The influence of dietary calcium and phosphorus on intestinal calcium transport in rats given vitamin D metabolites.

A new method for the simple analysis of methylated amino acids based on autoradiography is introduced. With this technique a survey of protein methylation in a prokaryote, Escherichia coli, and a eukaryote, fibroblasts in culture, was carried out in an attempt to identify, quantitate, and determine the subcellular localization of all the methylated amino acids found in the proteins of these organisms.In mammalian cells using an established mouse fibroblast line (3T3), we have found that nuclei-free and mitochondria-free cytoplasm contain readily detectable amounts of four identifiable methylated amino acids: N^?,N^?-dimethyllysine, N^?,N^?,N^?-trimethyllysine, N^G,N^G-dimethylarginine (or N^G-methylarginine), and N^G,N′^G-dimethylarginine. The crude nuclear pellet also contains these methylated amino acids, but in addition contains N^?-methyllysine and a new as yet unidentified methylated compound. Histones purified from these nuclei contain essentially the same array of methylated compounds.The ribosomal subunits of the mammalian cells contained only small amounts of the methylated amino acids; the 40S subunit contained a substantial amount of just one, N^G,N^G-dimethylarginine (or N^G-methylarginine), and smaller amounts of N^G,N′^G-dimethylarginine, and an as yet unidentified methylated compound. The 60S subunit contained even smaller amounts of methylated amino acids, 50% of which was N^?,N^?,N^?-trimethyllysine and smaller amounts of N^?-methyllysine, N^?,N^?-dimethyllysine, and N^G,N^G-dimethylarginine. These subunits also contained an as yet unidentified methylated compoundThese results were in marked contrast to those that we obtained with the prokaryote, Escherichia coli. Only the proteins of the 50S ribosomal subunit of the bacteria contained methylated amino acids. Of those present 50% was N^?,N^?,N^?-trimethyllysine, with the remainder distributed about equally between N^?-methyllysine and three unknowns, one of which is apparently the same as that found in the 60S subunit of the mouse fibroblasts. All of the N^?-methyllysine was apparently in the small acidic proteins, L7 and L12.     

Recommended method for the analysis of amino acids in biological materials

Fifty-five ninhydrin-positive compounds in physiological fluids were determined with a Hitachi Model KLA-5 amino acid analyzer by a two-column chromatographic procedure. Both columns were packed with Hitachi Custom 2618 ion-exchange resin. The total analysis time was 9.5 h.In this procedure, particularly glucosamine, mannosamine and galactosamine were separated completely from normal “protein” amino acids, and N^G-monomethylarginine, N^G,N^G-dimethylarginine and N^G,N′^G-dimethylarginine, which were present in the myelin basic protein of several species and excreted in human urine, were separated from other basic amino acids. The method is useful for various applications with biological materials.     

METHYLATED AMINO ACID RESIDUES OF PROTEINS OF BRAIN AND OTHER ORGANS

—Methods for the determination of methyl-lysine, methyllarginine and methylhistidine residues of tissue proteins are described. They consist of preliminary purification of basic amino acids, enzymic removal of lysine, arginine and histidine followed by amino acid analysis. Recovery rates and specificities of the method were satisfactory. The contents of methylamino acids in proteins of mammalian organs were determined. The distribution of proteins containing the methylamino acids in human brain showed that the concentrations of methyl-lysine and N^G,N′^G-dimethylarginine were highest in the gray matter of the cerebellar cortex and relatively high in regions rich in gray matter, while those of N^G-mono- and N^G,N′^G-dimethylarginine were highest in the white matter. The following findings suggest that most of the N^G-mono- and N^G,N′^G-dimethylarginine was associated with the myelin basic protein. The distribution of the methylarginine residues of acid-soluble proteins in bovine brains coincided with the cerebroside pattern. The concentrations of the amino acids in acid-soluble proteins of rat brain increased concomitantly with the increase of cerebroside. The methylamino acid content in proteins increased during the purification of the myelin basic protein from the white matter of human and bovine brains. Proteins containing N^G,N^G-dimethyiarginine and di- and trimethyl-lysine are concentrated in cell nuclei. The first amino acid was found mainly in nucleoplasmic proteins and the other two were found in histones. The concentration of 3-methylhistidine residue, highest in muscular proteins, is low in cerebral proteins and is probably derived from proteins of walls of blood vessels in the brain.     

Methylation of ribosomal proteins in HeLa cells.

Methylated amino acids from both 40 and 60S subunit proteins of HeLa cytoplasmic ribosome were analyzed. It was observed that methylation of ribosomal proteins occurs in both subunits with N^G,N^G-dimethylarginine as the major methylated amino acid. The presence of N^G,N^G-dimethylarginine has been identified by high-voltage paper electrophoresis, by paper chromatography, and by amino acid analysis. In addition, both ribosomal subunits contain methylated lysines with ?-N-trimethyllysine being the predominant one, followed by ?-N-dimethyllysine. Little, if any ?-N-monomethyllysine was detected in either subunit. The cytoplasmic 60S ribosomal subunit contains much more ?-N-trimethyllysine compared to the 40S ribosomal subunit. The possible biological significance of methylation was discussed.     

Spliceosome Sm proteins D1, D3, and B/B' are asymmetrically dimethylated at arginine residues in the nucleus

We report a novel modification of spliceosome proteins Sm D1, Sm D3, and Sm B/B′. L292 mouse fibroblasts were labeled in vivo with [³H]methionine. Sm D1, Sm D3, and Sm B/B′ were purified from either nuclear extracts, cytosolic extracts or a cytosolic 6S complex by immunoprecipitation of the Sm protein-containing complexes and then separation by electrophoresis on a polyacrylamide gel containing urea. The isolated Sm D1, Sm D3 or Sm B/B′ proteins were hydrolyzed to amino acids and the products were analyzed by high-resolution cation exchange chromatography. Sm D1, Sm D3, and Sm B/B′ isolated from nuclear fractions were all found to contain ω-N^G-monomethylarginine and symmetric ω-N^G,N^G′-dimethylarginine, modifications that have been previously described. In addition, Sm D1, Sm D3, and Sm B/B′ were also found to contain asymmetric ω-N^G,N^G-dimethylarginine in these nuclear fractions. Analysis of Sm B/B′ from cytosolic fractions and Sm B/B′ and Sm D1 from cytosolic 6S complexes showed only the presence of ω-N^G-monomethylarginine and symmetric ω-N^G,N^G′-dimethylarginine. These results indicate that Sm D1, Sm D3, and Sm B/B′ are asymmetrically dimethylated and that these modified proteins are located in the nucleus. In reactions in which Sm D1 or Sm D3 was methylated in vitro with a hemagglutinin-tagged PRMT5 purified from HeLa cells, we detected both symmetric ω-N^G,N^G′-dimethylarginine and asymmetric ω-N^G,N^G-dimethylarginine when reactions were done in a Tris/HCl buffer, but only detected symmetric ω-N^G,N^G′-dimethylarginine when a sodium phosphate buffer was used. These results suggest that the activity responsible for the formation of asymmetric dimethylated arginine residues in Sm proteins is either PRMT5 or a protein associated with it in the immunoprecipitated complex.     

Identification of NG, NG-dimethylarginine in a nuclear protein from the lower eukaryote physarum polycephalum homologous to the major proteins of mammalian 40S ribonucleoprotein particles

A nuclear protein apparently homologous to the two major proteins of 40S heterogeneous nuclear ribonucleoprotein particles from mammalian cells has been isolated from the lower eukaryote $\text{Physarum polycephalum}$, purified, and found to contain a substantial amount of the unusual amino acid N^G, N^G-dimethylarginine. The apparent homology is based on similar molecular weights, basic isoelectric points and amino acid compositions including the dimethylarginine and a high content of glycine. The implications of the presence of this protein in $\text{Physarum polycephalum}$ and the possible significance of the N^G, N^G-dimethylarginine are discussed.     

PARTIAL CHARACTERIZATION OF BASIC PROTEINS OF CHICKEN, TURTLE AND FROG CENTRAL NERVOUS SYSTEM MYELIN

Myelin basic proteins were isolated from CNS tissues of chicken, turtle and frog and compared with the corresponding protein of bovine origin. At acid pH all four proteins had comparable mobilities in polyacrylamide gels. Upon electrophoresis at alkaline pH the submammalian proteins, like the bovine protein, were separated into multiple components. The components of the chicken and frog proteins had exceptionally high and low mobilities, respectively, while those of the turtle protein had mobilities comparable to those of the bovine protein. The chicken and turtle proteins were similar to the bovine protein in amino acid composition except for containing considerably more serine and valine and having higher proportions of histidine to lysine. The frog protein differed further in having an unusually high content of tyrosine (approx 9 mol/mol protein), an unusually high arginine: glycine ratio (1.09) and practically no methylated arginine (0-0.036 mol/mol protein). Like those of mammalian origin, the submammalian proteins each contained a single tryptophan and two methionines. Arginine, serine and glycine together accounted for approximately 40 per cent of the residues in each protein. The chicken and turfle proteins each contained roughly equal amounts of N^G-monomethyl- and N^G, N^G-dimethylarginine, the two derivatives together comprising 0.5-0.6 mol/mol protein. No N^G, N^G-dimethylarginine was detected in any of the proteins examined. The microheterogeneity observed in the chicken and turtle proteins upon electrophoresis at alkaline pH was reproduced upon alkaline pH chromatography on carboxymethylcellulose. Chromatographic fractions of the chicken protein which differed electrophoretically at alkaline pH had virtualy identical amino acid compositions and apparent molecular weights and all contained comparable amounts of both N^G-monomethyl- and N^G, N^G-dimethylarginine. Treatment of the submammalian proteins with BNPS-skatole yielded two fragments comparable in size, charge and staining characteristics to those similarly produced from the bovine protein (residues 1-116 and 117-170). Fragments produced from the frog protein by treatment with BrCN were comparable in size and charge to those similarly produced from the bovine protein; those produced from the chicken and turtle proteins were much different. In immunodiffusion studies the submammalian and bovine proteins showed reactions of identity when tested against rabbit anti-chicken basic protein serum.     

Ng-Methylated Arginines in Broad Bean Seed

l-N^g-Methylarginine, l-N^g,N^g-dimethylarginine and ethanolamine were isolated from basic amino acids fraction of broad bean (Vicia faba L.) seed. The presence of N^g,N′^g-dimethylarginine was also suggested.     

Distribution of NG, NG-dimethylarginine in nuclear protein fractions

Nuclear proteins have been fractionated into five distinct classes according to their extractability from rat liver nuclei at different pH and salt concentrations. The fractions have been analyzed for their amino acid composition which shows the presence of N^G, N^G-dimethylarginine, in sizable amount, in non-histone nuclear proteins (NHNP). This modification is most prominent in proteins which are found associated with rapidly-labeled heterogeneous RNA (HnRNP proteins).     

Fluorometric analysis of N alpha-methylamino acids using fluorescamine

The fluorometric amino acid analyzer based on fluorescamine has been utilized for quantitative determination of N^α-methylamino acids. N-Chlorosuccinimide (1 × 10^?3m in 0.05 m HCl) was continuously introduced into the column eluate to convert N^α-methylamino acids to fluorescamine-sensitive methylamine. As little as 100 pmoles of l-N-methylalanine was detectable with a linear fluorescence response up to 10.0 nmoles. Distinction of primary and secondary amino acids was achieved by carrying out duplicate analyses with and without the introduction of the N-chlorosuccinimide solution.     

Some new aspects of the metabolism of phenacetin in the rat

1. Four new metabolites of phenacetin in the urine of the rat are described; these are (i) N-acetyl-S-ethylcysteine, (ii) quinol, (iii) acetamide and (iv) probably N-acetyl-S-2-(4-ethoxyacetanilido)cysteine S-oxide. 2. Metabolites (i), (iii) and (iv) were characterized and estimated by g.l.c., by t.l.c., by paper chromatography, by chemical reactions or by radioactive techniques after administration to rats of [ethyl-¹⁴C]phenacetin and [acetyl-³H]phenacetin; metabolite (ii), which was excreted mainly as conjugates of sulphuric acid and glucosiduronic acid, was measured by paper chromatography and characteristic colour reactions after enzymic and chemical hydrolysis of the conjugates. 3. Small amounts of azoxy-4-[ethyl-¹⁴C]ethoxybenzene and an unknown metabolite were also found in the urine of rats after administration of [ethyl-¹⁴C]phenacetin. 4. The likely mechanisms and some biological implications of these metabolic reactions are discussed.     

NG-Methylarginines: Biosynthesis,biochemical function and metabolism

Summary N^G-Methylarginines (N^G-monomethylarginine, N^G, N^G-dimethylarginine and N^G, N^G-dimethylarginine) occur widely in nature in either proteinbound or in free states. They are posttranslationally synthesized by a group of enzymes called protein methylase I with S-adenosyl-L-methionine as the methyl donor. The enzymes are highly specific not only towards arginine residues but also towards the protein species. Since transmethylation reaction is energy-dependent in the form of S-adenosyl-L-methionine and is catalyzed a group of highly specific enzymes, it is quite logical to assume that the enzymatic methylation of protein-bound arginine residues play an important role in the regulation of the function and/or metabolism of the protein. When determined with histones asin vitro substrates, protein methylase I activity parallels closely the degree of cell proliferation, and the myelin basic protein (MBP)-specific protein methylase I activity decreases drastically in dysmyelinating mutant mouse brain during myelinating period, suggesting an important role played in the formation and/or maintenance of myelin. When the methylated proteins are degraded by intracellular proteolytic enzymes, free N^G-methylarginines are generated. Some of these free N^G-methylarginines, particularly N^G-monomethylarginine, are extensively metabolized by decarboxylation, hydrolysis, transfer of methylamidine and deimination reaction. Recent experiment demonstrates that some of the N^G-methylarginines may be involved in the neutralization of activity of nitric oxide (NO) which has attracted a great deal of attention as vascular smooth muscle relaxation factor.     

METHYLASES OF MYELIN BASIC PROTEIN AND HISTONE IN RAT BRAIN

—The two enzymes methylating myelin basic protein and histone were purified 170- and 250-fold respectively from the cell sap fraction of rat brain. These enzymes methylated only arginine residues of the two proteins. The enzyme activities were present in all organs tested. Testis has the highest, brain a moderate and liver the lowest activity. Most of the activities were present in the cell sap fraction in brain, liver and testis. Methylation of myelin basic protein and histone was examined in both the cell sap and solubilized nuclear fraction of rat brain during life span after birth. The myelin basic protein methylating activity in the cell sap fraction increased during myelination. Histone methylase from the nuclear fraction was highest at birth and dropped rapidly thereafter. The other activities remained unchanged. The natural occurrence of N^G-mono- and N^G,N^G-dimethylarginine residues in histones prepared from rabbit liver was demonstrated.     

Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60

Thermostable N-acylamino acid recemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40° C, was purified to homogeneity and characterized. The relative molecular mass (M _r) of the native enzyme and the subunit was estimated to be 300 000 and 40 000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50° C and 7.5 respectively. The enzyme was stable at 55° C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-l-or d-methionine, N-acetyl-l-valine, N-acetyl-l-tyrosine and N-chloroacetyl-l-valine. In addition, the enzyme also catalyzed the recemization of the dipeptide l-alanyl-l-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and athyl ester derivatives of N-acetyl-d- and l-methionine were not racemized. The apparent K _m values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co²⁺, Mn²⁺ and Fe²⁺ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH₂-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835–840] was above 80%.     

N(omega)-arginine dimethylation modulates the interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic acids

We studied the interaction between a synthetic peptide (sequence Ac-GXGGFGGXGGFXGGXGG-NH₂, where X = arginine, N^ω,N^ω-dimethylarginine, DMA, or lysine) corresponding to residues 676–692 of human nucleolin and several DNA and RNA substrates using double filter binding, melting curve analysis and circular dichroism spectroscopy. We found that despite the reduced capability of DMA in forming hydrogen bonds, N^ω,N^ω-dimethylation does not affect the strength of the binding to nucleic acids nor does it have any effect on stabilization of a double-stranded DNA substrate. However, circular dichroism studies show that unmethylated peptide can perturb the helical structure, especially in RNA, to a much larger extent than the DMA peptide.     

Identification and characterization of the packaging proteins of core 40S hnRNP particles

The proteins involved in protein-RNA and protein-protein interactions to form the core structure of nuclear 40S hnRNP particles in HeLa cells have been identified and characterized. Through complete analysis of nuclear extracts on sucrose density gradients and controlled salt dissociation of particle proteins, six lower molecular weight polypeptides are identified as the protein constituents of the 40S ribonucleoprotein complex which appears in the electron microscope as 210 A spherical particles. 40S hnRNP particles isolated from Chinese hamster lung fibroblasts show a strikingly similar protein composition to the human cells. The proteins are specifically associated with rapidly labeled nonribosomal nuclear RNA. Particle proteins from HeLa cells migrate in polyacrylamide gels as three groups of closely spaced doublets (groups A, B and C) and are present in a simple fixed stoichiometry. The group C proteins (C₁ and C₂ of 42,000 and 44,000 daltons) interact directly with RNA to form a smaller high salt-resistant RNP complex. The group A proteins (A₁ and A₂ of 32,000 and 34,000 daltons) are major nuclear proteins and constitute 60% total particle protein mass. These two proteins are basic with isoelectric points near 9.2 and 8.4, respectively, and are characterized by an unusual amino acid composition, including high glycine (25%) and the unusual modified basic residue identified as N^G,N^G-dimethylarginine. The major particle proteins (A₁ and A₂) interact electrostatically with nucleic acids and apparently function structurally in the packaging and stabilization of hnRNA in a manner analogous to the histones in chromatin υ bodies. The similarity in protein composition of core RNP particles from different cell types (especially in the basic proteins, A₁, A₂ and B₁) is consistent with a conserved particle structure and function in eucaryotes.     

Post-synthetic modifications of nuclear proteins. High mobility group proteins are methylated.

Calf thymus high mobility group proteins 1 and 2 (HMG-1 and HMG-2) were purified to homogeneity from a 0.35 M NaCl extract of chromatin by selective precipitation with trichloroacetic acid followed by ion exchange chromatography on CM-Sephadex. Amino acid analysis of an acid hydrolyzate of HMG-1 and HMG-2 proteins revealed the presence of a ninhydrin-positive peak identified as N^G,N^G-dimethylarginine. Radioactive tracer experiments confirmed the presence of methylated arginine in HMG proteins.     

Basic Amino Acids at the C-Terminus of the Third Intracellular Loop Are Required for the Activation of Phospholipase C by Cholecystokinin-B Receptors

Abstract: In common with other G_q protein-coupled receptors, the third intracellular loop of the cholecystokinin-B (CCK-B) receptor contains three basic amino acids (K333/K334/R335) at the C-terminal segment. To determine the importance of these conserved basic residues in G_q-protein activation and stimulation of phospholipase C, these basic amino acids were mutated. Subsequently, the ability of resulting mutant receptors to activate phospholipase C was investigated by measuring inositol phosphate formation in COS-7 cells and recording Ca²⁺-activated Cl^? currents from Xenopus oocytes. Site-directed mutagenesis was performed to mutate the three basic amino acids, K333/K334/R335, to neutral amino acids, M333/T334/L335. When the resulting mutant CCK-B receptors were expressed in COS-7 cells and Xenopus oocytes, sulfated cholecystokinin octapeptide (CCK-8) failed to induce inositol phosphate formation in COS-7 cells and evoke Ca²⁺-activated Cl^? currents from oocytes. Each basic amino acid was also mutated (K333M, K334T, and R335L). All three single-point mutations resulted in a significant reduction in CCK-8-induced inositol phosphate formation and CCK-8-activated Ca²⁺-dependent Cl^? currents. It is interesting that substituting the basic amino acids, K333/K334/R335, with three other basic residues, R333/R334/K335, did not change the maximal CCK-8-simulated inositol phosphate formation and the amplitude of CCK-8-evoked Ca²⁺-dependent Cl^? currents. Radioligand-binding studies showed that the above-mentioned mutations did not affect the affinity for CCK-8 and receptor expression level in COS-7 cells. These findings suggest that basic amino acids at the C-terminus of the third cytoplasmic loop are required for the signal transduction by CCK-B receptors.     

Enzymatic synthesis of β-lactam antibiotics: A thermodynamic background

The equilibrium constants and the respective standard Gibbs energy changes for hydrolysis of some β-lactam antibiotics have been determined. Native and immobilized penicillin amidase (EC 3.5.1.11) from Escherichia coli has been used as a catalyst. The values of standard Gibbs energy changes corresponding to the pH-independent product of equilibrium concentrations (ΔG⁰_c = ? RT ln K_c) have been calculated. The differences in the structure of the antibiotics nucleus hardly ever affect the value of the pH-independent component of the standard Gibbs energy change (ΔG⁰_c) and value of apparent standard Gibbs energy change at a fixed pH (ΔG^0′_c). At the same time, the value of ΔG⁰_c is more sensitive to the structure of the acyl moiety of the antibiotic; when ampicillin is used instead of benzylpenicillin, ΔG⁰_c increases by ～6.3 kJ mol^?1 (1.5 kcal mol^?1). pH-dependences of the apparent standard Gibbs energy changes for hydrolysis of β-lactam antibiotics have been calculated. The pH-dependences of ΔG^0′_c for hydrolysis of all β-lactam antibiotics have a similar pattern. The thermodynamic pH optimum of the synthesis of these compounds is in the acid pH range (pH < 5.0). The breakage of the β-lactam ring leads to a sharp decrease in the ΔG^0′_c value and a change in the pattern of the pH-dependence. For example, at pH 5.0 ΔG^0′_c decreases from 14.4 kJ mol^?1 for benzylpenicillin to ?1.45 kJ mol^?1 for benzylpenicilloic acid. The reason for these changes is mainly a considerable increase in the pK of the amino group of the nucleus of the antibiotic and, as a consequence, a decrease in the component of standard Gibbs energy change, corresponding to the ionization of the system. The thermodynamic potentials of the enzymatic synthesis of semisynthetic penicillins and cephalosporins on the basis of both free acids and their derivatives (N-acylated amino acids, esters) are discussed. It is shown that with esters of the acids, a high yield of the antibiotic can, in principle, be achieved at higher pH values.     

A gas chromatography-mass spectrometry method for the quantitation of N-nitrosoproline and N-acetyl-S-allylcysteine in human urine: Application to a study of the effects of garlic consumption on nitrosation

Biomarkers in urine can provide useful information about the bioactivation of chemical carcinogens and can be used to investigate the chemoprotective properties of dietary nutrients. N-Nitrosoproline (NPRO) excretion has been used as an index for endogenous nitrosation. In vitro and animal studies have reported that compounds in garlic may suppress nitrosation and inhibit carcinogenesis. We present a new method for extraction and sensitive detection of both NPRO and N-acetyl-S-allylcysteine from urine. The latter is a metabolite of S-allylcysteine, which is found in garlic. Urine was acidified and the organic acids were extracted by reversed-phase extraction (RP-SPE) and use of a polymeric weak anion exchange (WAX-SPE) resin. NPRO was quantified by isotope dilution gas chromatography-mass spectrometry (GC-MS) using [¹³C₅]NPRO and N-nitrosopipecolinic acid (NPIC) as internal standards. This method was used to analyze urine samples from a study that was designed to test whether garlic supplementation inhibits NPRO synthesis. Using this method, 2.4 to 46.0 ng NPRO/ml urine was detected. The method is straightforward and reliable, and it can be performed with readily available GC-MS instruments. N-Acetyl-S-allylcysteine was quantified in the same fraction and detectable at levels of 4.1 to 176.4 ng/ml urine. The results suggest that 3 to 5 g of garlic supplements inhibited NPRO synthesis to an extent similar to a 0.5-g dose of ascorbic acid or a commercial supplement of aged garlic extract. Urinary NPRO concentration was inversely associated with the N-acetyl-S-allylcysteine concentration. It is possible that allyl sulfur compounds found in garlic may inhibit nitrosation in humans.