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P Walde

Publications and source records attributed to P Walde.

28 records · Page 2Linked to original sources
PubMed1989

Differential effects of liposome-entrapped desferrioxamine on proliferation and erythroid differentiation of murine erythroleukemic Friend cells.

It is known that iron chelators (such as desferrioxamine) are potent inhibitors of both cell proliferation and erythroid differentiation. We have shown with in vitro studies that in the case of tumor cells desferrioxamine is even more efficient in inhibiting cell proliferation when entrapped in liposomes consisting of egg yolk phosphatidylcholine. At the same time liposome-entrapped desferrioxamine retains only a slight effect on hexamethylenebisacetamide induction of erythroid differentiation and hemoglobin accumulation of murine erythroleukemic Friend cells. Based on these findings, we propose liposome-entrapped desferrioxamine as potential antineoplastic agent as well as a specific chemical for the study of both iron metabolism and distribution in normal and neoplastic cells. In addition, unlike free desferrioxamine, the liposome-entrapped drug could also be used in combination with inducers of differentiation. With respect to this issue, it is possible that liposome-entrapped desferrioxamine, might permit erythroid differentiation of both neoplastic cells as well as normal stem cells.

Animals↗
PubMed1988

Structure and activity of trypsin in reverse micelles.

The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in chloroform/isooctane (1:1, by vol.). Three substrates have been used, namely N alpha-benzoyl-L-Arg ethyl ester, N alpha-benzoyl-L-Phe-L-Val-L-Arg p-nitroanilide (BzPheValArg-NH-Np) in AOT and N alpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester (ZLysO-Np) in CTAB. One of the main aims of the work was to compare the behaviour of trypsin in reverse micelles with that of alpha-chymotrypsin, for which an enhancement of kcat had been observed with respect to aqueous solutions. The pH profile is not significantly altered in reverse micelles with respect to water, however the kinetic parameters (kcat and Km) differ widely from one another, and are markedly affected by the micellar conditions, in particular by the water content wo (wo = [H2O]/[AOT]). Whereas in the case of BzPheValArg-NH-Np kcat is much smaller than in water, in the case of ZLysO-Np at pH 3.2 (but not at pH 6.0) a slight enhancement with respect to water is observed. On the basis of rapid kinetic spectrophotometry (stopped-flow) and solvent isotope effect studies, this enhancement is ascribed to a change in the rate-limiting step (acylation rather than hydrolysis). As in the case of alpha-chymotrypsin, the maximal activity is found for all substrates at rather small wo values (below 12), which is taken to suggest that the enzyme works better when is surrounded by only a few layers of tightly bound water. Spectroscopic studies [ultraviolet absorption, circular dichroism (CD) and fluorescence] have been carried out as a function of wo. Whereas the absorption properties are practically unchanged, the CD spectrum in AOT micelles has a lower intensity than in water, which is interpreted as a partial unfolding. The intensity is partly restored when Ca2+ ions are added, indicating that the micellar environment may cause a partial denaturation by depleting it of calcium ions. Fluorescence data show that the emission properties of the protein in reverse micelles match those in aqueous solution at around wo = 13 approx., whereas lambda max shifts towards the red by increasing wo, indicating an exposure of the tryptophan residues and probably an unfolding of the whole protein, at wo values above 15. Finally the reaction between trypsin and its specific macromolecular Kunitz inhibitor from soybeans is studied.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium Chloride↗
PubMed1987

The mechanism of liposomal damage by taurocholate.

The stability of small unilamellar vesicles formed by egg-yolk phosphatidylcholine (PC) has been examined in the presence of sodium taurocholate. The permeability of the vesicular membrane changes as the total taurocholate concentration increases, until a transformation from mixed bile salt/PC vesicles to mixed micelles occurs. Based on experiments in which the bile salt-induced release of either hydrophilic (carboxyfluorescein) or hydrophobic (Bromothymol blue) probes was studied, and on fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene and turbidity measurements, a two-step process for the initial stage of liposomal damage by taurocholate is postulated.

Bile Acids and Salts↗
PubMed1987

Activity of bile-salt-stimulated human milk lipase in the presence of liposomes and mixed taurocholate-phosphatidylcholine micelles.

(1) The interaction of bile-salt-stimulated human milk lipase and liposomal membranes has been investigated in the presence or absence of sodium taurocholate. Freshly purified enzyme enhances the permeability of liposomal membranes but thermally inactivated enzyme does not. (2) The ability of the enzyme to catalyze the hydrolysis of a relatively hydrophilic substrate, 4-nitrophenyl acetate, and a more hydrophobic substrate, 4-nitrophenyl palmitate, has also been measured in media containing small unilamellar vesicles of egg phosphatidylcholine in both the absence and presence of taurocholate, and also in the presence of free taurocholate in the absence of liposomes. (3) The enzyme-catalyzed hydrolysis of 4-nitrophenyl acetate is enhanced in all of these systems, but 4-nitrophenyl palmitate is protected from enzymic attack in the phosphatidylcholine-bile salt systems. If free taurocholate be present in the system before 4-nitrophenyl palmitate is added, then, and only then, is enzymic activity observed. (4) These results have been interpreted in terms of the importance of the microenvironment around the substrate and the role played by the bile salt surfactant in stimulating the enzyme.

Bile Acids and Salts↗
PubMedDate not supplied

Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes.

The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.

Cell Membrane Permeability↗
PubMedDate not supplied

Bile salt roles in bile-salt-stimulated lipase activity.

The hydrolysis of 4-nitrophenylacetate and phenylsalicylate, catalyzed by human milk lipase in the presence of a range of concentrations of sodium cholate, has been measured at pH 7.3 and 37.5 degrees C, and maximum activity was observed for both substrates at 1 mmole/dm-3 bile salt. Lineweaver-Burk plots for the enzyme-catalyzed hydrolysis of N-methylindoxyl myristate and 4-nitrophenyloctanoate yielded values of Km equal to 34 and 20 mumoles/dm-3, respectively. However, an increase in the concentration of the latter substrate beyond 10 mumoles/dm-3 was not accompanied by a corresponding increase in the rate of hydrolysis. Comparison of these hydrolysis data with literature data for a variety of hydrophobic substrates suggests that there are two roles for the bile salt in the enzyme-catalyzed reaction--the first involving binding onto several sites on the enzyme, and the second solubilization of oil-phase substrates.

Bile Acids and Salts↗
PubMedDate not supplied

Inactivation of bile-salt-stimulated human milk esterase: effect of storage and heat.

The activity of bile-salt-stimulated esterase was monitored in human milk during storage at room temperature, in a refrigerator (4 degrees C), in a freezer (-19 degrees C), and in milk that had been freeze dried and rehydrated after storage of the lypophilized milk at 4 degrees C and -19 degrees C. In all cases, there was some loss of activity, independent of measurement in the presence or absence of sodium taurocholate, and the loss was highest in milk stored at room temperature, approximating a 50% loss in 1-2 days. Activation and inactivation of solutions of the purified enzyme have been identified by measuring its esterase activity against a variety of 4-nitrophenylalkanoate esters at pH 7.3 within the temperature range 5-46.5 degrees C. The optimum temperature was 42 degrees C. The presence of bile salt partially serves to maintain the conformation of the bile-salt-binding site as the esterolytic binding site becomes inactivated at 50 degrees C. Bile salts thus exert a positive template effect on the enzyme.

Bile Acids and Salts↗