Role of lysines in mediating interaction of modified low density lipoproteins with the scavenger receptor of human monocyte macrophages. (49/78)

The ability of the scavenger receptor of human monocyte macrophages to recognize human low density lipoproteins (LDL) progressively modified by three lysine-specific reagents, malondialdehyde, acetic anhydride, or succinic anhydride, has been investigated. Regardless of the reagent utilized, receptor-mediated uptake was dependent upon modification of greater than 16% of the peptidyl lysines rather than upon the net negative charge of derivatized LDL. Rates of lysosomal hydrolysis of acetyl-LDL and succinyl-LDL increased as a function of progressive modification and reflected the amount of derivatized LDL binding to the receptor. Succinylation or acetylation of greater than 60% of the lysines was necessary to attain maximal ligand binding, internalization, and degradation. In contrast, modification of only 16% of the peptidyl lysines by malondialdehyde resulted in maximal levels of binding, uptake, and hydrolysis. The expression of receptor recognition site(s) appears to depend upon the charge modification of critical lysine residues of the LDL protein rather than the net negative charge of the lipoprotein complex. Malondialdehyde, a bifunctional reactant, may modify surface and sequestered lysines concomitantly and thus promote efficient formation of the recognition site(s).  (+info)

Structure-function relationships of sea anemone toxin II from Anemonia sulcata. (50/78)

Chemical modifications of sea anemone toxin II from Anemonia sulcata have been used to study the residues involved in its toxic action on crabs and mice and in its binding properties to the Na+ channel of rat brain synaptosomes. Guanidination of th epsilon-amino groups of lysines 35, 36, and 46 with O-methylisourea hydrogen sulfate did not change the net charge of the toxin molecule and had no effect upon its toxic and binding properties. Either acetylation or fluorescamine treatment of the toxin that destroyed the positive charges of the three epsilon-amino groups and of the alpha-amino function of Gly produced an almost complete loss of toxicity and a considerable decrease in the binding activity. Iodination of the toxin on His induced practically no loss of toxic or binding properties. Carbethoxylation of both histidines 32 and 37 with diethyl pyrocarbonate provoked an important decrease of both the toxicity and the binding activity. Modifications of the guanidine side chain of Arg with 1,2-cyclohexanedione fully destroyed both toxicity and binding of the toxin to the Na+ channel. Modification of the carboxylate functions of Asp, Asp, and of the COOH-terminal Gln with glycine ethyl ester in the presence of a soluble carbodiimide completely abolished the toxicity but left the affinity for the sea anemone toxin receptor unchanged. The antagonist character of this carboxylate-modified derivative was further confirmed by electrophysiological and Na+ flux experiments. The theoretical and practical significance of these results are discussed.  (+info)

Surface accessibility of 13C-labeled lysine residues in membrane-bound myelin basic protein. (51/78)

Surface-exposed regions of membrane-bound myelin basic protein--the major extrinsic membrane protein of central nervous system myelin--have been implicated as possible antigenic sites in diseased myelin. With the goal of determining the extent and nature of these regions, we have prepared basic protein modified with 13CH3-enriched acetyl groups at 7 of its 13 lysine residues. The resulting protein was placed in a membrane environment and studied by NMR spectroscopy to determine the location and rates of molecular motion of the labeled side chains with respect to lipid bilayers of the membrane. When 13C NMR spectra were obtained of the acetylated protein bound to multilamellar vesicles prepared from dimyristoylphosphatidic acid in the gel state (T = 33 degrees C), conditions under which reduced motion in the lipid bilayer broadens methylene and methyl 13C resonances of the membrane beyond detection (i.e. greater than 75-100 Hz), line widths of membrane-bound protein were measured to be 7.8 Hz, an increase of 4 Hz versus free protein. A reduction of 25-30% in integrated intensity observed in protein acetyl resonances upon membrane interaction was shown to be attributable to a population of protein-aggregated liposomes whose resonances were similarly too broad to be observed. Thus, the epsilon-acetyllysyl probes distributed throughout the protein do not penetrate the dimyristoylphosphatidic acid bilayer, but must reside in the interstitial aqueous spaces at or between membrane surfaces. These findings suggest an overall surface accessibility of membrane-bound myelin basic protein and are therefore incompatible with a model for the protein involving membrane-embedded loops or regions of functional significance.  (+info)

The conformation of cytochrome c in solution. Localization of a conformational difference between ferri- and ferrocytochrome c on the surface of the molecule. (52/78)

The conformation of cytochrome c in solution is believed to change depending on the oxidation-reduction state of the heme iron, since ferri- and ferrocytochrome c exhibit several different physicochemical properties, but so far it is unknown if the conformational difference(s) is (are) confined to a particular part or domain of the molecule. We have therefore applied the method of differential chemical modification (Bosshard, H. R. (1979) Methods Biochem. Anal. 25, 273-304) to compare the chemical reactivity toward acetic anhydride of the 19 lysine residues of ferri- and ferrocytochrome c from horse heart. The epsilon-amino groups of the spatially related residues 39, 53, and 55 were significantly less reactive in ferrocytochrome c as compared to their reactivity in ferricytochrome c. The difference of reactivity was pH-dependent and was shown to be due to an increase of the pK values of the three epsilon-amino groups in ferrocytochrome c. These results indicate a local conformational change on the surface of the cytochrome c molecule in an area to the lower left of and below the heme cleft (standard front view of the molecule facing the exposed edge of the prosthetic group). The local conformational change might be instrumental in the cytochrome c-mediated electron shuttle between cytochrome c1 and a in the final segment of the mitochondrial electron transport chain.  (+info)

A rapid micro-assay method for gelatinolytic activity using tritium-labeled heat-denatured polymeric collagen as a substrate and its application to the detection of enzymes involved in collagen metabolism. (53/78)

A rapid micro-assay method for gelatinolytic activitiy has been developed using 3H-labeled heat-denatured polymeric collagen (gelatin) as a substrate to investigate enzymes involved in the post-collagenase catabolism of collagen. The method is based on the incubation of gelatin with enzyme followed by determination of the enzyme digestion products soluble in 67% dioxane. It is sensitive enough to detect microgram levels of gelatin fragments, and can be employed over wide ranges of pH and ionic strength. By applying the method to an embryonic chick skin culture system, three gelatinolytic enzyme fractions which showed high, limited and no caseinolytic activities were demonstrated to be separable by gel chromatography.  (+info)

Permeability properties of chemically modified porin trimers from Escherichia coli B. (54/78)

The pore-forming protein of the outer membrane of Escherichia coli, porin, was chemically modified with acetic anhydride, succinic anhydride, and glycinamide. Extensive modification of amino groups of the functional porin trimers caused reduced diffusion rates of the negatively charged solutes such as p-nitrophenyl phosphate and AMP, but did not reduce significantly the diffusion of positively charged molecules carbobenzoxy-glycyl-prolyl-arginine-p-nitranilide and tosyl-glycyl-prolyl-arginine-p-nitranilide. Modification of carboxyl groups of trimers caused decreased diffusion rates of the positively charged solutes more significantly than the diffusion rates of negatively charged solutes. The results suggest that the ionic interactions play an important role for the diffusion of charged solutes through the porin pore. The diffusion of p-nitrophenyl alpha-D-glucoside, an uncharged solute, ws not influenced significantly by modification of either amino or carboxyl groups. This observation suggests that modifications only occurred in areas outside of the narrowest portion of the pore or, alternatively, that amino and carboxyl groups are exclusively located at noncylindrical area of the pore. The structural integrity of the acetylated and the succinylated trimers seemed well preserved. On the other hand, modification of carboxyl groups decreased the thermal stability of trimers and extensive modifications caused the dissociation of trimers into monomers at 37 degrees C.  (+info)

Fate of vaccines of Propionibacterium acnes after phagocytosis by murine macrophages. (55/78)

Stationary-phase (48 h) cells of Propionibacterium acnes VPI 0009, a potent stimulator of the reticuloendothelial system, persist unchanged within phagocytes for at least 24 h after ingestion. In contrast, exponential-phase (12 h) cells of the same strain (which do not induce splenomegaly) are extensively degraded within 5 h of phagocytosis. Suspensions of P. granulosum VPI 6500, which fails to induce splenomegaly in mice, also show considerable degradation after phagocytosis. Stationary-phase cells of strain VPI 0009 treated with sodium metaperiodate or with trichloroacetic acid, although without ability to induce splenomegaly, resist destruction almost as well as untreated vaccines. However, bacteria inactivated by acetic anhydride show about 50% breakdown in 24 h.  (+info)

Chemical reactivity of the functional groups of insulin. Concentration-dependence studies. (56/78)

A modification to the competitive labelling procedure of Duggleby and Kaplan [(1975) Biochemistry 14, 5168-5175] was used to study the reactivity of the N-termini, lysine, histidine and tyrosine groups of insulin over the concentration range 1 X 10(-3)-1 X 10(-7)M. Reactions were carried out with acetic anhydride and 1-fluoro-2,4-dinitrobenzene in 0.1 M-KCl at 37 degrees C using Pyrex glass, Tefzel and polystyrene reaction vessels. At high concentrations all groups had either normal or enhanced reactivity but at high dilution the reactivities of all functional groups became negligible. This behaviour is attributed to the adsorption of insulin to the reaction vessels. The histidine residues show a large decrease in reactivity in all reaction vessels in the concentration range 1 X 10(-3)-1 X 10(-5)M where there are no adsorption effects and where the reactivities of all other functional groups are independent of concentration. With polystyrene, where adsorption effects become significant only below 1 X 10(-6)M, the reactivity of the phenylalanine N-terminus also shows a decrease in reactivity between 1 X 10(-5) and 1 X 10(-6)M. In 1 M-KCl insulin does not absorb to Pyrex glass and under these conditions the histidine reactivity is concentration-dependent from 1 X 10(-3) to 5 X 10(-6)M and the B1 phenylalanine alpha-amino and the B29 lysine epsilon-amino reactivities from 5 X 10(-6) to 1 X 10(-7)M, whereas the reactivities of all other groups are constant. These alterations in reactivity on dilution are attributed to disruption of dimer-dimer interactions for histidine and to monomer-monomer interactions for the phenylalanine and lysine amino groups. It is concluded that the monomeric unit of insulin has essentially the same conformation in its free and associated states.  (+info)