Efraín Toro-Goyco

Structural studies on pinguinain. Changes induced by carboxamidomethylation

Abstract Pinguinain, a thiol-dependent plant protease, was purified by Sephadex G-100 and DEAE-cellulose chromatography. Complete reduction of the enzyme with a 50-fold excess of β-mercaptoethanol in the presence of 0.15% SDS at 96°C for 2 h yielded 4.6–4.9 SH groups per 19 000 daltons, as determined by titration with 5,5′-dithiobis-2-nitrobenzoic acid. Complete carboxamidomethylation accomplished by reaction of reduced enzyme with iodoacetamide in 6 M guanidine hydrochloride at pH 6.0 led to drastic changes in the structure of the enzyme as indicated by a markedly diminished solubility in water, alterations in the column chromatographic behavior in Sephadex G-75 columns (decreased KD), diminished capacity to react immunochemically with antibodies against the native enzyme and marked alteration in the fiuorescence properties. Determination of tryptophan by two different methods indicated that carboxamidomethylation does not alter the tryptophan content of the enzyme. It is concluded that the integrity of two disulfide bonds is essential for the tertiary structure of the native enzyme and that their disruption leads to the observed changes in immunochemical and physicochemical properties. It is also postulated that the amino acid residues constituting the antigenic determinants are not closely localized in the primary structure but that these come together by virtue of the conformation of the native enzyme.

(Na+ + K+)ATPase inhibition by PALYTHOA extracts: Chemical nature of the inhibitor and kinetics of inhibition

Abstract Crude toxic extracts obtained by ethanol extraction from the coelenterate Palythoa caribaeorum were shown to possess strong (Na+ + K+)ATPase inhibitory activity on enzyme preparations from the electroplax of Etectrophorus electricus. The toxic and inhibitory effects were foun to be separable. Chromatographie, spectrophotofluorimetric, electrophoretic and biological data demonstrate that the inhibitor is serotonin. It is a non-competitive inhibitor for Na+ and ATP but is a competitive inhibitor for K+. In enzyme preparations of a specific activity of 1.5 μM Pi/min, I50 is of the order of 1 mM.

Schistosoma mansoni. I. Chemical composition of eggs.

Abstract Eggs of Schistosoma mansoni have been isolated from tissues of infected mice by digestion with the proteolytic enzyme pinguinain. The yield of eggs has been high enough to permit chemical characterization. These have been analyzed for carbohydrate, lipid, and protein contents after sonic disintegration. The water-soluble fraction of the sonicate contains a lipid-rich lipoprotein fraction which appears to be the only protein detectable in significant amounts. Glucose seems to be the most abundant hexose, while esterified cholesterol and neutral fats are the most abundant among the lipid entities.

Schistosome Ova: Rapid Separation from Mouse Tissue by Digestion with Pinguinain.

High yields of ova of Schistosoma mansoni were obtained from the livers and intestines of infected mice by digestion of the homogenized tissue with the proteolytic enzyme pinguinain.

Immunochemical studies on pinguinain, a sulfhydryl plant protease

Abstract Pinguinain, a sulfhydryl plant protease was purified by Sephadex G-100 gel filtration, tested for homogeneity by polyacrylamide gel electrophoresis, and tested immunochemically for structural similarities with papain and fruit bromelain. This study demonstrated appreciable structural similarities with fruit bromelain but no gross similarity to papain. Chemical modification of the sulfhydryl groups by 5,5′ dithio-bis-2-nitrobenzoic acid and N -ethyl maleimide markedly diminish its enzymatic activity but do not alter appreciably its immunochemical behavior. Combination of antibodies with the native enzyme does not abolish its enzymic activity on the low molecular weight substrates benzoyl arginine ethyl ester and benzoyl arginine amide but lowers its enzymatic activity on human serum albumin. From this data we have concluded that the active site is structurally distinct from the antigenic determinant sites.

Human Lipoproteins: Role in Transport of Thyroid Hormones

Lipoproteins from patients receiving therapeutic doses of iodine-131 were isolated by density-gradient techniques. The binding of circulating thyroid hormones by beta lipoproteins (those of low density) was negligible. Alpha lipoproteins (those of high density) bound appreciable amounts. The bulk, however, was bound by proteins of density higher than 1.23 g/ml.

Circumoval Precipitins: Localization by Gel Filtration in Serum of Humans infected with S. mansoni.

Summary The gel filtration technique was used to fractionate samples of sera from 6 individuals infected with S. mansoni. The gel used, Sephadex G-200, allows the separation of serum into 3 fractions. The individuals studied showed a marked increase in the 7 S globulins. The specific antibody which was present in the serum of these individuals and which has been found to react with the S. mansoni ova yielding a positive circumoval precipitin test was found to be a normal size gamma globulin with a sedimentation coefficient of 6.6 S and a molecular weight close to 200,000. Partial enzymatic hydrolysis of this protein was found to alter its immunologic properties, rendering the circumoval test negative after incubation with reactive ova, indicating that the chemical integrity of this protein is needed to yield a positive circumoval reaction.

Effect of (-) delta 9 THC on ATPase systems from various sources.

Chromatographically pure Δ9-tetrahydrocannabinol (Δ9THC) and other cannabinoids were tested for inhibitory activity on sodium and potassium stimulated ATPase (ATP phosphohydrolase, EC 3.6.1.3) from various sources. We found out that in addition to inhibiting Na+ and K+ dependent ATPases, Mg+ + ATPases were inhibited as well. Rat brain ATPases were much more sensitive to Δ9THC than to equal concentrations of ouabain. Inhibition by Δ9THC was shown to occur both in situ and in vitro. In vitro, an I50 of 3 × 10-6 M was calculated for enzyme preparations of specific activity of 1,200–1,800 nmoles Pi/min/mg protein. The drug was found to bind to the enzyme and to cause a marked increase in Km for Na+, but no change in the Km for K+. These results suggest that Δ9THC inhibits (Na + K) ATPases by interfering with the phosphorylation in the sequence of reactions leading to ATP hydrolysis. Phosphatidyl ethanolamine was found to reverse the inhibition caused by Δ9THC in these enzyme preparations. In Ehrlich ascites tumor cells (107 cells/flask) 60 μM Δ9THC inhibited nucleoside ([3H] thymidine) incorporation without exerting any significant effect on the viability of cells as measured by O2 consumption. Particulate ATPases isolated from cells exposed to Δ9THC had significantly lower activity than cells in control media, 123 ± 36 vs. 172 ± 16 nmoles Pi/min/mg protein (p < 0.005, n = 16). Thus a correlation seems to exist between ATPase inhibition and nucleoside uptake. A thorough study of the effect of cannabinoids on membrane bound ATPases deserves to be undertaken.

Binding of [3H]phenycyclidine to rat and human blood constituents.

The binding of [3H]phencyclidine (PCP) to rat serum and human plasma was studied using equilibrium dialysis. [3H]PCP bound with a relatively low affinity to both rat serum (KD = 1.5 X 10(-5) M) and human plasma (KD = 6.2 X 10(-6) M). However, the binding capacity was quite large for rat serum (5.7 nmoles/ml) and human plasma (5.6 nmoles/ml). Binding was readily reversible as shown by the efflux of [3H]PCP from a dialysis bag containing the rat serum-drug complex. In addition, the [3H]PCP-human serum complex appeared to dissociate completely when analyzed by Sephadex gel filtration chromatography. The low affinity of PCP for serum appeared to account in large part for the high tissue-to-plasma ratios that are observed in animals and humans injected with this drug. In vitro equilibration of [3H]PCP between rat serum and tissue homogenates resulted in at least a 10-fold accumulation of [3H]PCP in the homogenates. [3H]PCP was found to bind weakly to the major protein components of human serum (macroglobulins, immunoglobulins and albumins). The weak nature of the binding to serum proteins coupled with the relatively high capacity of binding probably account for the failure of other drugs to compete for PCP binding.

Effect of (-)-delta 9 tetrahydrocannabinol on nucleoside and amino acid uptake in Reuber-H-35 hepatoma cells.

(-)-Δ9-tetrahydrocannabinol (Δ9THC) was found to be a noncompetitive inhibitor for the uptake of thymidine into TCA soluble material by Reuber H-35 hepatoma cells. It also reduced uptake rates of adenosine, guanosine and cytidine. A 50% inhibition of thymidine uptake was produced with Δ9THC concentrations of 70 µM while cytidine uptake was inhibited to the same extent at 30 µM Δ9THC. The drug had no significant effect on the uptake of uridine, leucine and proline. Intracellular thymidine-containing nucleotide pools, isolated by thin layer chromatography after pulse labeling of the Δ9THC treated cells with 3H thymidine, were significantly diminished. Concentrations of 70 µM Δ9THC reduced radioactive dTMP, dTDP and dTTP levels to 50% of those of the control cells. As thymidine kinase activity was not inhibited by Δ9THC, it is concluded that Δ9THC inhibits thymidine uptake at some step prior to formation of dTMP. ACKNOWLEDGMENTS We thank Professor L. S. Harris, Department of Pharmacology, Medical College of Virginia for his gift of the Δ9THC, Mrs. Esther Torres for clerical help and Mr. Juan Coloca for the photographic work.