N-acetylated Forms Research Articles

Binding of the anti‐tubercular drug isoniazid to the arylamine N‐acetyltransferase protein from Mycobacterium smegmatis

Isoniazid is a frontline drug used in the treatment of tuberculosis (TB). Isoniazid is a prodrug, requiring activation in the mycobacterial cell by the catalase/peroxidase activity of the katG gene product. TB kills two million people every year and the situation is getting worse due to the increase in prevalence of HIV/AIDS and emergence of multidrug-resistant strains of TB. Arylamine N-acetyltransferase (NAT) is a drug-metabolizing enzyme (E.C. 2.1.3.5). NAT can acetylate isoniazid, transferring an acetyl group from acetyl coenzyme A onto the terminal nitrogen of the drug, which in its N-acetylated form is therapeutically inactive. The bacterium responsible for TB, Mycobacterium tuberculosis, contains and expresses the gene encoding the NAT protein. Isoniazid binds to the NAT protein from Salmonella typhimurium and we report here the mode of binding of isoniazid in the NAT enzyme from Mycobacterium smegmatis, closely related to the M. tuberculosis and S. typhimurium NAT enzymes. The mode of binding of isoniazid to M. smegmatis NAT has been determined using data collected from two distinct crystal forms. We can say with confidence that the observed mode of binding of isoniazid is not an artifact of the crystallization conditions used. The NAT enzyme is active in mycobacterial cells and we propose that isoniazid binds to the NAT enzyme in these cells. NAT activity in M. tuberculosis is likely therefore to modulate the degree of activation of isoniazid by other enzymes within the mycobacterial cell. The structure of NAT with isoniazid bound will facilitate rational drug design for anti-tubercular therapy.

The synthesis of some deoxygenated analogues of early intermediates in the biosynthesis of glycosylphosphatidylinositol (GPI) membrane anchors

Syntheses are described of 2-azido-4,6-di- O-benzyl-2,3-dideoxy- d- ribo-hexopyranosyl fluoride, 6- O-acetyl-2-azido-3- O-benzyl-2,4-dideoxy- d- xylo-hexopyranosyl fluoride and 2-azido-3,4-di- O-benzyl-2,6-dideoxy- d-glucopyranosyl fluoride. These glycosyl donors were coupled with the acceptor 1 d-2,3,4,5-tetra- O-benzyl-1- O-(4-methoxybenzyl)- myo-inositol and the α-coupled products were transformed into α- d-3dGlc pN-PI, α- d-4dGlc pN-PI and α- d-6dGlc pN-PI by way of the H-phosphonate route. Brief mention is made of the biological evaluation of these deoxy-sugar analogues and their N-acetylated forms as candidate substrate/inhibitors of the N-deacetylase and α-(1→4)- d-mannosyltransferase activities present in trypanosomal and HeLa (human) cell-free system.

The Bacillus subtilis Gne (GneA, GalE) protein can catalyse UDP-glucose as well as UDP- N-acetylglucosamine 4-epimerisation

Mutations in the Bacillus subtilis gene that affect the activity of the uridine diphosphate (UDP)- N-acetylglucosamine (GlcNAc) 4-epimerase (EC 5.1.3.7) were shown to map to galE, the structural gene of the UDP-glucose (Glc) 4-epimerase (EC 5.1.3.2). This genetic evidence that the same enzyme can catalyse the epimerisation of hexoses as well as of their N-acetylated forms is confirmed by in vitro assays with purified enzyme. It appears that in B. subtilis, Gne (GneA, GalE) is involved in two distinct and essential functions, i.e., cell detoxification under certain growth conditions and the biosynthesis of anionic cell wall polymers. We discuss the evidence that such enzymes capable of utilizing both UDP-hexoses and UDP- N-acetylhexosamines are present in other organisms.

Characterization of the Acid Stability of Glycosidically Linked Neuraminic Acid: USE IN DETECTING DE-N-ACETYL-GANGLIOSIDES IN HUMAN MELANOMA

The glycosidic linkage of sialic acids is much more sensitive to acid hydrolysis than those of other monosaccharides in vertebrates. The commonest sialic acids in nature are neuraminic acid (Neu)-based and are typically N-acylated at the C5 position. Unsubstituted Neu is thought to occur on native gangliosides of certain tumors and cell lines, and synthetic de-N-acetyl-gangliosides have potent biological properties in vitro. However, claims for their natural existence are based upon monoclonal antibodies and pulse-chase experiments, and there have been no reports of their chemical detection. Here we report that one of these antibodies shows nonspecific cross-reactivity with a polypeptide epitope, further emphasizing the need for definitive chemical proof of unsubstituted Neu on naturally occurring gangliosides. While pursuing this, we found that alpha2-3-linked Neu on chemically de-N-acetylated G(M3) ganglioside resists acid hydrolysis under conditions where the N-acetylated form is completely labile. To ascertain the generality of this finding, we investigated the stability of glycosidically linked alpha- and beta-methyl glycosides of Neu. Using NMR spectroscopy to monitor glycosidic linkage hydrolysis, we find that only 47% of Neualpha2Me is hydrolyzed after 3 h in 10 mm HCl at 80 degrees C, whereas Neu5Acalpha2Me is 95% hydrolyzed after 20 min under the same conditions. Notably, Neubeta2Me is hydrolyzed even slower than Neualpha2Me, indicating that acid resistance is a general property of glycosidically linked Neu. Taking advantage of this, we modified classical purification techniques for de-N-acetyl-ganglioside isolation using acid to first eliminate conventional gangliosides. We also introduce a phospholipase-based approach to remove contaminating phospholipids that previously hindered efforts to study de-N-acetyl-gangliosides. The partially purified sample can then be N-propionylated, allowing acid release and mass spectrometric detection of any originally existing Neu as Neu5Pr. These advances allowed us to detect covalently bound Neu in lipid extracts of a human melanoma tumor, providing the first chemical proof for naturally occurring de-N-acetyl-gangliosides.

N-Acetylcarnosine, a natural histidine-containing dipeptide, as a potent ophthalmic drug in treatment of human cataracts

A study was designed to document and quantify the changes in lens clarity over 6 and 24 months in 2 groups of 49 volunteers (76 eyes) with an average age of 65.3 ± 7.0 enrolled at the time of diagnosis of senile cataracts of minimal to advanced opacification. The patients received N-acetylcarnosine, 1% sol (NAC) (26 patients, 41 eyes = Group II), placebo composition (13 patients, 21 eyes) topically (two drops, twice daily) to the conjunctival sac, or were untreated (10 patients, 14 eyes); the placebo and untreated groups were combined into the control (reference) Group I. Patients were evaluated upon entry, at 2-month (Trial 1) and 6-month (Trial 2)-intervals for best corrected visual acuity (b/c VA), by ophthalmoscopy and the original techniques of glare test (for Trial 1), stereocinematographic slit-image and retro-illumination photography with subsequent scanning of the lens. The computerized interactive digital analysis of obtained images displayed the light scattering/absorbing centers of the lens into 2-D and 3-D scales. The intra-reader reproducibility of measuring techniques for cataractous changes was good, with the overall average of correlation coefficients for the image analytical data 0.830 and the glare test readings 0.998. Compared with the baseline examination, over 6 months 41.5% of the eyes treated with NAC presented a significant improvement of the gross transmissivity degree of lenses computed from the images, 90.0% of the eyes showed a gradual improvement in b/c VA to 7–100% and 88.9% of the eyes ranged a 27–100% improvement in glare sensitivity. Topographic studies demonstrated less density and corresponding areas of opacification in posterior subcapsular and cortical morphological regions of the lens consistent with VA up to 0.3. The total study period over 24 months revealed that the beneficial effect of NAC is sustainable. No cases resulted in a worsening of VA and image analytical readings of lenses in the NAC-treated group of patients. In most of the patients drug tolerance was good. Group I of patients demonstrated the variability in the densitometric readings of the lens cloudings, negative advance in glare sensitivity over 6 months and gradual deterioration of VA and gross transmissivity of lenses over 24 months compared with the baseline and 6-month follow-up examinations. Statistical analysis revealed the significant differences over 6 and 24 months in cumulative positive changes of overall characteristics of cataracts in the NAC-treated Group II from the control Group I. The N-acetylated form of natural dipeptide L-carnosine appears to be suitable and physiologically acceptable for nonsurgical treatment for senile cataracts.

Structures of two cell wall-associated polysaccharides of a Streptococcus mitis biovar 1 strain. A unique teichoic acid-like polysaccharide and the group O antigen which is a C-polysaccharide in common with pneumococci.

The cell wall of Streptococcus mitis biovar 1 strain SK137 contains the C-polysaccharide known as the common antigen of a closely related species Streptococcus pneumoniae, and a teichoic acid-like polysaccharide with a unique structure. The two polysaccharides are different entities and could be partially separated by gel chromatography. The structures of the two polysaccharides were determined by chemical methods and by NMR spectroscopy. The teichoic acid-like polymer has a heptasaccharide phosphate repeating unit with the following structure: The structure neither contains ribitol nor glycerol phosphate as classical teichoic acids do, thus we have used the expression teichoic acid-like for this polysaccharide. The following structure of the C-polysaccharide repeating unit was established: where AAT is 2-acetamido-4-amino-2,4, 6-trideoxy-D-galactose. It has a carbohydrate backbone identical to that of one of the two structures of C-polysaccharide previously identified in S. pneumoniae. C-polysaccharide of S. mitis is characterized by the presence, in each repeating unit, of two residues of phosphocholine and both galactosamine residues in the N-acetylated form. Immunochemical analysis showed that C-polysaccharide constitutes the Lancefield group O antigen. Studies using mAbs directed against the backbone and against the phosphocholine moiety of the C-polysaccharide revealed several different patterns of these epitopes among 95 S. mitis and Streptococcus oralis strains tested and the exclusive presence of the group O antigen in the majority of S. mitis biovar 1 strains.

Reverse hydrolysis reaction of chitin deacetylase and enzymatic synthesis of β- d-GlcNAc-(1→4)-GlcN from chitobiose

We found that a chitin deacetylase from Colletotrichum lindemuthianum could acetylate free amino sugar residues into N-acetylated forms in the presence of 3.0 M sodium acetate. The result was analyzed using a β- N-acetylhexosaminidase-coupled assay system with p-nitrophenyl 2-amino-2-deoxy-β- d-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β- d-glucopyranoside as the substrate, and the liberation of p-nitrophenol was observed as a consequence of enzymatic N-acetylation of the glucosamine residue at the nonreducing end of the substrate. The chitin deacetylase also acetylated chitobiose and chitotetraose as substrates, which was evidenced by the decrease in the amount of free amino sugar residues in the chitooligosaccharides. The reaction product of chitobiose after the acetylation reaction was exclusively 2-acetamido-2-deoxy-β- d-glucopyranosyl-(1→4)-2-amino-2-deoxy- d-glucose [GlcNAcGlcN], the structure of which was determined by FABMS and NMR analyses. This study offers a novel method for enzymatic N-acetylation of amino sugars, and especially with chitobiose as substrate, a selectively N-acetylated product, GlcNAcGlcN, can be synthesized.

Antitumor benzothiazoles. 8. Synthesis, metabolic formation, and biological properties of the C- and N-oxidation products of antitumor 2-(4-aminophenyl)benzothiazoles.

2-(4-Aminophenyl)benzothiazoles 1 and their N-acetylated forms have been converted to C- and N-hydroxylated derivatives to investigate the role of metabolic oxidation in the mode of action of this series of compounds. 2-(4-Amino-3-methylphenyl)benzothiazole (1a, DF 203, NSC 674495) is a novel and potent antitumor agent with selective growth inhibitory properties against human cancer cell lines. Very low IC(50) values (<0.1 microM) were encountered in the most sensitive breast cancer cell lines, MCF-7 and T-47D, whereas renal cell line TK-10 was weakly inhibited by 1a. Cell lines from the same tissue origin, MDA-MB-435 (breast), CAKI-1 (renal), and A498 (renal), were insensitive to 1a. Accumulation and metabolism of 1a were observed in sensitive cell lines only, with the highest rate of metabolism occurring in the most sensitive MCF-7 and T-47D cells. Thus, differential uptake and metabolism of 1a by cancer cell lines may underlie its selective profile of anticancer activity. A major metabolite in these sensitive cell lines has been identified as 2-(4-amino-3-methylphenyl)-6-hydroxybenzothiazole (6c). Hydroxylation of 1a was not detected in the homogenate of previously untreated MCF-7, T-47D, and TK-10 cells but was readily observed in homogenates of sensitive cells that were pretreated with 1a. Accumulation and covalent binding of [(14)C]1a derived radioactivity was observed in the sensitive MCF-7 cell line but not in the insensitive MDA-MB-435 cell line. The mechanism of growth inhibition by 1a, which is unknown, may be dependent on the differential metabolism of the drug to an activated form by sensitive cell lines only and its covalent binding to an intracellular protein. However, the 6-hydroxy derivative 6c is not the 'active' metabolite since, like all other C- and N-hydroxylated benzothiazoles examined in this study, it is devoid of antitumor properties in vitro.

The First Total Synthesis of 6-Sulfo-de-N-acetylsialyl Lewis(x) Ganglioside: A Superior Ligand for Human L-Selectin.

Originally discovered as a minor by-product of 6-sulfo-N-acetylsialyl Lewis(x) , the de-N-acetylated form 1 is a superior L-selectin ligand to the N-acetyl form. To substantiate the extraordinary reactivity of 1, it was synthesized for the first time and its binding to L-selectin investigated. Compound 1 and related structures may be high-affinity endogenous ligands for L-selectin that are involved in the interaction of leukocytes with the vascular endothelium.

Kinetics of formation of fluorescent products from hexanal and L-lysine in a two-phase system.

Kinetics of formation of fluorescent condensation products from hexanal and L-lysine (or its N-acetylated forms) including mass-transfer has been studied in a two-phase system consisting of lysine (or lysine derivative) in an aqueous phosphate buffer and a 1-octanol solution of hexanal as model for formation of fluorophores between protein and carbonyl compounds in peroxidizing biological systems. The initial rate of formation of fluorescent products in the aqueous phase was found to be proportional to the concentration of hexanal and lysine and to increase in both phases with increasing pH in the aqueous phase, in contrast to a higher-order dependence on hexanal in the octanol phase. At pH = 6.8, the temperature dependence of the appearance of fluorescent products corresponds to apparent energies of activation of 63 kj.mol-1 and 87 kj.mol-1 in the aqueous phase and the octanol phase, respectively. Fluorescent condensation products appeared faster in the octanol phase. However, by a kinetic analysis, the fluorescent products were shown to be formed in the aqueous phase, corresponding to the lower energy of activation and to the simple second-order kinetics, and subsequently distributed between the aqueous phase and the octanol phase. L-Lysine reacted faster than N alpha-acetyl-L-lysine which reacted faster than N epsilon-acetyl-L-lysine. Using fluorescence quantum yields, determined to be 1.4.10(-2) in octanol and 8.10(-3) in water at pH 6.8, an apparent partition coefficient of 17 (octanol/water) was determined for the condensation product of L-lysine. The steady-state fluorescence in the octanol phase was attributed to two components with fluorescence lifetimes at 25 degrees C of 0.7 +/- 0.05 ns and 5.1 +/- 0.2 ns, assigned to hexanal and the condensation product, respectively. The emission spectra were resolved in the two components using phase-sensitive detection, and the condensation product had emission maximum at 405 nm.

Cadmium-Induced Metallothionein Expression during Embryonic and Early Larval Development of the MolluscCrassostrea virginica

Newly fertilized eggs of the oysterCrassostrea virginicawere exposed to 0.2 μMCd and sampled during the first 24 hr of embryonic and larval development for determination of the temporal patterns of total bioaccumulated and metallothionein (MT)-bound Cd concentrations and the concentrations of MT mRNA. In comparison with controls, exposure to this concentration of Cd resulted in delayed development to the D-stage veliger larval stage. Maternal MT mRNA, which was carried over into the egg, declined immediately after fertilization. In controls, levels of MT mRNA were stable after this initial decline, and little or no detectable Cd was bound to MTs. Accumulation of new MT mRNA was observed in the exposed group after 12 hr of exposure when increases due to induction by Cd had occurred. Total accumulated Cd concentrations, which ranged from 0.04 to 0.1 pmol Cd/μg DNA in unexposed individuals, increased to 13.5 pmol Cd/μg DNA after 24 hr in Cd-exposed individuals. There was an increase in MT-bound Cd in exposed individuals prior to MT induction, which may be explained in part as Cd binding to maternal MT. This was detected as binding to CvNAcMT, the expected N-acetylated form of the oyster MT. Amounts of Cd bound to this form of MT increased, thereafter, and was followed by increases in binding to CvMT, the unexpected, nonacetylated form of the oyster MT, which has been associated with Cd toxicity, possibly in relation to the effects of Cd on normal cotranslational processing.

Standardization of acid hydrolysis procedure for urinary 3-methylhistidine determination by high-performance liquid chromatography

The N-acetylated form of N-methylhistidine (3-methylhistidine, 3-meH), a non-invasive marker of proteolysis, accounts for 80–90% of total 3-meH excretion (acetylated+non-acetylated 3-meH) in the rat. To determine total 3-meH excretion, samples require acid hydrolysis prior to determination by high-performance liquid chromatography. This study evaluated the stability of 3-meH at various times and temperatures of hydrolysis and determined the optimal conditions for hydrolysis of samples. Increasing temperature (120°C) results in significant degradation of 3-meH with no appreciable change in concentration being noted at 80°C. Hydrolysis at 100°C for 1.5 to 4 h or 80°C for 8 to 12 h is recommended for determining total 3-meH concentrations in rat urine.

POMC-related products in the intermediate pituitary of the amphibian, Bufo marinus: differential subcellular processing in the Golgi and secretory granules.

In the intermediate pituitary of the anuran amphibian, Bufo marinus, the N-acetylation of ACTH(1-13)-NH2 to yield alpha-MSH occurs as a cosecretory processing event, whereas the N-acetylation of beta-endorphin occurs as a posttranslational processing event. To understand how these two N-acetylation reactions are segregated, B. marinus intermediate pituitary cells were analyzed by immunogold labeling electron microscopy, and by using an ultracentrifugation procedure. The immunogold labeling studies indicated that ACTH(1-13)-NH2-related immunoreactivity was colocalized with N-acetylated beta-endorphin-related immunoreactivity in secretory granules. Furthermore, ACTH(1-13)-NH2-related immunoreactivity was not detected in either the ER or the Golgi. N-Acetylated beta-endorphin-related immunoreactivity, however, was detected in the Golgi. Ultracentrifugation analysis revealed that in an ER/microsomal fraction, beta-LPH-sized and nonacetylated beta-endorphin-sized immunoreactive material were present in a molar ratio of 1:2. No N-acetylated forms of beta-endorphin were detected in the ER/microsomal fraction. In a Golgi/secretory granule fraction, the molar ratio of beta-LPH to beta-endorphin was 1:9 with 58% of the beta-endorphin being N-acetylated. Collectively, these data support the following hypotheses. The proteolytic cleavage of ACTH (1-39) to yield ACTH (1-13)-NH2 is a late processing event occurring in secretory granules. The cleavage of beta-LPH to yield nonacetylated beta-endorphin is an early processing event that may occur in the ER or the Golgi. Because N-acetylated beta-endorphin and nonacetylated ACTH(1-13)-NH2 are colocalized in secretory granules, it appears, therefore, that the N-acetylation of beta-endorphin is completed prior to loading into secretory granules. Thus, there is a spatial and temporal separation of the posttranslational processing events associated with the beta-LPH portion and ACTH portion of the POMC biosynthetic pathway in amphibian intermediate pituitary cells.

Pharmacokinetics of the garlic compound S-allylcysteine.

The pharmacokinetic behavior of S-allylcysteine (SAC), one of the biologically active transformation products from garlic, was investigated after oral administration to rats, mice, and dogs. SAC was rapidly and easily absorbed in the gastrointestinal tract and distributed mainly in plasma, liver, and kidney. The bioavailability was 98.2, 103.0, and 87.2% in rats, mice, and dogs, respectively. SAC was mainly excreted into urine in the N-acetyl form in rats; however, mice excreted both SAC and the N-acetyl form. The half-life of SAC was longer in dogs than in rats and mice.

Studies on biogenic amines and metabolites in nervous tissue and hemolymph of male cabbage looper moths—III. Fate of injected octopamine, 5-hydroxytryptamine and dopamine

Adult male cabbage looper moths, Trichoplusia ni, were injected with two dosages of octopamine, or 5-hydroxytryptamine, or dopamine. The dosages were 1 and 10 μg, the latter a dosage that was previously shown to induce changes in nocturnal locomotor activity (5-hydroxytryptamine) and sensitivity to sex pheromone (octopamine). Injections were made into the hemolymph in the head capsule dorsal to the supraesophageal ganglion. Males were injected 1 hr prior to the onset of scotophase, and then brains + optic lobes, thoracic ganglia and hemolymph were analyzed at five time intervals over the following 6 hr. Levels of the three amines and their N-acetylated metabolites were quantified using HPLC with electrochemical detection. With the 1 μg dosage of the three amines, there was a significant increase in hemolymph values, but no changes in levels in the nervous system. With the 10 μg dosage, significant increases in each amine were found in the hemolymph, thoracic ganglia and brain + optic lobes. Levels of injected amines then rapidly decreased from hemolymph and nervous tissue. Within 2.5 hr following injections, levels of octopamine and dopamine in the brain + optic lobes and thoracic ganglia stabilized at twice the level in saline injected controls, 5-hydroxytryptamine levels were below control levels and hemolymph levels of each amine were not significantly different from controls. Decreases in amine levels were accompanied by significant increases in the N-acetylated form of each amine. In no instance did we observe a measurable peak for metabolites that would suggest the presence of MAO activity.

Analysis of the Post-translational Processing of α-MSH in the Pituitaries of the Chondrostean Fishes, Acipenser transmontanus and Polyodon spathula

Multiple forms of α-MSH were isolated from the pituitaries of the white sturgeon, Acipenser transmontanus, and the paddlefish, Polyodon spathula, following fractionation of the pituitary extracts by reversed-phase HPLC. In both species, immunoreactive forms which eluted with the same retention times as synthetic mammalian ACTH(1-13)NH 2, monoacetylated α-MSH, and diacetylated α-MSH were detected. In the pituitary of the sturgeon, N-acetylated forms of α-MSH represented 91% of the total α-MSH isolated. However, in the pituitary of the paddlefish, N-acetylated forms of α-MSH accounted for only 45% of the total α-MSH isolated. The presence of N-acetylated forms of α-MSH in the pituitaries of representatives of the two extant genera of chondrostean fish coupled with the observation that N-acetylated forms of α-MSH are present in the pituitaries of cladistian and neopterygian fishes indicates that the α-MSH specific N-acetylation mechanism evolved prior to the radiation of the Actinopterygii. A conspicuous feature of the chondrostean α-MSH N-acetylation reaction was the low levels of diacetylated α-MSH produced relative to the levels of monoacetylated α-MSH. Similar observations have been made for the cladistian fish, Calamoichthys calabaricus, and the cartilaginous fish, Hydrolagus colliei. However, in holostean fishes, lungfishes, and tetrapods, the diacetylated form of α-MSH is the major end product produced in the intermediate pituitary. The phylogenetic implications of these observations will be discussed.

Structural characterization of three new AAL toxins produced by Alternaria alternata f. sp. lycopersici

Three new pairs of biologically active regioisomeric toxins (AAL toxins TC, TD, and TE) were isolated from liquid cultures of Alternaria alternata f. sp. lycopersici, purified using standard chromatographic procedures, and their structures elucidated following interpretation of spectra obtained from NMR and mass spectrometry experiments. Each of the toxin congeners is structurally similar to the AAL toxin TA, which was characterized earlier from culture filtrates of this fungus. AAL toxin TC resembles TA but differs in its lack of hydroxyl groups at C 4 and C 5 . AAL toxin TE is the N-acetylated form of TC [...]

Mass spectrometric analysis of rabbit and bovine trypsin-solubilized cytochrome b5

The sequence and blocking group of the amino-terminal 15 amino acids of rabbit trypsin-solubilized cytochrome b5 were determined by liquid secondary ion mass spectrometry (LSIMS) and tandem mass spectrometry (MS/MS). The molecular weights of peptides generated from a Staphylococcus aureus V8 protease digest of this protein were determined by LSIMS analysis and the two peptides containing the blocked amino-terminus were sequenced by tandem mass spectrometry to yield the sequence; N-acetyl-Ala-Ala-Glu-Ser-Asp-Lys-Asp-Val-Lys-Tyr-Tyr-Thr-Leu-Glu-Glu. Comparison of this sequence with a recently reported cDNA sequence (Dariush et al., 1988) indicates that Gln at position 3 is selectively deamidated, although no other discrepancies were found. Intact rabbit and bovine trypsin-solubilized cytochrome b5 were also analyzed by LSIMS on a high-field mass spectrometer equipped with a diode array detector. Mass measurement of the unresolved protonated molecular ion peak tops gave average molecular weights of 9462.2 +/- 2 and 9502.3 +/- 2 for bovine and rabbit trypsin-solubilized cytochrome b5, respectively. In both cases, these molecular weights correspond to a cytochrome b5 fragment consisting of amino acids Asp(7)-Arg(88). The average molecular weight for the rabbit amino-terminal-blocked form of trypsin-solubilized cytochrome b5 was found to be 10,144.5 +/- 2, which was consistent with the molecular weight predicted for the extended N-acetylated form (residues 1-88) of Mr 10,146.1.

Analysis of Carnosine, Homocarnosine, and Other Histidyl Derivatives in Rat Brain

Isocratic reverse-phase analytical HPLC has been used to examine naturally occurring imidazoles of rat brain. Elution of brain extracts with a phosphate buffer mobile phase from columns packed with Hypersil ODS (5 microns) resulted in good separation of the well-documented brain imidazole-containing dipeptides carnosine and homocarnosine. Measured concentrations corresponded to published values. Several further peaks observed had properties consistent with those of N-acetyl derivatives of compounds related to carnosine and homocarnosine. N-Acetyl forms not commercially available were prepared and their identities verified by nuclear magnetic resonance spectroscopy. A number of these had chromatographic properties identical to those of compounds in brain extracts. Fractions corresponding to some of the peaks were examined using staining systems specific for certain chemical features and compared with results obtained for commercial or synthetic standards. The results of these tests supported the chromatographic data. Thus, chromatographic and microchemical evidence is presented for the existence of N-acetyl forms of histidine, 1-methylhistidine, carnosine, anserine, and homocarnosine in rat brain.

Detection of N-acetylated forms of β-endorphin and nonacetylated α-MSH in the intermediate pituitary of the toad, Bufo marinus

Steady-state analysis of the acid extracts of the intermediate pituitary of the toad, Bufo marinus, revealed the presence of multiple forms of β-endorphin and α-MSH. Approximately 98% of the immunoreactive β-endorphin was N-acetylated. The major form of N-acetylated β-endorphin, which represented 81.5% of the total β-endorphin recovered from this tissue, had an apparent molecular weight of 1.2 kDa and a net charge of + 1 at pH 2.75. Approximately 98% of the immunoreactive α-MSH present in the Bufo intermediate pituitary had reverse phase HPLC properties similar to the nonacetylated form of α-MSH, ACTH(1–13)amide. These observations are in agreement with studies on the intermediate pituitary of the frog, Xenopus laevis, which have shown that the N-acetylation of α-MSH in this species is a cosecretory processing event, whereas the N-acetylation of β-endorphin is a posttranslational processing event (2, 5, 15). These observations indicate that the N-acetylation of β-endorphin and α-MSH occurs at distinct subcellular sites in intermediate pituitary cells of anuran amphibians. The Bufo intermediate pituitary will serve as a good model system for studying these novel N-acetyltransferase reactions.