Mechanism of lipid activation of Na,K, Mg-activated adenosine triphosphatase and K,Mg-activated phosphatase of bovine cerebral cortex

Summary Na⁺, K⁺, Mg⁺⁺-activated adenosine triphosphatase and K⁺, Mg⁺⁺-activatedp-nitrophenyl phosphatase prepared from a membrane fraction of bovine cerebral cortex were studied with regard to the manner of their activation by phospholipids, using phosphatidyl serine, lysolecithin, monodecyl and didecyl phosphates. The kinetic and chromatographic studies suggested the following. (1) When the enzyme proteins bind the phospholipids in a proper ratio, they attain the optimum activation. (2) The binding causes a simple conversion of the enzymes from an inactive form to a fully activated form. (3) The lipids in both micellar form and molecular dispersion activate the enzymes. (4) Of the proteins contained in the enzyme preparation, only a group of proteins possessing the ATPase and the phosphatase activities bind phospholipids, and the amount of the bound lipids is limited.     

Influence of temperature acclimatization on the ionic activation of goldfish intestinal adenosine triphosphatase

1. An adenosine triphosphatase membrane system, dependent on Mg²⁺ and activated further by Na⁺+K⁺, was prepared from goldfish anterior intestine by differential centrifugation of homogenized intestinal scrapings. 2. The affinity of this preparation for Na⁺ in the presence of K⁺+Mg²⁺, for K⁺ in the presence of Na⁺+Mg²⁺ and for Mg²⁺ alone, measured at 37°, did not depend on the previous environmental temperature of the fish. When Na⁺+K⁺ were added to preparations from 8°-acclimatized fish the affinity for Mg²⁺ increased; this was not seen with preparations from 30°-acclimatized fish. 3. Part of the Mg²⁺-activated adenosine triphosphatase was inhibited by Na⁺ and the amount of inhibition appeared to increase at high acclimatization temperatures. 4. This Na⁺-inhibited adenosine triphosphatase was separated from the (Na⁺+K⁺)-activated enzyme by centrifugation on sucrose density gradients. 5. Preparations from 8°-acclimatized fish contained less Mg²⁺-activated and more (Na⁺+K⁺)-activated adenosine triphosphatase than did similar fractions from 30°-acclimatized fish. 6. Acclimatization to different environmental temperatures might involve one form of adenosine triphosphatase changing to another. The origin of various membranes seen in microsomal fractions must, however, be established before this hypothesis can be tested further.     

The action of thiol reagents on the adenosine-triphosphatase activities of heavy meromyosin and L-myosin

1. The Ca²⁺-activated adenosine triphosphatase of heavy meromyosin is maximally stimulated by lower relative molar concentrations of phenylmercuric acetate than are required with myosin. 2. Stimulation of the Ca²⁺-activated adenosine triphosphatase of both heavy meromyosin and myosin by thiol reagents is markedly affected by ionic strength, the effects being greater with the former than with the latter. In particular, N-ethylmaleimide strongly inhibits the Ca²⁺-activated adenosine triphosphatase of heavy meromyosin at ionic strength below about 0·2. 3. The precise behaviour of the thiol reagents at low ionic strength is slightly modified by the age of the heavy meromyosin and myosin preparations. 4. Stimulation of the Mg²⁺-activated adenosine triphosphatase of heavy meromyosin by thiol reagents is relatively insensitive to ionic strength. 5. The adenosine triphosphatases of heavy meromyosin and myosin activated by potassium chloride in the absence of bivalent activators are inhibited by thiol reagents over the range of ionic strength at which stimulation occurs in the presence of calcium chloride as activator. 6. The modifying effects of potassium chloride and sodium chloride are qualitatively different when heavy-meromyosin adenosine triphosphatase is stimulated with phenylmercuric acetate. No such difference is observed when the enzyme is stimulated with N-ethylmaleimide.     

Purification and Regulatory Properties of Fructose 1,6-Diphosphatase from Hydrogenomonas eutropha

Fructose diphosphatase of Hydrogenomonas eutropha H 16, produced during autotrophic growth, was purified 247-fold from extracts of cells. The molecular weight of the enzyme was estimated to be 170,000. The enzyme showed a pH optimum of 8.5 in both crude extracts and purified preparation. The shape of the pH curve was not changed in the presence of ethylenediaminetetraacetic acid. The enzyme required Mg²⁺ for activity. The MgCl₂ saturation curve was sigmoidal and the degree of positive cooperativity increased at lower fructose diphosphate concentrations. Mn²⁺ can replace Mg²⁺, but maximal activity was lower than that observed with Mg²⁺ and the optimal concentration range was narrow. The fructose diphosphate curve was also sigmoidal. The purified enzyme also hydrolyzed sedoheptulose diphosphate but at a much lower rate than fructose diphosphate. The enzyme was not inhibited by adenosine 5′-monophosphate but was inhibited by ribulose 5-phosphate and adenosine 5′-triphosphate. Adenosine 5′-triphosphate did not affect the degree of cooperativity among the sites for fructose diphosphate. The inhibition by adenosine 5′-triphosphate was mixed and by ribulose 5-phosphate was noncompetitive. An attempt was made to correlate the properties of fructose diphosphatase from H. eutropha with its physiological role during autotrophic growth.     

Studies on the ultrastructural localization of adenosine triphosphatase activity in fracture callus

Summary The fine structural localization of Mg⁺⁺-activated and Mg⁺⁺-independent neutral adenosine triphosphatase (ATPase) was studied in fracture callus of the rat using EDTA-decalcified DMSO-treated tissues incubated in Wachstein-Meisel type lead-containing media, and N-ethylmaleimide, NaF, EDTA and histidine as inhibitors to test the specificity of the reaction. Final product was found to be deposited on the plasma membranes and associated structures (subplasmalemmal vesicles and vacuoles) of phagocytic monocytoid cells, fibroblasts, osteoblasts and ruffled border regions of osteoclasts when Mg⁺⁺ was present in the incubation medium; the most abundant precipitate was noted on the plasma membranes of osteoblasts. When Mg⁺⁺ was omitted from the medium, the ruffled borders of osteoclasts were the only plasmalemmal sites showing conspicuous activity. This apparently Mg⁺⁺-independent ATPase was also demonstrated in the lysosomes of all the different cell types in the callus and in the vacuoles and specific granules located beneath the ruffled border of osteoclasts; lack of inhibition with NaF suggested that the enzyme was not a conventional nonspecific acid phosphatase. Neither the Mg⁺⁺-activated nor the Mg⁺⁺-resistant ATPase were inhibited by EDTA or histidine, indicating that they were unrelated to non-specific alkaline phosphatase. Deposition of final product did not occur on the plasma membranes of chondroblasts, chondrocytes of osteocytes.     

Modification of sarcoplasmic reticulum adenosine triphosphatase by adenosine triphosphate magnesium

Lineweaver-Burk plots of Ca²⁺-activated adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum have been determined for a wide range of substrate concentrations. The plots measured at constant Mg²⁺ concentrations are normally nonlinear, but approach linearity either as the sarcoplasmic reticulum ages, or when small quantities of Triton-X100 are added. Titration with N-ethylmaleimide has the same effect on the activity of the ATPase measured either at high or low substrate concentrations. Lineweaver-Burk plots measured under conditions where the Mg²⁺ concentration is varied so as to be always equal to the ATP concentration are linear. These results have been interpreted as evidence that the adenosine triphosphatase has a single active site which uses MgATP as its substrate and which can be modified by free Mg²⁺.     

Adenosine triphosphatase in isolated membranes of Staphylococcus aureus

The preparation of cytoplasmic membranes from suspensions of Staphylococcus aureus lysed by an enzyme recently isolated in these laboratories is described. These membranes contained: protein, 34.4%; ribonucleic acid, 6.6%; lipids, 34.5%; and total phosphorus, 1.4%. Such membranes exhibited adenosine 5′-triphosphatase (E.C. 3.6.1.3) activity, liberating orthophosphate at an initial rate of 0.53 μmole per min per mg of protein under optimal conditions. The enzyme was Mg⁺⁺-dependent and K⁺- or Na⁺-stimulated. Maximal activity was observed with a molar adenosine 5′-triphosphate (ATP) to Mg⁺⁺ ratio of 1. One mole of orthophosphate was liberated per mole of ATP; the other product of digestion was adenosine 5′-diphosphate. Inorganic pyrophosphate and the 5′-triphosphates of guanosine, uridine, and cytidine were also attacked by membrane preparations, but more slowly than ATP. Ouabain, p-chloromercuribenzoate, and 2,4-dinitrophenol did not alter adenosine triphosphatase activity, whereas both Atebrine and chlorpromazine were inhibitory.     

Saure und alkalische Phosphatase sowie Adenosintriphosphatasen im Epithel der Pars proventricularis des Pferdemagens

Zusammenfassung Im Epithel der Pars proventricularis des Pferdes wurden mit licht- und elektronenmikroskopischen cytochemischen Methoden eine lysosomale saure Phosphatase sowie die Mg⁺⁺- und Ca⁺⁺-aktivierbaren ATPasen nachgewiesen und lokalisiert. Positive Reaktionen der unspezifischen alkalischen Phosphatase fanden sich dagegen ausschließlich an den subepithelialen Gefäßschlingen der Papillae occultae.Die ATPasen-Aktivität nimmt vom Stratum basale zum Stratum superficiale kontinuierlich ab. Die Reaktionsintensität ist nach 45 min-Inkubationszeit optimal und läßt sich nicht weiter steigern. Eine Erhöhung der Mg⁺⁺-aktivierbaren Grundaktivität des Enzyms durch Na⁺ und K⁺ ist cytochemisch nicht nachweisbar. Die Reaktionsprodukte haften an der äußeren Lamelle der Zellmembranen. Frei von Enzymaktivitäten sind lediglich die an der Bildung von Desmosomen und Halbdesmosomen beteiligten Plasmalemm-Abschnitte.Einige Probleme des aktiven Transportes, die sich aus den histochemischen Enzym-Befunden ergeben, werden diskutiert.

---

Histochemical and ultrastructural study of the acid phosphatase, alkaline phosphatase, and adenosine triphosphatase contents in the pars proventricularis epithelium of the horse stomach

Summary The activities and localization of a lysosomal acid phosphatase and of a Mg⁺⁺-and Ca⁺⁺-activated ATPase were shown in the pars proventricularis epithelium of the horse stomach using cytochemical methods and the light and electron microscopes. Activity of the non-specific alkaline phosphatase was exclusively demonstrated on the subepithelial vessel loops of the papillae occultae.The activity of ATPases gradually decreased from the stratum basale to the stratum superficiale. Reaction intensity was optimal after 45 min and did not increase further. An increase of the Mg⁺⁺-activated basal activity by Na⁺ and K⁺⁺ was not shown cytochemically. Reaction products adhered to the outer lamellae of plasmalemmata. Only the plasmalemmal regions forming desmosomes and hemidesmosomes were free of enzyme activities.Some problems of active transport related to activities of these enzymes have been discussed.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Histological and histochemical studies on the subfornical organ of the squirrel monkey

Summary Detailed studies have been made on the distribution of several enzymes in the subfornical organ (SFO) of the squirrel monkey. In this species, the nerve cells of the SFO show reactions of varying intensity for enzymes of the glycolytic and aerobic pathways. The nerve cells, glial cells and ependymal cells of the SFO and the choroid plexus are equipped with enzymes of the Embden-Meyerhof (EM) pathway, pentose cycle and tricarboxylic acid (TCA) cycle. Many nerve cells and oligodendroglia in the body of this organ are rich in enzymes of the TCA cycle and the pentose cycle and thus presumably have the capacity of producing adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH₂) [reduced triphosphopyridine nucleotide (TPNH)]. In the neurons, ATP is probably used as energy for synaptic transmission, active transport, secretion and various other metabolic processes, whereas NADPH₂ is used for synthetic processes such as the production of fatty acids and some amino acid conversion (e.g., conversion of phenylalanine into tyrosine). The SFO and its stalks contain both cholinergic and adrenergic neurons and fibers. The outermost layer of the perivascular sheath gives a positive reaction for enzymes of the gylcolytic pathways (EM pathway, pentose cycle and TCA cycle), whereas the inner layer of this sheath shows negligible activity for these enzymes. On the other hand, the whole sheath (inner and outer layers) exhibits strong staining for Mg⁺⁺-activated adenosine triphosphatase (ATPase), and moderate staining for Ca⁺⁺-activated ATPase. This sheath, rich in ATPase, may carry on active transport and such related functions. Since the outermost layer contains various enzymes of the glycolytic pathways, it is possible that the ATP required for these functions is produced in this layer.Visiting scientist from the Department of Anatomy, Tokyo Medical and Dental University, Tokyo, JapanT. R. Shanthaveerappa in previous publications.     

Inhibitory effects of mersalyl and of antibodies directed against smooth-muscle myosin on a calcium adenosine triphosphatase of the plasma membrane from mouse liver cells.

The effect of mersalyl and of antibodies, directed against smooth-muscle myosin and skeletal muscle myosin, on the (Ca²⁺ + Mg²⁺)-activated adenosine triphosphatase (Ca,Mg)ATPase) system of mouse liver plasma membranes has been studied. Antismooth-muscle myosin inhibited by 38.6% at optimum substrate concentration the (Ca,Mg)ATPase with a K_m of 0.88 × 10^?3m. Mersalyl (0.5 mm) also inhibited this enzyme, the percentage inhibition being 44.6% at optimal substrate concentration. These results suggest the presence of a smooth-muscle myosin-like protein in the plasma membrane of mouse liver cells which has an associated (Ca,Mg)ATPase activity.     

Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase. 2. The Mg²⁺,Ca²⁺-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA⁻) could not be re-activated by the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA⁻).     

Adenosine triphosphatase activity of cutaneous nerve fibers

Summary The histochemical study of Mg⁺⁺-activated adenosine triphosphatase (Mg⁺⁺-ATPase) activity was carried out on the peripheral nerves of mouse digital skin by light and electron microscopy. Under the light microscope, the ATPase activity was clearly demonstrated on the nerve fibers as a fine network in the subepidermal regions. Under the electron microscope, the reaction product of enzyme activity was located in the interspace between axolemma and the surrounding Schwann cells of the unmyelinated nerve fibers. No reaction product was observed in the space between the axolemma and the Schwann cells associated with myelinated nerve fibers. Demonstrable activity was absent at the nodes of Ranvier as well as on the para- and internodal regions of these myelinated axons. The part of the axolemma lacking a Schwann cell sheath failed to show a reaction product. The perineural epithelial cells surrounding the nerve fibers displayed reaction product in the caveolae. These results suggest a functional difference in the axon-Schwann interface of myelinated as compared to unmyelinated nerve fibers. The function of the perineural epithelial cell would be expected to be a regulatory one in transferring materials across the epithelium to keep the proper humoral environment around nerve fibers.     

Plasma membrane Ca2+ transport: Antagonism by several potential inhibitors

Summary Inside-out vesicles prepared from human red blood cells took up Ca²⁺ by an active transport process. Membranes from the same red blood cells displayed Ca²⁺-activated, Mg²⁺-dependent adenosine triphosphatase activity. Both the initial rate of Ca²⁺ transport and the (Ca²⁺+Mg²⁺)-adenosine triphosphatase activity were increased approximately twofold by the calcium binding protein, calmodulin. Activities in the absence of added calmodulin were termed basal activities. Calmodulin-activated Ca²⁺ transport and adenosine triphosphatase activities could be antagonized in a relatively selective fashion by the phenothiazine tranquilizer drug, trifluoperazine. High concentrations of trifluoperazine also inhibited basal Ca²⁺ transport and adenosine triphosphatase activity. By contrast, calmodulin binding protein from beef brain selectively antagonized the effect of calmodulin on Ca²⁺ transport with no inhibition of basal activity. Ruthenium red antagonized calmodulin-activated and basal activity with equal potency. The results demonstrate that although phenothiazines can act as relatively selective antagonists of calmodulin-induced effects, other effects are possible and cannot be ignored. Calmodulin-binding protein may be a useful tool in the analysis of calmodulin functions. Ruthenium red probably interacts with Ca²⁺ pump adenosine triphosphatase at a site not related to calmodulin.     

Evidence implicating a membrane ATPase in the control of passive permeability of excitable cells

The temperature sensitivity of the ATPase enzyme systems in a muscle microsomal preparation from the crayfish, Astacus pallipes, was studied. Preincubation of the enzyme preparation in the range 33–36°C produced a marked inactivation of the ATPases; the Mg⁺⁺-dependent ATPase was very much more sensitive to this treatment than the Na⁺-K⁺-Mg⁺⁺-dependent ATPase. Thus, the Arrhenius μ for the inactivation of the Mg⁺⁺-dependent ATPase produced by eight minute preincubation is > 100 Kcals. These results are compared with the changes that are observed during the heat death of the whole animal, where exposure to 35°C produces a dramatic change in Na⁺ permeability within five minutes. Arrhenius μ for heat death is also > 100 Kcals and operates over the identical critical temperature range. It is suggested that the Mg⁺⁺-dependent ATPase controls passive permeability in these excitable cells and the results also confirm the view that Mg⁺⁺ and Na⁺-K⁺-Mg⁺⁺ ATPases are separate enzymes.     

Histochemische und elektronenoptische Untersuchungen über Adenosintriphosphatasen des endometrialen Oberflächenund Drüsenepithels der Ziege (Capra hircus)

Zusammenfassung Im Oberflächen- und Drüsenepithel des nicht graviden Uterus der Ziege wurden licht- und elektronenoptische Untersuchungen über den Nachweis der Mg⁺⁺-, Ca⁺⁺- und der (Mg⁺⁺-Na⁺-K⁺)-aktivierbaren ATPasen durchgeführt.Spezifische Enzymaktivitäten lassen sich nur an Proben aus der Follikelphase nachweisen. In der Corpus luteum-Phase dagegen liegen diese Enzyme nicht in nachweisbarer Intensität vor.Die lichtmikroskopisch an den seitlichen Zellflächen nachgewiesenen Niederschläge der ATPasen finden sich elektronenoptisch als schwarze Granula an der Außenlamelle der lateralen Plasmalemm-Abschnitte. An apikalen und basalen Zellmembran-Bereichen in beiden Zyklusphasen zu beobachtende Reaktionsprodukte sind das Ergebnis einer Spontanhydrolyse des Substrats ATP bzw. von Spaltungen aufgrund der unspezifischen alkalischen Phosphatase. Haftkomplexe und Desmosomen sind regelmäßig frei von Bleisalz-Niederschlägen. Es können weder regionale Unterschiede in der Reaktionsintensität der Enzyme noch Differenzen im Hinblick auf die Verteilung und ultrastrukturelle Lokalisation von Niederschlägen der Mg⁺⁺-bzw. der Ca⁺⁺-aktivierbaren ATPase festgestellt werden.

---

Histochemical and electron microscopic studies of the adenosine triphosphatases in the endometrial surface and glandular epithelium of the goat (Capra hircus)

Summary Light and electron microscopic studies were performed on the Mg⁺⁺-, Ca⁺⁺-, and the (Mg⁺⁺-Na⁺-K⁺)-activated ATPases in the surface and glandular epithelium of the non-pregnant uterus of the goat.Specific enzyme activities have been shown only in the epithelial samples collected during the follicular phase. In the luteal phase, none of these enzymes have been demonstrated with certainty. Precipitates of the ATPases which have been shown with the light microscope on the sides of the cell surface, can electron optically be observed as black granules on the outer lamella of the lateral plasmalemma. Reaction products on the apical and basal cell membrane regions observable in both cyclic phases, result from spontaneous hydrolysis of the substrate ATP, i.e. splitting caused by non-specific alkaline phosphatase. Junctional complexes and desmosomes are regularly free from lead salt precipitates. Neither regional differences in reaction intensity of the enzymes nor differences in the distribution and ultrastructural localization of either Mg⁺⁺-or Ca⁺⁺-activated ATPase precipitates can be observed.

Frau E. Merl danken wir für sorgfältige technische Mitarbeit.     

Ecto-ATPase

Summary An ecto-adenosine triphosphatase (E.C. 3.6.1.4 ATP-phosphohydrolase) is shown to be localised on the outer surface of varieties of cell membrane. The enzyme is different from the ATPase involved in biological energy transduction and ion transport mechanism. The characteristic of the enzyme lies in having a very broad substrate specificity and is inhibited by EDTA and higher concentration of ATP. The enzyme is dependent on bivalent metal ions, Mg⁺⁺ or Ca⁺⁺ for its optimum activity. The enzyme is highly sensitive to SH-reagents but insensitive to inhibitors of mitochondrial ATPase or Na⁺−K⁺-ATPase. The possible functions of the enzyme in being oriented outside the cell membrane is discussed.     

Effect of bivalent cations on the adenosine triphosphatase of actomyosin and its modification by tropomyosin and troponin

1. After removal of tropomyosin and troponin from the `natural'' actomyosin complex, the adenosine triphosphatase activity of the resulting `desensitized'' actomyosin is stimulated to the same extent by various bivalent cations with an ionic radius in the range 0·65–0·99å when tested at optimum concentration of the metal ion in the presence of 2·5mm-ATP at low ionic strength and pH7·6. Under identical conditions the adenosine triphosphatase activity of myosin alone is stimulated to an appreciable extent only by Ca²⁺ (ionic radius 0·99å). 2. Tropomyosin narrows the range of size of the stimulatory cations by inhibiting specifically the adenosine triphosphatase activity of `desensitized'' actomyosin when stimulated by Ca<sup>2+</sup> or the slightly smaller Cd<sup>2+</sup> (ionic radius 0·97å). Tropomyosin has no effect on the adenosine triphosphatase activity of `desensitized'' actomyosin when stimulated by the smaller cations, nor on the Ca²⁺-activated adenosine triphosphatase activity of myosin alone. 3. The adenosine triphosphatase activity of the `natural'' actomyosin system (containing tropomyosin and troponin) stimulated by the smallest cation, Mg²⁺ (ionic radius 0·65å), is low when the system is deprived of Ca²⁺ but high in the presence of small amounts of Ca²⁺. This sensitivity to Ca²⁺ seems to be a unique feature of the Mg²⁺-stimulated system. 4. The changes in specificity of the myosin adenosine triphosphatase activity in its requirement for bivalent cations caused by interaction with actin, tropomyosin and troponin primarily concern the size of the metal ions. The effects on enzymic properties of myofibrils due to tropomyosin and troponin can be demonstrated at low and at physiological ionic strength.     

Protein synthesis in rabbit reticulocytes XXII+: a heat stable dialyzable factor (EIF-I*) modulates Met-tRNAf binding to EIF-1.

The peptide chain initiation factor EIF-1 forms a ternary complex, Met-tRNA_f·EIF-1·GTP in the absence of Mg⁺⁺ and the preformed complex is stable to Mg⁺⁺. However, with homogeneous preparations of EIF-1, addition of Mg⁺⁺ during the initial formation of the ternary complex strongly inhibits the complex formation.A heat stable dialyzable factor (EIF-1¹) which mostly remains associated with the high molecular weight protein complex, EIF-2 (TDF) during purification of the peptide chain initiation factors, has been purified using a phenol extraction procedure. EIF-1¹ restores the Met-tRNA_f binding activity of EIF-1 in the presence of 1 mM Mg⁺⁺; in the presence of EIF-1¹, Met-tRNA_f binding by EIF-1 shows a sharp Mg⁺⁺ optimum around 1 mM. EIF-1¹ is heat stable, alkali stable, dialyzable and pronase sensitive. The same EIF-1¹ preparation also strongly inhibits Met-tRNA_f binding to EIF-1 in the absence of Mg⁺⁺ and stimulates protein synthesis in a mRNA-dependent rabbit reticulocyte lysate system.     

Induction, Purification and Characterization of α-1, 3-Glucanase from Brassica juncea L Infected with Albugo candida

Infection of Brassica juncea plants with Albugo candida caused the induction of β-1,3-glucanase in the leaf tissues following inoculation; the induction being more pronounced in the resistant cultivars. The enzyme purified from infected leaves of resistant cultivar RC-781 had a molecular mass of 35.4 kD. The enzyme exhibited its optimum activity at 50°C and at pH 5.5 with K_m value of 0.55 mg laminarin ml^?1. The enzyme activity was slightly stimulated by Cl^? and Mg⁺⁺, while inhibited by Hg⁺⁺ and Mn++ at higher concentrations only     

Increased Membrane-bound Adenosine Triphosphatase Activity Accompanying Development of Enhanced Solute Uptake in Washed Corn Root Tissue

Washing of excised corn (Zea mays L., variety WF9×M14) root tissue is accompanied by an increase in (Mg²⁺ + K⁺)-stimulated adenosine triphosphatase. This is the adenosine triphosphatase described by Fisher, Hansen, and Hodges as positively correlated with ion accumulation rates. The increase in activity is confined to the microsomal fraction. A close parallel exists between increases in adenosine triphosphatase and phosphate absorption, and they respond similarly to inhibitors of RNA and protein synthesis. However, the amplitude of change is much smaller in adenosine triphosphatase. Possible reasons for this discrepancy are discussed.