Mandelonitrile beta-glucuronide: synthesis and characterization.

Mandelonitrile beta-glucuronide, the compound patented as Laetrile, has been synthesized from rabbit liver uridine diphosphate-glucuronosyl transferase immobilized on beaded sepharose, has been analyzed by thin-layer chromatography, nuclear magnetic resonance, and gas chromatography-mass spectrometry, and has been tested for cytotoxicity and mutagenic activity with Salmonella typhimurium strains TA 98 and TA 100. Several commercial laetrile preparations contained no glucuronide; they contained amygdalin and neoamygdalin instead. Mandelonitrile, mandelonitrile glucuronide, and a mixture of amygdalin and neoamygdalin were each found to be mutagenic.

Proteomic strategies for rapid characterization of micro-organisms.

Bioinformatic considerations are offered to illustrate strengths and limitations of the characterization of Bacillus spores based on proteomic interpretation of matrix-assisted laser desorption/ionization spectra. In particular, species-specific biomarkers are evaluated in the context of both experimental access and uniqueness in silico.

Identification of phosphorodiamidic acid mustard as a human metabolite of cyclop hosphamide.

An active metabolite of cyclophosphamide, N,N-bis(2-chloroethyl)phosphorodiamidic acid, has been confirmed as a circulating and excreted metabolite in patients receiving the drug in therapy, by selected ion monitoring on a gas chromatograph mass spectrometer.

Alkylating properties of phosphoramide mustard.

The relative alkylating activities of two of the cytotoxic metabolites of cyclophosphamide, phosphoramide mustard and nornitrogen mustard, have been studied at pH 4.6 and 7.4. The products formed on alkylation of ethanethiol by these metabolites have been identified, confirming that phosphoramide mustard undergoes alkylation reactions as an intact molecule. Deuterated analogs of the two metabolites have been synthesized, namely N,N-bis(2,2-dideutero-2-chloroethyl)-phosphorodiamidic acid and N,N-bis(2,2-dideutero-2-chloroethyl)amine and used to determine that alkylation proceeds directly via an aziridinium intermediate rather than a direct SN2 displacement of the chlorine atom.

Quantitation by gas chromatography-chemical ionization mass spectrometry of cyclophosphamide, phosphoramide mustard, and nornitrogen mustard in the plasma and urine of patients receiving cyclophosphamide therapy.

Unambiguous and sensitive methods based on gas chromatography-chemical ionization mass spectrometry have been developed to quantitate cyclophosphamide and two alkylating and cytotoxic metabolites, phosphoramide mustard and nornitrogen mustard. The levels of these materials have been determined in the plasma and urine of five patients receiving cyclophosphamide, 60 or 75 mg/kg i.v. Peak plasma levels of phosphoramide mustard of 50 to 100 nmoles/ml were found at 3 hr after cyclophosphamide administration. Variable levels of nornitrogen mustard were found in the plasma. This product may be arising in part from the decomposition of other metabolites during sample storage and preparation.

Identification of aldophosphamide as a metabolite of cyclophosphamide in vitro and in vivo in humans.

Aldophosphamide (NSC 254), a putative key metabolite of cyclophosphamide, has now been isolated as a cyanohydrin derivative from an incubation mixture of cyclophosphamide with mouse liver microsomes in vitro and from the plasma of a cyclophosphamide-treated patient. The cyanohydrin has been shown to be identical with an authenic synthetic sample by mass spectrometry and combined gas chromatography-mass spectrometry.

Quantitation by gas chromatography-chemical ionization-mass spectrometry of phenylalanine mustard in plasma of patients.

An unambiguous and sensitive method based on gas chromatography-chemical ionization-mass spectrometry has been developed to quantitate L-phenylalanine mustard and has been applied to measure levels in plasma of five patients receiving 0.15 to 0.25 mg/kg (10 to 17 mg) of the drug p.o. Peak plasma levels of 50 to 190 ng/ml were found to occur between 0.7 and 2.3 hr after ingestion. The time for the plasma level to fall to one-half of the peak value varied from 0.6 to 3 hr, and very low levels (less than 2 ng/ml) were present by 24 hr.

Primary structure of molluscan metallothioneins deduced from PCR-amplified cDNA and mass spectrometry of purified proteins.

The primary structure of metallothioneins (MT) of a mollusc, the oyster Crassostrea virginica, was determined by molecular cloning and mass spectrometry of purified proteins. The cloning strategy included PCR amplification of the responsible cDNAs from total cDNA using completely degenerate oligonucleotides (derived from the N-terminal amino acid sequence) and oligo(dT)20 as primers. Primer extension off mRNA was used as an independent determination of the nucleotide sequence represented by the degenerate PCR primers. The deduced amino acid sequence was consistent with characteristics of class I MT. Twenty-one cysteine residues, were arranged in nine Cys-X-Cys motifs, five as Cys-Lys-Cys. A single Cys-X-X-Cys motif was also observed. Two MTs that differ only in the presence or absence of an N-acetyl group exist in this organism. Masses of tryptic peptides of purified MTs corresponded with those of peptides predicted from tryptic cleavages of the deduced amino acid sequence. Allowing for known N-terminal modifications, 96% of the deduced sequence was confirmed by mass spectrometry. Comparison (FASTA algorithm) of the primary structure of the oyster MTs with those of other species indicated a higher similarity with vertebrate MTs than with those of other invertebrates.

Structure and time-dependent behavior of acetylated and non-acetylated forms of a molluscan metallothionein.

Cadmium-induced metallothionein in a mollusc, the oyster Crassostrea virginica, occurs in both blocked and unblocked forms (Roesijadi, G., Kielland, S.L. and Klerks, P. (1989) Arch. Biochem. Biophys. 273, 403-413). The block, which is the sole difference in the structure of the two proteins, was identified as an acetyl group with use of tandem mass spectrometry. The blocked and unblocked proteins carried N-acetylserine and serine, respectively, at the N-terminus and were designated CvNAcMT and CvMT. Only CvNAcMT was detected under basal conditions. Both forms were induced by Cd. Pulse-labeling with [35S]cyteine at specified times during exposure showed that the rate of CvNAcMT synthesis in gills increased rapidly, initially exceeding that of CvMT, then declined to the rate attained by CvMT. Turnover rates for Cd-induced CvMT and CvNAcMT were similar to each other. They appeared to be faster when measured in the absence of Cd in the external medium (k = 0.18 and 0.16/day, respectively), than in its presence (k = 0.03 and 0.06/day, respectively).

Primary sequence of duck metallothionein.

Only one metallothionein appears in domesticated duck upon zinc induction. The complete amino acid sequence has been elucidated. This metallothionein has the same sequence as the chicken metallothionein, as determined by chemical sequencing of overlapping peptides produced by selective proteinase digestion and confirmed by mass spectrometry. The observation that animals of divergent origins share a common gene product presents an example of extreme conservation of a stress-inducible protein.

Mass spectrometric analysis of proteins.

The past year has seen greatly increased acceptance and application of the analytical capabilities of mass spectrometry by the biochemical community. The technique has been used to provide accurate mass determinations of non-covalently bound protein complexes, rapid mapping of molecular weights of altered peptides in protease digests, sequencing by collisional activation in tandem mass spectrometry, characterization of glycosylation and other modifications, and quantitation of peptides used in clinical diagnostics.

Monitoring metal ion flux in reactions of metallothionein and drug-modified metallothionein by electrospray mass spectrometry.

The capabilities of electrospray ionization mass spectrometry are demonstrated for monitoring the flux of metal ions out of and into the metalloprotein rabbit liver metallothionein and, in one example, chlorambucil-alkylated metallothionein. Metal ion transfers may be followed as the reactions proceed in situ to provide kinetic information. More uniquely to this technique, metal ion stoichiometries may be determined for reaction intermediates and products. Partners used in these studies include EDTA, carbonic anhydrase, a zinc-bound hexamer of insulin, and the core domain of bacteriophage T4 gene 32 protein, a binding protein for single-stranded DNA.

The gene encoding glyoxalase I from Pseudomonas putida: cloning, overexpression, and sequence comparisons with human glyoxalase I.

The gene encoding glyoxalase I (GlxI) from Pseudomonas putida has been cloned into the high-expression plasmid pBTacI. In the presence of IPTG, JM109 cells transformed with this vector give expression levels of GlxI 4000-fold higher than wild-type Escherichia coli. Contrary to a previous report, the nucleotide sequence of the gene encodes a 173-amino-acid polypeptide. Edman analysis indicates that the predicted N-terminal methionine is lost post-translationally to yield a 19407-Da protein. Mass spectrometry of the intact protein, and of the peptides generated from treatment with CNBr, does not indicate any additional post-translational modifications of the enzyme. Contrary to previous conclusions, there are no major regions of dissimilarity between the human and bacterial enzymes.

Solid-phase synthesis of drug glucuronides by immobilized glucuronosyltransferase.

Rabbit liver glucuronosyltransferase immobilized on beaded agarose has been used to synthesize glucuronic acid conjugates of meprobamate, diethylstibestrol, bilirubin, borneol, benzioc acid, and p-nitrothiophenol. The immobilized enzyme exhibited a high degree of specificity for UDPGA as cofactor when p-nitrophenol is used as substrate. Other cofactors tested were less effective, all producing less than 10% conjugation relative to UDPGA. The effects on agarose-bound enzyme activity of a variety of cosolvents and emulsifiers have been studied. Ethanol, dimethyl sulfoxide, propylene glycol, and bovine serum albumin are among the cosolvents and emulsifiers which can be used within limited concentration ranges to sulubilize lipophilic substrates for conjugation. Concentrations of calcium and magnesium cations between 1.5 and 10.0mM were found to enhance glucuronosyltransferase activity of the immobilized enzyme.

Immobilized glucuronosyltransferase for the synthesis of conjugates.

Partially purified rabbit liver UDPglucuronosyltransferase is immobilized on agarose by the cyanogen bromide activation method. Both soluble and matrix-bound enzyme preparations display very similar Km and pH optimum. The storage stability of the immobilized enzyme at 4 degrees is 5-10 times improved over the soluble preparations. The agarose-bound UDPglucuronosyltransferase is successfully used in the synthesis of p-nitrophenyl glucuronide in an overall yield of 50-70%. The matrix-bound enzyme is reusable over an extended period of time and offers an easy and convenient synthetic tool for various drug glucuronides.

Identification of an N-acetylated microsomal glutathione S-transferase by mass spectrometry.

Microsomal glutathione S-transferase (mGST) was purified to homogeneity from male Sprague-Dawley rat liver, as determined by SDS-PAGE. Removal of Triton X-100 and further separation by reversed phase HPLC revealed two proteins, mGST 1 and mGST 2, in a 1:3 ratio. Analysis of mGST 1 and mGST 2 by electrospray ionization mass spectrometry determined their molecular weights to be 17,354.2 +/- 6.6 and 17,397.9 +/- 6.6, respectively. mGST 1 was in close agreement with the calculated molecular weight of 17,348, as predicted by the previously reported cDNA sequence. Cyanogen bromide digestion and peptide mapping by fast atom bombardment mass spectrometry (FAB-MS) localized the mass increase to the N-terminal peptide, 1-7. FAB-tandem mass spectrometry of this peptide in conjunction with Edman reactions on the intact protein demonstrated the N-terminal alanine to be acetylated.

In vitro formation of glutathione conjugates of the dimethylester of bilirubin.

Rat hepatic microsomes catalyzed the formation of two distinct glutathione conjugates of bilirubin dimethylester (DMB). The two conjugates were identical to those isolated from the bile of Gunn rats infused with DMB. The microsomal reaction was dependent on NADPH, oxygen and glutathione and was inhibited by nitrogen and the cytochrome P450 inhibitors metyrapone, 1-benzyl-imidazole, and alpha-naphthoflavone. Conjugate formation was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but not phenobarbital pretreatment. The rate of formation of conjugates was not affected by washings of the microsomal pellet or by the presence of superoxide dismutase and/or catalase. Cation fast atom bombardment mass spectrometry (FAB/MS) of the conjugates indicated a molecular ion of 937 atomic mass units (amu). Fragmentation revealed a loss of 307 amu, consistent with glutathione, and a residual mass of 629 amu suggesting a hydroxylated derivative of DMB (612 amu). Cation FAB/MS/MS of conjugates formed in vitro under an atmosphere of oxygen-16 and oxygen-18 demonstrated the incorporation of molecular oxygen by a difference of 2 amu in the respective molecular ions. Our results suggest that DMB is oxidized by the cytochrome P450 IA gene family to an epoxide intermediate which is then subsequently conjugated with glutathione.

Proton affinities of polyglycines assessed by using the kinetic method.

The proton affinities of a series of peptides, chosen to show the effects of chain length, were measured by the kinetic method using amines as standard reference bases. Proton affinities of polyglycines with residues ranging from 2 to 10 were measured and the values were found to increase as the number of residues increases.

Endothermic ion molecule reactions.

Endothermic ion-molecule reactions in a tandem mass spectrometer have been used for a number of years for determining thermodynamic quantities, such as heats of formation and proton affinities, for gaseous ions. Recently, the reactive, endothermic collision has been exploited as an analytical technique for the structural analysis of peptides and other biomolecules. The technique is based upon the endothermic transfer of protons associated with amide bonds to ammonia. This reaction proceeds via a long-lived collision complex. When additional beam energy is supplied, other dissociation channels are opened up, leading to the production of sequence ions for the mass-selected, protonated analyte that are normally observed in high energy collision-induced dissociation spectra. The advantage, however, is that such spectra can be produced at very low beam energies. In this article, the rationale for developing this scheme, and its roots in previous ion-molecule studies, are explored.

Oligopeptide fragmentation viewed as constant neutral loss.

Spectra were recorded of all fragment ions formed by elimination of 28-u neutral fragments in fast atom bombardment spectra of peptides in the mass range 1000-1800 u, This approach can provide less complex spectra than either conventionally scanned spectra or product ion scans from collisionaIly activated four-sector experiments, and spectra that contain information that is both overlapping and complementary to those from the other techniques. Constant neutral loss spectra may provide a reading frame for distinguishing sequence ion series in tandem or single analyzer spectra.