THE EFFECTS OF OUABAIN AND THE ACTIVATION OF NEURAL MEMBRANE ATPase BY BIOGENIC AMINES

Abstract— The effects of 10⁻⁵ m -noradrenaline (NA), 5-hydroxytryptamine (5-HT) and dopamine (DA) on the activities of Na⁺-K⁺ ATPase (EC 3.6.1.3) were studied in synaptic membranes from 6 regions of the rabbit brain. NA and 5-HT stimulated the synaptic membrane Na⁺-K⁺ ATPase from the cerebrum, but none of the amines influenced the activity of this enzyme in the other brain regions. The Na⁺-K⁺ ATPase activity of the cerebral synaptic membrane isolated at the 0.8/0.9 m & 0.9/1.0 m interphase of a sucrose density gradient was increased two-fold by 10⁻⁵ m -NA and 5-HT. The Na⁺-K ⁺ ATPase recovered at the 1.0/1.2 m interphase was not influenced by NA, DA or 5-HT. NA, DA and 5-HT did not activate the Mg ATPase of synaptic membranes from any of the 6 brain regions or whole brain synaptic vesicles. The cortex synaptic membrane (Na⁺-K⁺) ATPase is postulated to have a direct role in the uptake of the biogenic amines. An indirect role is proposed for this enzyme in amine uptake into brain stem.     

ATPASE AND PHOSPHATIDYLINOSITOL KINASE ACTIVITIES OF ADRENAL CHROMAFFIN VESICLES

Abstract— The hypothesis that the ATPase and phosphatidyhnositol (PI) kinase activities of chromaffin vesicle membranes are catalysed by same enzyme was investigated. The two activities exhibited entirely different responses to variations in Mg²⁺ or Mn²⁺ concentrations. In the presence of 1 mM ATP, maximal ATPase activity occurred with 1 mM Mg²⁺ while maximal PI kinase activity required 100 mM Mg²⁺ Similar differences were observed with Mn²⁺ with the exception that maximal ATPase activity occurred with 0.5 mM Mn²⁺ and maximal PI kinase activity occurred with 5 mM Mn²⁺ Mn²⁺ was more effective than Mg²⁺ in stimulating PI kinase activity at low concentrations, but at optimal concentrations of each, the maximal activity obtained with Mg²⁺ was 5-fold greater than the maximal activity obtained with Mn²⁺ The heat stabilities of the two enzymes are vastly different. At 50°C the ATPase activity of the intact membranes was stable for up to 20 min while the t _l/2 of PI kinase was less than 2 min. After solubilization in Lubrol PX or at higher temperatures both enzymes were less heat stable, but PI kinase was still inactivated at a much greater rate than the ATPase. The evidence suggests that the ATPase and the PI kinase are different proteins.
The major phosphorylated product was diphosphatidylinositol and once formed, it was stable. Phosphorylation of membrane protein accounted for less than 10% of the total ³²P-incorporated into chromaffin vesicles. SDS gel electrophoresis of the solubilized membranes showed the presence of at least 2 major phosphorylated high molecular weight components.     

The seven NAD(H)-glutamate dehydrogenase isoenzymes exhibit similar anabolic and catabolic activities

The specific activities of aminating NADH- and deaminating NAD⁺-glutamate dehydrogenase (GDH, EC 1.4.1.2) varied considerably in crude extracts of grapevine ( Vitis vinifera L. cv. Sultanina) callus and were dependent on the nitrogen source of the culture medium. However, dialysis of the enzyme preparations resulted in a significant decrease in the deaminating GDH specific activity while the aminating activity was not affected. The presence of malate in the crude extract resulted in erroneous overestimation of the NAD⁺-GDH activity through the malate dehydrogenase reaction. Thus, in dialysed extracts, the ratio of the NADH-GDH/NAD⁺-GDH specific activities remained relatively constant irrespective of the nitrogen source. In view of this evidence, we now have modified methods for staining both the NADH-GDH and NAD⁺-GDH activities on gels in order to compare the aminating and deaminating activities of each of the 7 GDH isoenzymes. The results from the staining of NADH-GDH and NAD⁺-GDH activity of enzyme preparations from calluses revealed the same isoenzyme profile. Furthermore, separated leaf isoenzymes showed similar activity ratios and kinetic properties. These results may suggest that each one of the 7 isoenzymes have similar in vitro anabolic and catabolic activities.     

ATPase Activity in Isolated Flagella from Euglena gracilis

SYNOPSIS. The ATPase activity of isolated flagella was studied in Euglena gracilis strain Z in the presence of Mg⁺⁺ or Ca⁺⁺. With Mg⁺⁺, the optimum activity was at pH 7 and with Ca⁺⁺, at pH 9. The K _m values were respectively 6.6 × 10⁻⁴ and 3.6 × 10⁻⁴. Activity was influenced also by temperature and ionic strength. Results with inhibitors of membrane ATPase suggest the presence of a specific contractile system in the flagella. Our results are compatible with a multicomponent enzymic system containing 2 active ATPases.     

CHOLINERGIC STIMULATION OF PHOSPHOLIPID LABELLING FROM [32P]ORTHOPHOSPHATE IN GUINEA-PIG CORTEX SYNAPTOSOMES IN VITRO: SUBSYNAPTOSOMAL LOCALIZATION

Abstract— Subsynaptosomal localization of stimulation of phospholipid labelling by cholinergic agents was investigated. Synaptosomes prepared from guinea-pig cortex were incubated with [³²P]orthophosphate in the presence or absence of 10⁻³ m carbamylcholine. Following incubation and osmotic shock, lysed synaptosomes were subjected to density gradient fractionation. Subsynaptosomal fractions were examined by electron microscopy and analysed for enzyme activities and ³²P-labelled lipids.
In the absence of carbamylcholine, labelled phosphatidate and phosphatidylinositol were recovered in layers and interfaces A, B, C and D formed over 0.9, 1.1, 1.2 and 1.3 m sucrose, with highest amounts of label in fractions C and D for both lipids. Carbamylcholine induced the greatest increment in these two labelled lipids in fractions A and B. This distribution correlated with the presence of acetylcholinesterase activity and membrane ghosts. No correlation was found among the four fractions between the induced increase in labelling and succinic dehydrogenase activity or with the abundance of mitochondria, synaptic vesicles, or cytoplasmic fragments identified by electron microscopy. In contrast with the increases seen in phosphatidylinositol and phosphatidate labelling, carbamylcholine caused a decrease in ³²P-labelling of the polyphosphoinositides, and this effect was seen primarily in the heavier subsynaptosomal fractions, C and D.     

Development of Vacuoles and Vacuolar H+-ATPase Activity under Extremely High CO2 Conditions in Chiorococcum littorale Cells

Abstract: The number and cross-sectional area of vacuoles in Chlorococcum littorale cells visualized with a differential interfer ence fluorescence microscope increased after their transfer from air to 40% CO₂. An immunological observation indicated that the level of subunit B of vacuolar H*ATPase also increased under 40% CO₂ conditions. The activity of nitrate-sensitive ATP-ase associated with the vacuolar membrane was 2–fold higher in 40% CO₂-_-grown cells than in air-grown cells. The effects of inhi bitors on the ATPase activity confirmed that these activities were derived from vacuolar-type H-ATPase. These results sug gest that vacuole development associated with that of vacuolar H⁺-ATPase occurred during the acclimatization of C. littorale cells to extremely high CO₂ conditions.     

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membrane after treatment of intact tissues and cell free extracts with triacontanol

Ca²⁺- and Mg²⁺-dependent ATPase activity (EC 3.6.1.3) in a plasma membrane-enriched fraction increased rapidly after in vivo application of physiologically active concentrations of triacontanol (TRIA) to the roots of barley ( Hordeum vulgare L. cv. Conquest) seedlings. Ca²⁺- and Mg²⁺-dependent ATPase activity was 64 and 85% higher, respectively, in the roots of seedlings germinated in the presence of growth-promoting concentrations of TRIA compared to controls. The increase in vivo was concentration dependent, with the greatest increase obtained at 2.3 n M TRIA. Maximal stimulation of ATPase activity of excised tissue treated with TRIA coincided with the temperature at which the barley was grown. At this temperature the plasma membrane is primarily in a mixed gel/liquid crystalline state. Pretreatment of barley roots with cyclohexamide did not alter ATPase stimulation by TRIA. Two to three times more [¹⁴C]-TRIA (mg membrane protein)⁻¹ was found associated with plasma membrane-enriched vesicles treated with TRIA than with vesicles enriched for mitochondrial membranes or for vesicles enriched for tonoplast, Golgi and rough endoplasmic reticulum. Both Ca²⁺- and Mg²⁺-dependent ATPase activity increased by 40–60% within 30 min of the addition of 2.3 n M TRIA to cell-free extracts of barley roots. The addition of octacosanol, the C₂₈ analogue of TRIA, to cell-free extracts did not affect metal-dependent ATPase activity. Consistent with many studies in the green-house, simultaneous additions of equimolar amounts of TRIA and octacosanol to cell-free extracts resulted in inhibition of ATPase stimulation by TRIA. TRIA may directly affect plasma membrane function in barley roots.     

Inorganic nitrogen assimilation in the non-N2-fixing cyanobacterium Phormidium laminosum. I. Cellular levels of glutamine synthetase and NADPH-dependent glutamate dehydrogenase

Cell-free extracts of nitrate-grown as well as of ammonium-grown cells of the filamentous non-nitrogen-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) showed detectable levels of both glutamine synthetase (GS, EC 6.3.1.2) and NADPH-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) activities. The GS level of nitrate-grown cells was higher than that of ammonium-grown cells, whereas the GDH level was higher in ammonium-grown cells and depended on the external ammonium concentration. When nitrate-grown cells were transferred to an ammonium-containing medium, a decrease of GS and an increase of GDH specific activities occurred, even in the presence of nitrate. Conversely, when ammonia-grown cells were transferred to a nitrate-containing medium, an increase of GS and a decrease of GDH-specific activities took place. Both these effects were inhibited by chloramphenicol and were probably mediated by de novo protein synthesis. When either cell type was transferred to a medium without nitrogen source, the specific activities of both enzymes increased. When nitrate-grown cells were transferred to nitrate medium with L-methionine-DL-sulphoximine (MSX) added, the specific activity of GDH also increased. Here we present some evidence that, under certain conditions of nitrogen availability, GDH would play a minor role in ammonium assimilation.     

Characterisation of the H+-ATPase in plasma membranes isolated from the green alga Chlamydomonas reinhardtii

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two-phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P-type ATPase. This was verified by using antibodies against P-type ATPase from Arabidopsis , which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (K_i= 0.9 μ M ) but not affected by inhibitors specific for F- or V-type ATPases. demonstrating the purity of the plasma membranes. Mg-ATP was the substrate, and the rate of ATP-hydrolysis followed simple Michaelis-Menten kinetics giving a K_m= 0.46 m M . Free Mg²⁺ stimulated the activity, K_1/2= 0.68 m M . Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20-fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right-side-out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H⁺-pumping as shown by the optical pH probe acridine orange. H⁺-pumping was dependent on the presence of valinomycin and K⁺ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside-out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.     

CYTOCHEMICAL DIFFERENCES IN KIDNEYS FROM WINTER-HIBERNATING AND AROUSED BATS (MYOTIS LUCIFUGUS), WITH PARTICULAR REFERENCE TO THE GOLGI ZONE

Kidneys from winter bats (Myotis lucifugus) were removed and fixed in cold formalin-calcium while the animals were in the following states: (a) natural hibernation; (b) arousal from hibernation for 24 hours; (c) laboratory maintained hibernation; and (d) no hibernation since the previous winter. With fixed frozen sections, the lead salt method of Wachstein and Meisel with adenosine triphosphate as substrate (pH 7.2) showed enzymic activity localized in large vacuoles and smaller vesicles or droplets in the Golgi region of distal and proximal tubular epithelial cells of kidneys from hibernating bats. No ATPase activity was detected in the basal lamellae of tubular epithelium from hibernating bats. ATPase activity in the Golgi region was not seen in cells from kidney tubules of bats aroused from hibernation 24 hours previously or of animals that had not hibernated, whereas activity for ATPase was present in the basal infoldings of tubular epithelium from these animals. Inosine di- and triphosphatase and calcium activated ATPase activities were also detected in the Golgi region of hibernating bats but were not present in the basal infoldings of tubular epithelium from active animals. There was little or no activity toward the mono- and diphosphates of adenine, thiamine pyrophosphate, and the di- or triphosphates of guanidine, cytidine, or deoxyadenosine. The loss of enzymic activity from the Golgi region of the tubular epithelium from hibernating bats and its increase in the region of the basal infoldings of tubular epithelium in aroused bats suggests that the Golgi region plays a role in the synthesis of enzymic protein usually identified with the external cell membrane.     

Polymorphic glutamate dehydrogenase in lilac vitroplants as revealed by combined preparative IEF and native PAGE: Effect of ammonium deprivation, darkness and atmospheric CO2 enrichment upon isomerization

The activity and polymorphism of glutamate dehydrogenase (GDH) were studied in basal callus of lilac ( Syringa vulgaris L.) vitroplants. Native PAGE alone revealed seven bands staggered at regular intervals. Preparative liquid-vein IEF allowed the separation of six to ten GDH fractions with charges ranging between 5.18 and 7.08. Analysis of these GDH fractions in native PAGE indicated that up to seven GDH bands can be detected for each fraction. This suggests the existence of seven isoforms of the enzyme with subunits presenting different isoelectric points. Dark- and ammonium-controlled forms were found to be the more acidic and faster migrating ones in native PAGE. The results support for the first time that atmospheric CO₂ enrichment increases GDH activity dramatically and modifies isomerization of the enzyme.     

Effect of calcium and other metal ions on pectic activities from autolysed cultures of Alternaria alternata

Abstract The effect of increasing concentrations of Ca²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Na⁺ and EDTA on the pectic enzymic activities (polymethylgalacturonase, endopectinase and pectin-lyase) present in the autolytic complex from Alternaria alternata has been studied. In all cases the divalent metal ions and EDTA produced an increased inhibition correlated with increasing concentration of each ion. An opposite effect was shown by the Na⁺ ion, which produced an increase in pectic enzymic activities, principally at low concentrations.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Aquaporin functionality in relation to H+-ATPase activity in root cells of Capsicum annuum grown under salinity

As water and nutrient uptake should be related in the response of plants to salinity, the aim of this paper is to establish whether or not aquaporin functionality is related to H⁺-ATPase activity in root cells of pepper ( Capsicum annuum L.) plants. Thus, H⁺-ATPase activity was measured in plasma membrane vesicles isolated from roots and aquaporin functionality was measured using a cell pressure probe in intact roots. Salinity was applied as 60 m M NaCl or 60 m M KCl, to determine which ion (Na⁺, K⁺ or Cl⁻) is producing the effects. We also investigated whether the effects of both salts were ameliorated by Ca²⁺. Similar results were obtained for cell hydraulic conductivity, Lp_c, and H⁺-ATPase activity, large reductions in the presence at NaCl or KCl and an ameliorative effect of Ca²⁺. However, fusicoccin (an activator of H⁺-ATPase) did not alter osmotic water permeability of protoplasts isolated from roots. Addition of Hg²⁺ inhibited both ATPase and aquaporins, but ATPase also contains Hg-binding sites. Therefore, the results indicate that H⁺-ATPase and aquaporin activities may not be related in pepper plants.     

ATPase activity of isolated plasma membrane vesicles of leaves of Elodea as affected by thiol reagents and NADH/NAD+ ratio

The goal of this study was to test the hypothesis that the plasma membrane-bound ATPase activity is influenced by the redox poise of the cytoplasm. Purified plasma membrane vesicles from leaves of Elodea canadensis Michx. and E. nuttallii (Planch.) St. John were isolated using an aqueous polymer two-phase batch procedure. The distribution of marker enzyme activities confirmed the plasma membrane origin of the vesicles. The vesicles exhibited NADH-ferricyanide reductase activity, indicating the presence of a redox chain in the plasma membrane. The K⁺, Mg²⁺-ATPase activity associated with these vesicles was inhibited by the sulfhydryl reagents N-ethylmaleimide and glutathione (GSSG). Furthermore the activity was inhibited by NAD⁺. This inhibition by NAD⁺ was relieved by increasing the NADH/NAD⁺ ratio. The possibility that the ATPase activity is regulated by the cytoplasmic NAD(P)H/ NAD(P)⁺ ratio is discussed, as well as the role of a plasma membrane-bound redox chain.     

Phospholipid and detergent effects on (Ca2+ + Mg2+)ATPase purified from human erythrocytes

(Ca2+ + Mg2+)ATPase (EC 3.6.1.3) was solubilized from human erythrocyte membranes by detergent extraction with Triton N-101 (0.5 mg/mg membrane protein) and purified by calmodulin affinity chromatography. ATPase activity was assayed in mixtures of Triton N-101 and phospholipid, without reconstitution into bilayer vesicles. At low levels of phospholipid (5 micrograms/ml), the ATPase activity was highly sensitive to the detergent concentration, with maximal activity occurring at or near the critical micelle concentration of the detergent. With increased amounts of phospholipid (50 micrograms/ml), detergent concentrations greater than the critical micelle concentration were required for maximal activity. Detergent alone did not support ATPase activity. Sonicated phospholipid in the form of vesicles was equally ineffective. Activity seemed to be dependent on the presence of detergent/phospholipid mixed micelles. The acidic phospholipids, phosphatidylserine and phosphatidylinositol, as well as the commercial phospholipid preparation, Asolectin, gave activities five to eight times greater than the same amount of phosphatidylcholine. Mixtures of phosphatidylserine and phosphatidylcholine produced intermediate ATPase activities, with the maximal value dependent on the phosphatidylserine concentration. Addition of phosphatidylcholine to fixed concentrations of phosphatidylserine caused a rise in activity that was independent of the ratio of the two phospholipids or the total phospholipid concentration. Phosphatidylcholine may therefore be irreplaceable for some aspect of ATPase function. The number of phospholipid molecules present in mixed micelles at maximal ATPase activity was calculated to be near 50. This value implied that the hydrophobic surface of the ATPase molecule must be completely coated by a single layer of phospholipid molecules for maximum activity to occur.     

Proton-ATPase activity in cells of lactobacilli grown in the presence of propionate

Lactobacillus helveticus ATCC 15009 and CRL 581, and Lact. casei LC3 were grown in a complex medium with and without 15 mmol 1^-1 of neutralized propionic acid and assayed for proton-translocating ATPase activity. The enzyme activity was higher when the medium contained fatty acid than in its absence for all strains studied. Characteristics of this increased ATPase were identical to those of the enzyme located on the membrane of normal cells. The substrate consumption rate of resting cells was increased by propionate. This effect was reverted by the specific H⁺-ATPase inhibitor N,N '-dicyclohexylcarbodiimide indicating that the increment of fermentative activity was related to the H⁺-ATPase activity. These results suggest that the amplification of H⁺-ATPase activity could be involved in the inhibition of lactobacilli growth in cultures where propionic acid is unavoidably present, such as some mixed cultures with propionibacteria.     

Phospholipid protection against proteolysis of D-beta-hydroxybutyrate dehydrogenase, a lecithin-requiring enzyme

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apodehydrogenase, i.e. the purified enzyme devoid of lipid, has been purified from beef heart mitochondria and as such is inactive. It can be reactivated by insertion into phospholipid vesicles containing lecithin. Proteolytic digestion with different proteases has been carried out to obtain insight into the orientation of the enzyme in the membrane and to assess the extent of immersion of the protein into the phospholipid bilayer. Digestion of the apodehydrogenase with either trypsin, chymotrypsin, Staphylococcus aureus protease, thermolysin, carboxypeptidases A and Y, or Pronase (from Streptomyces griseus) leads to loss of activity, as assayed with phospholipid. Limited digestion with carboxypeptidase results in complete inactivation. Of the proteases tested, only Pronase and chymotrypsin cleave and inactivate the enzyme inserted into phospholipid vesicles (enzyme-phospholipid complex). For the enzyme-phospholipid complex, the loss of activity with Pronase digestion follows a single exponential decay to less than 10% of the initial activity. With chymotrypsin digestion, the staining intensity of the original approximately 31,500-dalton polypeptide decreases more rapidly than the loss of enzymic activity. The enzyme-phospholipid complex, after limited cleavage with chymotrypsin, retains enzymic activity and resonance energy transfer from protein to bound NADH and an approximately 26,000-dalton polypeptide is observed. Phospholipid alters the cleavage pattern with both chymotrypsin and Pronase, and the rate of inactivation of the enzyme-phospholipid complex is slowed in the presence of NAD(H). Moreover, the rate of inactivation of the apodehydrogenase with chymotrypsin is diminished approximately 3-fold in the presence of NAD+. Digestion of submitochondrial vesicles with either trypsin, chymotrypsin, or Pronase rapidly inactivates D-beta-hydroxybutyrate dehydrogenase; the addition of NAD+ or NADH, together with dithiothreitol and increased salt (to 50 mM), decreases the rate of inactivation, and with trypsin, virtually eliminates inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Target size of D-beta-hydroxybutyrate dehydrogenase. Functional and structural molecular weight based on radiation inactivation

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apoenzyme has been purified to homogeneity from beef heart; it is devoid of lipid and inactive. It can be functionally reconstituted with lecithin or phospholipid mixtures containing lecithin. The active form of the enzyme is the enzyme-phospholipid complex. Classical target analysis of radiation-inactivation data has now been used to determine the molecular size of the enzyme both in the native membrane (submitochondrial vesicles) and in the reconstituted enzyme inserted into phospholipid vesicles containing lecithin. For both forms of the enzyme, we find the same molecular size, approximately 110,00 daltons. This size is consistent with a tetramer. Radiation results in fragmentation of the polypeptide and the destruction of the polypeptide correlates with loss of enzymic function. A similar size is obtained when purified D-beta-hydroxybutyrate dehydrogenase is inserted into a nonactivating mixture of phospholipid (i.e. in the absence of lecithin). We conclude that: 1) the native enzyme in submitochondrial vesicles and the purified active enzyme in phospholipid vesicles are the same size, approximating a tetramer; 2) radiation of D-beta-hydroxybutyrate dehydrogenase results in loss of activity and fragmentation of the polypeptide; and 3) the role of lecithin in activation of D-beta-hydroxybutyrate dehydrogenase is unrelated to determining oligomeric size of the enzymes since both active and nonactive forms exhibit the same structural size.     

Regulation of glutamate dehydrogenase activity by amino acids in maize seedlings

Root or secondary leaf segments from maize ( Zea mays L. cv. Ganga safed-2) seedlings were incubated with 9-amino acids and two amides separately, each at 5 m M for 24 h, to study their effects on glutamate dehydrogenase (GDH) activity. Most of the compounds tested inhibited the specific activity of NADH-GDH and increased that of NAD⁺-GDH in the roots in the presence as well as in the absence of ammonium. In the leaves, such effects were recorded only with a few amino acids. Total soluble protein in the root and leaf tissues increased with the supply of most of the amino compounds. The effect of glutamate on enzyme activity and protein was concentration dependent in both tissues. When the enzyme extracts from root or leaf tissues were incubated with some of the amino acids, NADH-GDH declined while NAD⁺-GDH increased in most cases. The inhibition of NADH-GDH increased with increasing concentration of cysteine from 1 to 5 m M . The experiments demonstrate that most of the amino acids regulated GDH activity, possibly through some physicochemical modulation of the enzyme molecule.