Analysis of the subsite specificity of rat insulysin using fluorogenic peptide substrates.

Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K(m) of 13 microm and a V(max) of 2.6 micromol min(-1) mg(-1). Derivatives of this peptide in which the P(2) leucine or the P(2)' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P(2) residue produced a 4-fold range in K(m) and a 7-fold range in k(cat). The nature of the P(2) residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond. Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the R-K bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and R-K were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P(2)' residues produce less of a change in both K(m) and k(cat) and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the R-K bond, produce significant cleavage at the L-R bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site.

Insulysin hydrolyzes amyloid beta peptides to products that are neither neurotoxic nor deposit on amyloid plaques.

Insulysin (EC. 3.4.22.11) has been implicated in the clearance of beta amyloid peptides through hydrolytic cleavage. To further study the action of insulysin on Abeta peptides recombinant rat insulysin was used. Cleavage of both Abeta(1-40) and Abeta(1-42) by the recombinant enzyme was shown to initially occur at the His(13)-His(14), His(14)-Gln(15), and Phe(19)-Phe(20) bonds. This was followed by a slower cleavage at the Lys(28)-Gly(29), Val(18)-Phe(19), and Phe(20)-Ala(21) positions. None of the products appeared to be further metabolized by insulysin. Using a rat cortical cell system, the action of insulysin on Abeta(1-40) and Abeta(1-42) was shown to eliminate the neurotoxic effects of these peptides. Insulysin was further shown to prevent the deposition of Abeta(1-40) onto a synthetic amyloid. Taken together these results suggest that the use of insulysin to hydrolyze Abeta peptides represents an alternative gene therapeutic approach to the treatment of Alzheimer's disease.

New fluorogenic substrates for N-arginine dibasic convertase.

N-Arginine dibasic (NRD) convertase is a recently described peptidase capable of selectively cleaving peptides between paired basic residues. The characterization of this unique peptidase has been hindered by the fact that no facile assay procedure has been available. Here we report the development of a rapid and sensitive assay for NRD convertase, based on the utilization of two new internally quenched fluorogenic peptides: Abz-GGFLRRVGQ-EDDnp and Abz-GGFLRRIQ-EDDnp. These peptides contain the fluorescent 2-aminobenzoyl moiety that is quenched in the intact peptide by a 2, 4-dinitrophenyl moiety. Cleavage by NRD convertase at the Arg-Arg sequence results in an increase of fluorescence. NRD convertase cleaves these peptides efficiently and with high specificity as observed by both HPLC and fluorescence spectroscopy. The rate of hydrolysis of the fluorogenic substrates is proportional to enzyme concentration, and obeys Michaelis-Menten kinetics. The kinetic parameters for the fluorescent peptides (Km values of approximately 1.0 microM, and Vmax values of approximately 1 microM/(min. mg) are similar to those obtained with peptide hormones as substrates.

Mass spectrometry at low and high mass.

The introduction of novel methods as well as expanding applications to diverse areas highlight truly impressive progress in mass spectrometry. These developments are illustrated here by two seemingly different areas of research: new methods designed for the determination of isotopic enrichment and novel ionization methods; and mass analyzers which have enabled the precise determination of the molecular weight of proteins and large oligonucleotides.

Evaluating the role of niacin in human carcinogenesis.

Our understanding of the role of ADP-ribose polymer metabolism in limiting carcinogenic events and the dependence of this metabolism on cellular NAD levels predicts that niacin deficiency leading to reduced NAD levels may enhance carcinogenesis. This prediction has led us to initiate studies to evaluate the potential of niacin as a preventive factor in human cancer. The first approach involves development of a method to assess biochemically niacin status in humans using intracellular NAD derived from whole blood, primarily erythrocytes, as the relevant marker of niacin status. We have shown that erythrocyte NAD content varies by as much as 12-fold within a population and can be modulated readily by supplementation. A second approach to testing this hypothesis involves understanding the relationship of dietary niacin, circulating levels of NAD precursors (nicotinamide and nicotinic acid) and NAD in target tissues for human cancer. Current analytical methods for quantification of plasma levels of nicotinic acid and nicotinamide following intake in the dietary range are not sufficient. Thus, we have developed a GC-MS method for the rapid, sensitive, and selective determination of both nicotinamide and nicotinic acid in plasma. These methods will now allow assessment of niacin metabolism in humans that could lead to a new understanding of niacin in prevention of cancer.

Inhibitors of sterol synthesis. Submicromolar 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol causes a major modification of the sterol composition of CHO-K1 cells and a marked change in cell morphology.

Incubation of Chinese hamster ovary cells (CHO-K1) with 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol (0.1 microM) in lipid-deficient medium led to a major change in cellular sterol composition, which was characterized by a very marked accumulation of C30 sterols (lanosterol and 24,25-dihydrolanosterol). The accumulation of C30 sterols was associated with a striking change in cell morphology. The change in cell shape (elongation) was similar to that described previously (A. W. Hsie and T. T. Puck, 1971. Proc. Natl. Acad. Sci. USA. 68: 358-361; and confirmed herein) for CHO-K1 cells incubated in the presence of dibutyryl cAMP (1 mM). This change in morphology, induced by dibutyryl cAMP, was not accompanied by a change in cellular sterol composition. The cell elongation and accumulation of C30 sterols, induced by the 14 alpha-ethyl diol, were prevented by the addition of cholesterol (10 microM or 100 microM) and were reversed by removal of the 14 alpha-ethyl diol from the incubation medium. Incubation of the cells with the 14 alpha-ethyl diol had no effect on the levels of cAMP under the conditions studied. Incubation of the cells with miconazole (10 microM) or with lanosterol (10 microM) was also associated with the accumulation of C30 sterols and an elongation of the cells. 24,25-Dihydrolanosterol (10 microM) also induced similar changes in cellular morphology. The results presented herein demonstrate that marked changes in the sterol composition of CHO-K1 cells can be effected by incubation of the cells with 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol, miconazole, or lanosterol. In addition, the findings reported herein indicate an important role of sterols in the control of the shape of these cells.

A novel UDP-Glc-specific glucosyltransferase catalyzing the biosynthesis of 6-O-glucosides of bile acids in human liver microsomes.

Two active site-directed photoaffinity analogs, 5-[beta-32P]azido-UDP-glucuronic acid and 5-[beta-32P]azido-UDP-glucose, were used for the characterization of UDP-sugar-utilizing enzymes in human liver microsomes. Both compounds were recognized by human microsomal proteins: major photolabeled bands of 50-56 kDa were detected. Both photoincorporations were competitively decreased by increasing concentrations of either UDP-Glc or UDP-GlcUA, indicating a high affinity for both nucleotides. The patterns of photoaffinity labeling in the 50-56-kDa range by the two probes were significantly different, indicating the presence of different UDP-GlcUA- and UDP-Glc-specific enzymes of similar molecular mass. The presence of a UDP-Glc-dependent transferase was confirmed by the identification of an enzymatic activity catalyzing the formation of glucosides of the 6 alpha-hydroxylated bile acid hyodeoxycholic acid (3 alpha, 6 alpha-diOH (HDCA)) in the presence of UDP-Glc. The specific activity of 1.5-3.2 nmol/min/mg of protein was similar to that of 6 alpha-glucuronidation of HDCA. The apparent Km for UDP-Glc estimated with HDCA was 280 microM, and the formation of HDCA glucosides was strongly inhibited by UDP-GlcUA (apparent Ki = 7 microM). Evidence is presented that HDCA-specific UDP-glucuronosyltransferase (clone UGT2B4) expressed in V79 cells is not involved in glucosidation of HDCA and is not photolabeled with 5-[beta-32P]azido-UDP-Glc. Rigorous structure identification of the biosynthetic product proved that HDCA was glucosidated at the 6-position. Thus, this UDP-Glc-dependent activity catalyzing the biosynthesis of 6-O-glucosides of 6 alpha-hydroxylated bile acids represents a new pathway in the metabolism of these bile acids.

2,3-Dihydrofarnesoic acid, a unique terpene from trichomes ofLycopersicon hirsutum, repels spider mites.

Lycopersicon hirsutum, a wild relative of the tomato, is highly resistant to arthropod herbivores. Both botanic forms ofL. hirsutum, L. hirsutum f.glabratum (C.H. Mull.) andL. hirsutum f.typicum (Humb. & Bonpl.), are resistant to two-spotted spider mites,Tetranychus urticae Koch. However, leaves and trichome secretions from f.typicum repel mites more so than those from f.glabratum. We have previously demonstrated that trichome secretions from LA 1363 and LA 1927, accessions of f.typicum, repelled mites. In this paper we report the identification of the primary component of trichome secretions responsible for repellency. Leaflet washes having compositions similar to trichome secretions were collected and separated into neutral and acid fractions; repellency was mainly associated with the acid fraction, which, when applied to nonrepellent leaflets of f.glabratum, rendered them repellent. Separation of leaflet washes by HPLC allowed purification and subsequent identification by gas chromatography-mass spectrometry and nuclear magnetic resonance of 2,3-dihydrofamesoic acid (3,7,11-trimethyl-6, 10-dodecadienoic acid) as the primary chemical component responsible for repellency. Application of this acid to leaflets ofL. esculentum rendered them repellent. Other volatile compounds present in minor amounts in the acid fractions were farnesoic acid and 16∶0, 16∶3, 18∶0, 18∶2, and 18∶3 fatty acids. This is the first report of the natural occurrence of 2,3-dihydrofarnesoic acid.

Hepatic metabolism of short-chain bile acids. Inversion of the 3-hydroxyl group of isoetianic acid (3 beta-hydroxy-5 beta-androstane-17 beta-carboxylic acid) by the adult rat.

The stereospecificity of mechanisms for hepatic transport of short-chain bile acids has been examined by following the hepatic metabolism and biliary secretion of 3 beta-hydroxy-5 beta-androstane-17 beta-carboxylic acid (isoetianic acid) administered in two different labeled forms to rats prepared with an external biliary fistula. While 93% of the administered [2,2,4,4-3H]isoetianic acid was recovered in bile after 20 h, only 18% of a similar dose of [3 alpha-3H]isoetianic acid was secreted in bile over the same time period. The recovered radioactivity of the latter compound was mainly associated with bile water. With the [2,2,4,4-3H]isoetianic acid, the bulk of the biliary isotope was determined to be in the form of two glucuronide conjugates. Spectral analysis identified these metabolites as the hydroxyl-linked (major) and carboxyl-linked (minor) beta-glucuronides, not of the 3 beta-hydroxy compound administered, but of 3 alpha-hydroxy-5 beta-androstane-17 beta-carboxylic acid (etianic acid), i.e., the products of hydroxyl group inversion. It is concluded that isoetianic acid is efficiently cleared from plasma and conjugated with glucuronic acid after its epimerization to etianic acid. The prevalent, but not complete, loss of the 3-tritium atom and the retention of the 2- and 4-tritium atoms probably indicates a 3-oxo-5 beta-androstane-17 beta-carboxylic acid intermediate with partial return of the label via a limited labeled pool of reduced nicotinamide cofactor.

Inhibitors of sterol synthesis. Characterization of side chain oxygenated derivatives formed upon incubation of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one with rat liver mitochondria.

3 beta-Hydroxy-5 alpha-cholest-8(14)-en-15-one, a potent inhibitor of sterol biosynthesis, was incubated with rat liver mitochondrial preparations in the presence of NADPH. The following four major products were isolated and characterized by nuclear magnetic resonance and mass spectrometry: (25R)- and (25S)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one (4:1 ratio), 3 beta-hydroxy-15-oxo-5 alpha-cholest-8(14)-en-26-oic acid, and 3 beta,25-dihydroxy-5 alpha-cholest-8(14)-en-15-one. In addition, 3 alpha,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one and 3 beta,24-dihydroxy-5 alpha-cholest-8(14)-en-15-one were identified as minor products by capillary gas chromatography-mass spectrometry.

Vulpecholic acid (1 alpha, 3 alpha, 7 alpha-trihydroxy-5 beta-cholan-24-oic acid): a novel bile acid of a marsupial, Trichosurus vulpecula (Lesson).

A novel trihydroxylated C24 bile acid was isolated from the gallbladder bile of the Australian opossum, Trichosurus vulpecula (Lesson). This acid, for which the name vulpecholic acid is proposed, was identified as 1 alpha, 3 alpha, 7 alpha-trihydroxy-5 beta-cholan-24-oic. The structure proof included mass spectral and 1H and 13C nuclear magnetic resonance characterization of all crucial derivatives obtained by: oxidation of the methyl ester to a triketone with the enolizable 1,3-diketone function; methylation of this triketone to two isomeric methyl enol ethers; and reductive removal of oxygen functions from this triketone to give 5 beta-cholan-24-oic and 7-oxo-5 beta-cholan-24-oic acids. Vulpecholic acid was found in the bile in the unconjugated form; it accounted for more than 60% of the solid bile material. The marsupial T. vulpecula is the first example of a mammal secreting a 1 alpha-hydroxylated bile acid as well as the first example of a mammal secreting the major bile acid in a free form.