An analog of the human albumin N-terminus (Asp-Ala-His-Lys) prevents formation of copper-induced reactive oxygen species.

Copper mobilization and redox activity form damaging reactive oxygen species (ROS) and are implicated in the pathogenesis of ischemia-reperfusion injury, chronic inflammation, Alzheimer's disease, aging, and cancer. Protein sequestration of Cu(II) ions has been shown to prevent ROS-generating reactions. The first four amino acids of the N-terminus of human albumin, Asp-Ala-His-Lys (DAHK), form a tight binding site for Cu(II) ions. We synthesized several analogs, including the enantiomer d-DAHK, to study their effects on copper-induced hydroxyl radical and superoxide formation in the presence of ascorbate. d-DAHK prevented thiobarbituric acid-reactive species (TBARS) formation within physiological and acidic pH ranges (7.5-6.5) and inhibited low-density lipoprotein lipid peroxidation. A d-DAHK/Cu complex exhibited superoxide dismutase-like activity by significantly inhibiting superoxide formation. These in vitro results suggest that d-DAHK may shift the Cu(II)-binding equilibrium from the exchangeable Cu(II) pool to the tightly-bound, nonexchangeable pool, prevent ROS formation, and potentially provide therapeutic benefit for ROS-related diseases.

Asp-Ala-His-Lys (DAHK) inhibits copper-induced oxidative DNA double strand breaks and telomere shortening.

Both DNA and the telomeric sequence are susceptible to copper-mediated reactive oxygen species (ROS) damage, particularly damage attributed to hydroxyl radicals. In this study, ROS-induced DNA double strand breaks and telomere shortening were produced by exposure to copper and ascorbic acid. Asp-Ala-His-Lys (DAHK), a specific copper chelating tetrapeptide d-analog of the N-terminus of human albumin, attenuated DNA strand breaks in a dose dependent manner. d-DAHK, at a ratio of 4:1 (d-DAHKCu), provided complete protection of isolated DNA from double strand breaks and, at a ratio of 2:1 (d-DAHKCu), completely protected DNA in Raji cells exposed to copper/ascorbate. Southern blots of DNA treated with copper/ascorbate showed severe depletion and shortening of telomeres and Raji cell treated samples showed some conservation of telomere sequences. d-DAHK provided complete telomere length protection at a ratio of 2:1 (d-DAHKCu). The human albumin N-terminus analog, d-DAHK, protects DNA and telomeres against copper-mediated ROS damage and may be a useful therapeutic adjunct in ROS disease processes.

Secretion of glycosylated human erythropoietin from yeast directed by the alpha-factor leader region.

The pre-pro alpha-factor leader region of the yeast MF alpha 1 gene was used to direct the secretion of the human glycoprotein, erythropoietin (EPO), into the culture medium. An examination of the role of expression level on secretion of biologically active EPO indicated that there are several rate-limiting steps. These include processing of the alpha-factor-EPO precursor protein by the KEX2-encoded endoproteinase and transport of the protein through the secretory pathway. The rate-limiting steps for transport were early in the secretory pathway, probably from the endoplasmic reticulum to the Golgi apparatus.

Purification and preliminary characterization of a monomeric glutathione S-transferase from Tetrahymena thermophila.

Cell extracts of the protozoan Tetrahymena thermophila contain high levels of glutathione S-transferase (EC 2.5.1.18). The level of the enzyme varied with the growth cycle, increasing in early stationary phase. It was localized in the cytoplasm. Only one major molecular form of the enzyme was detected by ion-exchange and gel filtration chromatography and isoelectric focusing (pI 7.0), and it was purified to homogeneity. The molecular weight of the enzyme was estimated to be 35,000 from gel filtration and 33,000 from sodium dodecyl sulfate-gel electrophoresis, indicating the enzyme is a monomer. The enzyme was specific for glutathione as the thiol substrate, and was most active with 1-chloro-2,4-dinitrobenzene as the electrophilic substrate. It also exhibited glutathione peroxidase activity. The apparent Km values for glutathione and 1-chloro-2,4-dinitrobenzene were 3.2 and 1.5 mM, respectively. The enzyme catalyzed an S-aryltransfer with pentachloronitrobenzene (Km = 7.2 microM) consistent with the view that this pesticide is metabolized in part via S-conjugation pathways in T. thermophila.

Expression of a biologically active analogue of somatomedin-C/insulin-like growth factor I.

A synthetic gene coding for an analogue of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) was synthesized by solid support phosphoramidite chemistry and subsequently cloned and expressed in Escherichia coli as a fusion protein. The gene, designed with a threonine codon substituted for a methionine codon at position 59 was expressed fused to an eight-amino acid leader peptide under the direction of the E. coli tryptophan promoter. The fusion protein, termed L0-[Thr59]-Sm-C/IGF-I was purified extensively (greater than 97%) and found to be 60% as active as native Sm-C/IGF-I in a radioimmunoassay and 50% as potent as native Sm-C/IGF-I in a radioreceptor assay. Like native Sm-C/IGF-I it was also mitogenic for Balb/c 3T3 cells. After removal of the eight amino acid leader peptide by cyanogen bromide treatment, the resulting threonine analogue, termed [Thr59]-Sm-C/IGF-I was 80% as potent as native Sm-C/IGF-I in both the RIA and the radioreceptor assays. It was also mitogenic in Balb/c 3T3 cells. These two analogues, therefore, display biological activities similar to human-derived Sm-C/IGF-I.

Simplified liquid chromatographic assay for epoxide hydrolase.

A simplified liquid chromatographic assay for epoxide hydrolase (E.C. 3.3.2.3) is described. The enzyme substrate employed, 3-(p-nitrophenoxy)-1,2-propene oxide, is commercially available and is readily resolved by reversed-phase liquid chromatography from the enzymatic hydration product, 3-(p-nitrophenoxy)-1,2-propane diol. As little as 100 pmol of the diol can be detected by absorbance monitoring at 315 nm. This assay has been employed to measure epoxide hydrolase in rat liver microsomes from animals treated with the dietary antioxidant, 2(3)-tert.-butyl-4-hydroxyanisole, a known inducer of the enzyme. The assay can also be used to measure rat liver cytosolic epoxide hydrolase.

Bovine kidney 3-methylcrotonyl-CoA and propionyl-CoA carboxylases: each enzyme contains nonidentical subunits.

3-Methylcrotonyl-CoA carboxylase (MCase; EC 6.4.1.4) and propionyl-CoA carboxylase (PCase; EC 6.4.1.3) have been obtained in highly purified form from bovine kidney mitochondria. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that each enzyme is composed of nonidentical subunits, including a smaller biotin-free subunit (Mr 62,000 and 58,000 for MCase and PCase, respectively), and a larger biotin-containing subunit (Mr 80,000 and 74,000 for MCase and PCase, respectively). The possibility that these subunits were derived from a single, larger precursor polypeptide via proteolysis was explored by purification and electrophoresis of each enzyme in the presence of protease inhibitors, but no evidence for proteolysis was obtained. Specific antisera directed towards each enzyme were prepared. The anti-PCase preparation was used to precipitate crossreacting PCase from a pig heart extract. Analysis of the immunoprecipitate obtained revealed a biotin-containing polypeptide (Mr 78,000) and a biotin-free polypeptide (Mr 55,000), suggesting that pig heart PCase also contains nonidentical subunits analogous to those seen in the kidney mitochondrial MCase and PCase. A bipartite subunit structure may be a common feature in mammalian MCase and PCase.

Biodegradation of acyclic isoprenoids by Pseudomonas species.

The ability of various pseudomonads to utilize acyclic isoprenoids as a sole carbon source was investigated. Tests for utilization of acyclic isoprenols such as citronellol and geraniol were complicated by toxic effects of these alcohols, and most species tested were killed by exposure to citronellol or geraniol (0.1%, vol/vol) in liquid culture. In the case of Pseudomonas citronellolis, sensitivity to isoprenols is reduced by prior induction of the isoprenoid degradative pathway via either growth on succinate in the presence of citronellol or growth on citronellic acid. For this species, citronellic acid proved to be the best isoprenoid growth substrate tested. Geraniol utilization as a taxonomic indicator for different subgroups of pseudomonads is discussed. Only a few of the species tested were able to utilize acyclic isoprenoids. Two species which utilize C10 acyclic isoprenoids, P. aeruginosa and P. mendocina, were shown to contain the inducible enzyme geranyl-coenzyme A carboxylase, one of the unique enzymes in the isoprenol degradative pathway known to occur in P. citronellolis. Of the species which utilized geranitol, none showed definite growth on the homologous C15 and C20 isoprenols.

Photoaffinity labeling of acyl-coenzyme A:glycine N-acyltransferase with p-azidobenzoyl-coenzyme A.

A photolabile reagent, p-azidobenzoyl-CoA, has been synthesized and tested as a photoaffinity label for acyl-CoA:glycine N-acyltransferase (EC 2.3.1.13) from beef liver. p-Azidobenzoyl-CoA is an active-site-directed reagent for this N-acyltransferase, since it is an alternate substrate (Km = 26 micronM, when [glycine] = 100 mM). Ultraviolet irradiation of a mixture of p-azidobenzoyl-CoA and the N-acyltransferase produces irreversible inhibition. Benzoyl-CoA protects the enzyme from inhibition by photoactivated p-azidobenzoyl-CoA. Acyl-CoA:glycine N-acyltransferase is composed of a single polypeptide with a molecular weight of about 35 000. Photolabeling experiments show that there is one active site per molecule of enzyme.

High-performance liquid chromatographic determination of L-aspartyl-L-phenylalanine methyl ester in various food products and formulations.

A simple, rapid, and specific high-performance liquid chromatographic (HPLC) procedure is described for the analysis of the chemical sweetener L-aspartyl-L-phenylalanine methyl ester (aspartame). Using a strong cation exchange column and pressures less than 1000 psig, an analysis can be performed in less than 15 min. The technique has been applied to a wide range of food products and formulations. No interferences were found in the samples studied. Recoveries are quantitative, and the coefficients of variation for replicate analyses are less than or equal to 2.5%.

Controlled drug release from polymeric delivery devices IV: in vitro--in vivo correlation of subcutaneous release of norgestomet from hydrophilic implants.

The in vitro and in vivo releases of norgestomet from hydrophilic implants were found to follow a matrix-controlled (Q - t1/2) process. The sorption of drug onto the implants was observed to obey the same mechanism but with a much smaller magnitide of the Q/t1/2 value. The effect of the extent of cross-linking on the magnitude of drug release (Q/t1/2) profiles was analyzed both theoretically and experimentally. The release of norgestomet from hydrophilic implants was found to be an energy-linked process. Two energy terms were calculated; the activation energy for matrix diffusion was 7.71 kcal/mole, and the heat of drug crystal solvation was 25-28.6 kcal/mole.

PIP: The mechanisms and rates of the controlled release of norgestomet from hydrophilic implants were analyzed and the correlations of in vivo and in vitro drug release profiles were investigated. Both in vitro and in vivo releases of norgestomet from the implants followed a matrix-controlled process. The sorption of drug onto the implants obeyed the same mechanism but with a smaller magnitude. Release of norgestomet from the implants was an energy-linked process. The activation energy for matrix diffusion was 7.71 kcal/mole and heat of drug crystal solvation was 25-28.6 kcal/mole. It is concluded that regardless of the difference in the physicochemical nature of polymer between hydrophilic hydrogel and lipophilic silicone delivery devices, the same theoretical model is followed for the release of drug molecules dispersing throughout the polymeric matrices.