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Enzymatic activation of Clostridium perfringens epsilon prototoxin removed a small basic part of the molecule, causing a slight change in molecular weight (32,700 to 31,200) and a large change in isoelectric point (from pH 8.02 into fractions of 5.36 and 5.74).
Acanthamoeba myosin IB is a single-headed enzyme containing one heavy chain of 125,000 daltons, one light chain of 27,000 daltons, and one light chain of 14,000 daltons. The 125,000- and 27,000-dalton polypeptides are consistently found in a molar ratio of 1:1. The content of the 14,000-dalton peptide is usually only 0.1 to 0.2, and always less than 0.5, relative to the other two chains and might be a contaminant or a degradation product of one of the other chains. The specific activities of the Ca2+-ATPase, (K+, EDTA)-ATPase, and (after phosphorylation of its heavy chain by a specific kinase) actin-activated Mg2+-ATPase of Acanthamoeba myosin IB are similar to those of rabbit skeletal muscle myosin. After treatment of the enzyme with 2 M LiCl, the 125,000-dalton heavy chain of Acanthamoeba myosin Ib can be obtained, by chromatography on Sephadex G-200, essentially free of the 14,000-dalton peptide and more than 90% free of the 27,000-dalton peptide. This isolated heavy chain has the same specific ATPase activities as the original enzyme. Therefore, the heavy chain of Acanthamoeba myosin IB contains the ATPase catalytic site, the actin-binding site, and the phosphorylation site and is fully active enzymatically in the absence of light chains.
A protein antigenically related to the simian virus (SV 40) A gene product has been purified to near homogeneity from cells infected with the adenovirus-SV 40 hybrid virus Ad2(+)D2 and shown to contain ATPase (ATP phosphohydrolase, EC 3.6.1.3) and protein kinase (ATP:phosphotransferase, EC 2.7.1.37) activity. Both enzymatic activities copurify with the protein through six stages including one gel filtration column, two ion exchange columns, and a heparin affinity column. Analogous fractions from extracts of cells uninfected or infected with adenovirus 2 alone do not contain these enzymatic activities. The D2 hybrid protein resolves into two forms (I and II) during ion exchange chromatography. Form I, the major species (85%) of the D2 hybrid protein, elutes from DEAE-Sephadex in 0.37 M NaCl and is able to catalyze the hydrolysis of ATP to ADP + P(i) at a rate of 3 mumol/hr per mg. The remaining 10-15% of the D2 hybrid protein consists of form II which elutes from DEAE-Sephadex in 0.29 M NaCl and is able to hydrolyze ATP as well as to incorporate phosphorus from ATP into either the D2 hybrid protein itself or other protein acceptors such as phosvitin. Although both forms are able to bind DNA, the ATPase activity of form I cosediments with SV 40 DNA more efficiently than does the protein kinase activity of form II during glycerol gradient centrifugation. The ATPase activity of form I is efficiently inhibited by addition of anti-T gamma globulin to the reaction mixture whereas control gamma globulin has no effect. Similarly, the phosphorylation of the D2 hybrid protein by form II is inhibited by anti-T gamma globulin. By contrast, phosphorylation of phosvitin is specifically inhibited by antibody only when the immune complex is removed from the reaction mixture. Thus, it appears likely that one and possibly two enzymatic activities are carried out by the D2 hybrid protein. These findings are discussed in terms of mechanisms of SV 40 DNA replication and virally induced transformation.
The injection of proteolytic inhibitors changes phagocytosis and metabolism of leucocytes in different ways. The polyvalent inhibitor, inhitril, increased phagocytosis but does not change digestive activity of leucocytes. Activities of acid phosphatase, peroxidases and succinatedehydrogenase as well as glycogen contents are seen decreased, whereas activity of glycerophosphate dehydrogenase increases, and that of lactate dehydrogenase does not change. Anti-tryptic serum decreases phagocytosis, both digestive and enzymatic activities of leucocytes, glycogen accumulation being observed. Anti-kallikrein serum decreases phagocytosis and activity of lactate dehydrogenase, and does not change glycogen accumulation and activities of digestion and of acid phosphatase. In addition, activities of succinate-, glycerophosphate dehydrogenase and peroxidase increase.
Determination of serum enzymatic activities in hepatosplenic bilharziasis (H.S.B.) were conducted in 100 bilharzial patients, in different stages and 30 controls SGot, SGPT, ALK pH, ALD and SLDH with its both fractions heat stable and labile.). Early elevation of serum enzymatic activities of SGOT, SGPT and SALK. pH, may be considered as a sensitive parameter for functional changes in H.S.B. rather than other conventional liver function tests. The elevated enzymic activity of total LDH in this study was associated with elevation in its both fractions. In particular, the changes in the total activity was in parallel with that of its heat labile fraction. The latter may be considered as a confirmatory test for marked deterioration of liver functions in H.S.B. The changes in the heat stable fraction was inconstant and may be attributed to extrahepatic bilharzial dissimilation. Significantly high serum enzymatic activity of SALD was found in cases of H.S.B. particularly those showing striking muscle wasting.
The requirements for choleragen activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] were investigated by using an enzyme preparation solubilized with Triton X-100 from an extensively washed brain particulate fraction and partially purified with DEAE-cellulose. Unlike the particulate enzyme, this preparation was not activated after incubation with choleragen plus dithiothreitol, ATP, and NAD. Addition of the purified protein activator of cyclic nucleotide phosphodiesterase and calcium to the partially purified enzyme increased basal activity somewhat, but choleragen activation was minimal. When cyclase was incubated with GTP plus the protein activator (and calcium), choleragen markedly increased the activity 3- to 6-fold. When GppNHp and protein activator were incubated with the cyclase prior to assay, activity was elevated but no effect of choleragen was observed. GTP and GppNHp had relatively small effects on cyclase activity in the absence of protein activator or if they were added directly to the assay. Boiled brain supernatant was consistently more effective than protein activator (plus calcium) and GTP, suggesting that other factors are required for maximal cyclase activity after choleragen treatment. It appears that the cyclase system is dissociable into several components, all of which may be necessary for optimal regulation of activity. It is probable that one of these is the heat-stable calcium-dependent protein activator of cyclic nucleotide phosphodiesterase and adenylate cyclase that we have found is required along with GTP for demonstration of choleragen activation of partially purified brain adenylate cyclase.
The enzymatic activities of two mitochondrial enzymes, i.e. succinate dehydrogenase and NADH-cytochrome c reductase were investigated in the brain of rats at different stages of post-natal development. In addition, the effect of the pharmacological treatment with two drugs, nicergoline and bamethan, able to interact with the alpha or the beta receptors respectively, was evaluated. The results show that both the enzymatic activities rapidly increase in the first days of extra-uterine life, thus indicating an adaptation of mitochondrial oxidative processes to post-natal environmental conditions. The pharmacological treatment with the two drugs does not induce any changes in the enzymatic activities tested.
Ligandin binds several classes of compounds, has glutathione-S-transferase activity and is postulated to function in the intracellular transport of substances which bind to it and/or are substrates for its enzymatic activity. The effects of a group of organometals reported to inhibit the enzymatic activity of ligandin on the biliary excretion of sulfobromophthalein have been investigated to determine what role the enzymatic activity of ligandin has with respect to the biliary excretion of the dye. Triethyllead reduced the rate of dye excretion into the bile without affecting blood pressure, blood or liver sulfhydryl compounds or the volume of bile flow. The organometal had no effect on the initial rate of plasma dye clearance. Inhibited biliary sulfobromophthalein excretion by triethylead-treated rats correlated with relative increases in liver and bile unconjugated dye, decreases in liver and bile conjugated dye and reduced glutathione-S-aryltransferase activity in supernatant fractions isolated from the liver. The results clearly demonstrated that the tested organometals can inhibit the enzymatic activity of ligandin in vivo and suggested that, if ligandin has a role in the translocation of the dye from the blood to the liver, the enzymatic activity of the protein may not be involved.
RNase U2 was purified and crystallized from the enriched culture medium (ammonium sulfate-urea-corn meal) of Ustilago sphaerogena and its characteristics were investigated. Chemical modification of RNase U2 was conducted with monoiodoacetic acid to carboxymethylate Glu-61 and with 2-methoxy-5-nitrotropone to nitrotroponylate the amino terminal residue. The amino terminal residue was modified reversibly by this reagent. Comparison of the 2'-AMP binding in the modified enzyme and the native one showed that Glu-61 is essential for the formation of the enzyme-substrate complex, while the amino terminal residue plays no important role in the enzymatic activity. The enzymatic activity and the structure of RNase U2 in aqueous organic solution were also investigated. The affinity of the enzyme for 2'-AMP, the inactivation by monoiodoacetic acid and the fluorescence intensity were examined. The profiles of the changes in the properties of the enzyme protein were consistent with those in the enzymatic activity. Fluorescence studies of the enzyme suggest that the tryptophan residue is closely related to the activity.
Activity of ATPases was studied in erythrocytes of patients with phobia neurosis of the 11 degree and in erythrocytes of mentally healthy patients. The total Mg2+, Na+, K+-dependent ATPase activity of patients with the neurosis was unaltered in blood. But the Mg2+-ATPase activity was increased and the Na+, K+-ATPase activity was decreased in the patients as compared with the control group. Addition of lithium carbonate into incubation mixtures exhibited different effect on the Na+, K+-stimulated ATPase depending upon the initial activity of the enzyme. In blood of patients with low initial values of the enzymatic activity it was increased. But the Na+, K+-ATPase activity was decreased by lithium carbonate in those cases when the initial enzymatic activity was high. Quantitatively the effect of lithium on the enzymatic activity was similar in the both groups of neurotic patients studied (although the direction of changes was oposite).
The changes induced by phenobarbital in cerebral enzymatic activities of the Krebs' cycle (citrate synthase, malate dehydrogenase) and electron transfer chain (total NADH-cytochrome c reductase and cytochrome oxidase) were studied. In addition, the activity of lactate dehydrogenase of acetylcholine esterase and of glutamate dehydrogenase was also studied. These enzymatic activities were evaluated in the homogenate in toto and in a crude mitochondrial fraction from rat brain. The modifications in some of these activities indicate that several new metabolic situations occur in brain tissue after phenobarbital treatment.
Hamsters were intratracheally instilled with saline solutions containing a high dose (145 to 220 micrograms) or a low dose (1.3 to 1.5 micrograms) of 3H-methylated pancreatic elastase or N-acetyl-(L-alanyl)3-L-alanine chloromethyl ketone-inactivated 3H-methylated pancreatic elastase. Only the lysyl residues of the elastase molecule were methylated and radiolabeled in a nonlabile manner. The 3H-methylated elastase preparation exhibited esterolytic and elastolytic activity, spectral properties, and emphysema-inducing properties indistinguishable from those of unmodified pancreatic elastase. There was no detectable hemorrhagic or emphysematous reaction with the inactivated 3H-methylated elastase, and this material was cleared from the lungs 11 times faster than the corresponding enzymatically active high dose of 3H-methylated elastase and 18 times faster than the corresponding enzymatically active low dose of 3H-methylated elastase. There were correspondingly higher amounts of radioactivity in the urine of hamsters treated with the inactivated elastase. All of the 3H radioactivity recovered from the urine was associated with epsilon-N-methyllysyl and epsilon-N,N-dimethyllysyl residues. Significant levels of radioactivity were found in the cells, primarily alveolar macrophages, lavaged from the lungs. The low dose of enzymatically active elastase caused neither detectable hemorrhage nor emphysema, both of which were associated with the high dose. At 144 days significant radioactivity (1,200 cpm) remained in the lungs of animals treated with high or low doses of enzymatically active elastase, whereas virtually no radioactivity (100 cpm) was found in the lungs of those treated with high or low doses of inactivated elastase. The data presented support the hypothesis that the formation of elastase complexes with alpha 1-protease inhibitor and alpha 2-macroglobulin is associated with the slower clearance and the retention of significant amounts of radioactivity in the lungs. Some of the residual radioactivity found in the hamster lungs might represent enzymatically active elastase complexed with alpha 2-macroglobulin and might offer an explanation for the progressive nature of emphysema induced by a single dose of elastase.
The influence of incubation with D-penicillamine on pure enzyme preparations and on enzymatic activities of serum and skin homogenates was investigated. Three of the nine enzymatic activities studied underwent significant changes. Such effects of D-penicillamine must be taken into consideration if therapeutic or unwanted actions of this drug are to be fully understood; they are elicited by concentrations reached under conditions used for human therapy.
The present study provides data on the properties of Ca2+-dependent Atpase of sarcoplasmic reticulum in states intermediary between the fully detergent-solubilized and vesicular form. After solubilization of ATPase vesicles by dodecyloctaoxyethylene glycol monoether (C12E8), the protein is mainly present as a monomer exhibiting enzymatic activity. Gel chromatography in presence or absence of Tween 80 gives rise to formation of oligomers of various size and smaller amounts of monomeric ATPase. Only the oligomeric species retain enzymatic activity (half-life, 3 to 4 days), while the gel chromatographic monomer is enzymatically inactive. Teteramers or trimers of ATPase, containing approximately 22 mol of phospholipid/mol of ATPase, are the smallest enzymatically active units after gel chromatography. Formation of larger sized particles and vesicles of ATPase appears to depend on the presence of sufficient lipid to make a cohesion between the tetrameric or trimeric units. The protein appears to be partially deaggregated by a relatively high Tween 80 concentration in the eluant (0.5 mg/ml) and under these conditions, phospholipid binding is reduced to a low level (approximately 11 mol/mol of protein). The data indicate that any bonds between ATPase polypeptide chains are easily disrupted by detergent and that lipid also may play a role in mediating contact between individual polypeptide chains in the tetrameric or trimeric units. Phospholipid analysis and exchange experiments indicate that the phospholipid left on ATPase after solubilization has a similar composition to that of the whole membrane. The binding of Tween 80 by soluble ATPase above the critical micellar concentration is 0.23 to 0.29 g/g of protein. The inactive monomer of ATPase binds phospholipid and Tween 80 to about the same extent, but has a slightly different circular dichroism spectrum, than oligomeric ATPase.
Some enzymatic activities were studied in long ter cultures of human liver cells : glucose-6-phosphatase, U.D.P. glucuronosyltransferase, phenylalanine 4-hydroxylase and tyrosine aminotransferase. Only weak tyrosine aminotransferase activity has been found in 12 subcultures, and it has not been increased by addition of corticoids. This tyrosine aminotransferase activity has been measured at different passages of the culture. Our results are compared with those found in literature. The different reasons which could explain the absence of liver specific biochemical functions have been discussed.
Even a small amount of formaldehyde is shown to induce a drop in the RNase A enzymatic activity. This drop is rapid from the start and then begins to be slower. A supposition was made on nature of the enzyme activity. Comparison of the effects of formaldehyde on the enzymatic and the destabilizing activity of RNase A was made. The effect of formaldehyde on the enzymatic activity does not correlate with its effect on the ability of RNase to destabilize the DNA double helix.
beta-Bungarotoxins have been shown to be presynaptic blockers of neuromuscular transmission. This paper reports experiments using the most positively charged beta-bungarotoxin that elutes from a CM-Sephadex C-25 column. The toxin is shown to be a single polypeptide with a molecular weight of approximately 11,000 and has phospholipase A2 activity. The application of the enzymatically active toxin to the frog sciatic nerve-sartorius muscle preparation results in an initial decrease in the average endplate potential amplitude followed by a temporary rebound in endplate potential amplitude, and finally a complete inhibition of endplate potentials. Similarly, minature endplate potential frequency is initially reduced upon toxin application but then increases dramatically. After the phospholipase A2 of the toxin is inactivated, treatment with the toxin results in only the initial decrease in transmitter release. There results suggest that this beta-bungarotoxin acts in two functionally separate steps: (i) by binding to a specific presynaptic site possibly associated with calcium entry, and (ii) by perturbing the presynaptic membrane by its enzyme action, which results in an increase and then a failure in transmitter release.
In order to investigate the roles of the physical states of phospholipid and protein in the enzymatic behavior of the Ca2+ -ATPase from sarcoplasmic reticulum, we have modified the lipid phase of the enzyme, observed the effects on the enzymatic activity at low temperatures, and correlated these effects with spectroscopic measurements of the rotational motions of both the lipid and protein components. Replacement of the native lipids with dipalmitoyl phosphatidylcholine inhibits ATPase activity and decreases both lipid fluidity, as monitored by EPR spectroscopy on a stearic acid spin label, and protein rotational mobility, as monitored by saturation transfer EPR spectroscopy on the covalently spin-labeled enzyme. Solubilization of the lipid-replaced enzyme with Triton X-100 reverses all three of these effects. Ten millimolar CaCl2 added either to the enzyme associated with the endogenous lipids or to the Triton X-100 soulbilized enzyme inhibits both ATPase activity and protein rotational mobility but has no detectable effect on the lipid mobility. These results are consistent with the proposal that both lipid fluidity and protein rotational mobility are essential for enzymatic activity.