It is well accepted that papain and cathepsin B work by stabilizing the transition state of the substrate formed during catalysis. This species carries a formal negative charge which, in the serine proteases family, is stabilized by an oxyanion hole formed by the enzyme. The same structure has been proposed to exist for cysteine proteases. Because the side chain of a Gln residue contributes one stabilizing hydrogen bond to the transition state in the oxyanion hole of papain and cathepsin B, site directed mutagenesis was used in this work to change this residue to Ala and Ser. It was found that this Gln contributes between 2.5 and 3.7 kcal/mol to the transition state stabilization in papain and 3.7 kcal/mol for cathepsin B. The pH profile of cathepsin B is also shifted to higher pH indicating a destabilization of the catalytic ion-pair caused by the mutation. These results demonstrate that the oxyanion hole plays an active role in catalysis by cysteine proteases. (Abstract shortened by UMI.)

<p> The objective of this study is to develop a &ldquo;nanovehicle&rdquo; to transport resveratrol in the plasma and to targeted intracellular sites. Resveratrol is a bioflavonoid possessing a variety of biological activities. We incorporated resveratrol into reconstituted high-density lipoproteins (rHDL), which are water-soluble lipid/protein complexes. rHDL was prepared using apolipoprotein E3 (apoE3) N-terminal domain and phospholipids. The role of apoE3 NT domain is to mediate cellular uptake of lipoproteins via the low-density lipoprotein receptor (LDLr). Biochemical, biophysical and functional studies using glioblastoma cells indicate that (i) resveratrol has partitioned into the hydrophobic milieu of rHDL, and is shielded from the aqueous environment, and, (ii) the presence of resveratrol does not alter the overall structural integrity of rHDL and the ability of LDLr to mediate cellular uptake of rHDL. Our results suggest that rHDL containing apoE3 can serve as an effective &ldquo;nanovehicle&rdquo; to transport and potentially deliver resveratrol to targeted intracellular sites.</p>

No description available.

It was shown, previously that N. crassa contains an endo-exonuclease which exist in two forms, an active form and an inactive form activated by trypsin treatment in vitro. Both of these forms were found in the cytosol and in mitochondria, but only the active form is present in the vacuoles. In the present work, it has been shown that both forms are also present in the nuclei bound to the chromatin. / Active endo-exonuclease was previously implicated in DNA repair and in this study additional evidence was obtained in further support of such a role: (i) the mutagen sensitive uvs-3 mutant of N. crassa was found to contain only 10% of the level of active enzyme in the two DNA-containing organelles, in comparison to the levels found in the wild-type, (ii) in response to low doses of the DNA-damaging agent 4-nitroquinoline 1-oxide (4-NQO), the inactive enzyme decrease and the active form increase in the DNA-containing organelles indicating that the inactive enzyme may have been converted proteolytically to the active form. This response did not occur in the uvs-3 mutant. / Under a different stress, namely heat shock, endo-exonuclease was regulated differently in the DNA-containing organelles. Active enzyme was released from the nuclei, mitochondria, and vacuoles into the cytosol where it was nearly completely inhibited by a specific inhibitor induced by heat shock. This heat shock-induced inhibitor shared some properties in common with a constitutive endo-exonuclease inhibitor recently isolated from this laboratory. However, there were also some distinct differences between these two inhibitors. At least one difference may be explained by phosphorylation of the heat shock inhibitor.

The mechanism of protein translocation into mitochondria has been investigated by studying properties of precursor proteins destined for the mitochondrial matrix. Characterization of the amphiphilic properties of the signal sequence for pre-ornithine carbamyltransferase has led to the conclusion that precursors can not translocate across the inner membrane via a lipid route alone (i.e. in the absence of proteins). A correlation was established between the rate of precursor import and the degree of hydrophobicity of a short region in the presequence, suggesting that precursor binding to the two-dimensional phospholipid surface of the outer membrane may enhance the rate of diffusion to the translocation apparatus. / The conformations of the mature portions of two hybrid proteins, pOCAT and pODHFR, were examined at various steps on the import pathway. The bulk population of these precursors remained in a near-native conformation prior to precursor engagement of the import apparatus. Unfolded polypeptide translocation intermediates, the formation of which requires ATP, an intact signal sequence, and a protease-sensitive component of the outer mitochondrial membrane, have been detected in association with submitochondrial fractions containing sites of contact between the inner and outer mitochondrial membranes.

The binding of selected bile acids and bile pigments by peptides and quaternary amines has been studied by adsorption and NMR experiments. Novel adsorbents with quaternized peptide-containing functional groups for bile acids have been prepared by solid phase peptide synthesis techniques. The adsorption studies, conducted in aqueous buffer solutions, show that these resins have an enhanced capacity, on a per active site basis, and improved specificity over cholestyramine and colestipol. The interaction between bile acid anions and the pendants is predominantly ionic linkage, although hydrophobic and other interactions are also important. An NMR study of the binding between bile acids and various ligands, including peptides, by the determination of carbon-13 spin-lattice relaxation times, confirms the ionic and hydrophobic interactions which occur cooperatively and simultaneously. / New adsorbents for bilirubin have been prepared by covalently coating a water-swellable polyamide resin with polypeptides. These resins have much higher capacities for bilirubin in aqueous buffer solution than cholestyramine and improved capacities over the resins with attached oligopeptide pendants. The binding behavior of the resin coated with poly- sc D-lysine is the same as that of poly- sc L-lysine. The amount of bilirubin adsorbed by these resins is directly proportional to the number of lysine residues on the resin, which is consistent with the formation of an ionic linkage. This is confirmed by a study of the interaction of bilirubin with an oligopeptide, sc L-lysyl- sc L-lysine, by measurements of proton and carbon-13 NMR spin-lattice relaxation times combined with nitrogen-15 NMR experiments. The $ sp{15}$N NMR spectra of bilirubin and some related bile pigments have also been assigned by two-dimensional $ sp{15}$N-$ sp1$H heteronuclear correlation experiments.

Light emission in marine bacteria is under the control of growth dependent induction. Associated with the induction is the increase in synthesis of the components of the two enzymes which catalyze the light emitting reaction, luciferase and fatty acid reductase. Large DNA fragments from three marine bacteria Vibrio fischeri, Vibrio harveyi and Photobacterium phosphoreum, were isolated from genomic libraries of these strains. Escherichia coli, harboring a recombinant plasmid containing a 16 kbp fragment of V. fischeri DNA, were found to express growth dependent luminescence. E. coli clones harboring analogous sequences of V. harveyi or P. phosphoreum lux DNA only expressed a low constitutive level of light which was dependent on the presence of aldehyde in the growth media. Cell extracts of E. coli expressing the lux genes from V. fischeri contained a fatty acid reductase activity analogous to the activity first identified in P. phosphoreum. This result showed that the genes for luciferase and fatty acid reductase were linked inV. fischeri. By differential acylation of the fatty acid reductase subunits in extracts of V. fischeri and the E. coli clone, the products of the luxC, luxD and luxE genes of V. fischeri were identified as the reductase, transferase and synthetase components, respectively. In addition, the lux genes from a fatty acid stimulatible dark mutant of V. harveyi were cloned and it was demonstrated that the luxD gene from this mutant synthesized a non-functional transferase polypeptide. / The lux structural genes are coordinately expressed to different levels during induction of luminescence. The lux polypeptides of V. fischeri were shown to be differentially synthesized when expressed under the control of a T7 promoter in E. coli. This result indicated that postranscriptional events were regulating the differential expression of the lux genes. A set of overlapping transcripts encoding the lux genes were shown to be induced during luminescence induction, suggesting that segmental differences in mRNA stability may be related to differential expression of the lux genes. / The luxA and luxB genes of V. harveyi encoding the alpha and beta subunits of luciferase, respectively, were fused by site-directed mutagenesis. This fused gene synthesized a monocistronic luxAB mRNA capable of programming the synthesis of a functional single subunit luciferase enzyme in E. coli, in a rabbit reticulocyte lysate and in Saccharomyces cerevisiae. This fused gene has potential applicability for studying gene expression in both prokaryotic and eukaryotic systems.

No description available.

No description available.

Diverse eukaryotic proteins require the post-translational addition of S-acyl chains to cysteine residues for proper function, a process known as S-palmitoylation, or S-acylation. To study the effects of this lipid moiety, various complex methods have been developed for the preparation of synthetic lipopeptides. In order to facilitate this task, a novel technique employing readily-prepared long-chain acyl thioesters has been devised. Using S-phenylmercapto-palmitoyl thioester as well as other acyl thioesters, the fluorescent-labeled peptide, myristoyl-GCG-caBim, was S-acylated to high stoichiometry at halftimes as short as 20 min. (initial rate of S-acylation of 179.8 +/- 24.7%/hr) in homogeneous solution, without the presence of micelles or vesicles. The chemical reaction occurred regioselectively on cysteine side-chains without modification of serine or lysine derivatives of the peptide. This method was also utilized to selectively S-acylate the fully deprotected Po peptide, IRYCWLRR-NH2. Such an innovative technique should provide a useful scheme for the general synthesis of S-acylated peptides.