethylsulfonyl fluoride (PMSF) as well as chymostatin are strong inhibitors. We infer that the induced proteinase is probably identical with proteinase B of yeast.     

EFFECTS OF HYPOXIA ON TYROSINE HYDROXYLASE ACTIVITY IN RAT CAROTID BODY

Abstract— Tyrosine hydroxylase (TH) activity was measured in the carotid body. superior cervical ganglion and adrenal glands of the rat under normal conditions and at 48 h following exposure of the animals for 1-3 h in a low O₂ atmosphere. Basal TH levels were 5-6 nmol/h/mg tissue for both the carotid body and the ganglion. Forty-eight hours after hypoxia, there was an increase in enzyme activity in both tissues which paralleled the severity of the hypoxia but was greater in the carotid body than the superior cervical ganglion. Thus, following exposure to 5% O₂ in N₂ for two 30-min periods (20-min interim), TH activity had increased by 50% in the carotid body and 33% in the ganglion; after exposure to 10% O₂ in N₂ for 3 h (continuous), TH levels were increased by 37% in the carotid body and 12% in the ganglion. In the adrenal gland, basal TH activity was 3.42 ± 1.87 nmol/h/mg tissue, and this value was unchanged following either level of hypoxia.     

PROPERTIES OF RAT BRAIN NAD-KINASE

Abstract— NAD-kinase was purified from rat brain acetone powder according to the method of W ang and K aplan (1954). The acetate buffer supernatant showed only very low specific activity but was largely free of the factors that interfere with the enzyme assay. The Michaelis constants for both substrates were determined, the values were 0·5 m m for NAD and 4·0 m m for ATP. The optimal pH was 7·4 in tris-HCl buffer and the highest NAD-kinase activity was observed in the hyaloplasm fraction. NADH₂ inhibited the enzyme whereas NADPH₂ did not. Finally, the reversible inhibition of SH-binding compounds is described and the observed properties of rat brain NAD-kinase compared with the properties of NADP synthesizing enzymes from pigeon liver and rat liver.