Inhibition of enzymic incision of thymine dimers by covalently bound 2-[N-[(deoxyguanosin-8-yl)acetyl]amino]fluorene in deoxyribonucleic acid

The effects of DNA adducts of the carcinogen 2-[N-(acetoxyacetyl)amino]fluorene on enzymic incision of thymine dimers was investigated. Escherichia coli DNA labeled with [3H]thymidine was reacted with the carcinogen. Thymine dimers were then introduced into the modified DNA by irradiation with monochromatic 254-nm light in the presence of the photosensitizer silver nitrate. This DNA containing both types of damages, mainly 2-[N-[(deoxyguanosin-8-yl)acetyl]fluorene and thymine dimers, was then used as substrate for pyrimidine dimer-DNA glycosylase, purified from E. coli infected by bacteriophage T4. Activity was assayed by measuring release of free labeled thymine after photoreversal of the enzyme-reacted DNA by 254-nm light. The Vmax of the enzyme was decreased when it was reacted with the extensively arylamidated substrate. This inhibition of incision of pyrimidine dimers was increased with the number of carcinogen-DNA adducts, although no enzymic activity against modified guanines was present. Therefore, carcinogen-modified purine moieties can interfere with initiation of excision repair of ultraviolet-induced pyrimidine dimers. This suggests an indirect pathway by which modified DNA bases can be mutagenic.     

Human placental estradiol 17 beta-dehydrogenase. Identification of a single histidine residue affinity-labeled by both 3-bromoacetoxyestrone and 12 beta-bromoacetoxy-4-estrene-3,17-dione

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity-labeled at pH 6.3 by 3-bromo[2'-14C]acetoxyestrone and 12 beta-bromo-[2'-14C] acetoxy-4-estrene-3,17-dione (both are substrates) in separate incubations. The affinity-alkylated enzyme samples were then treated separately as described below. Amino acid compositions of both samples revealed radioactive 3-carboxymethylhistidine. Tryptic digests of each sample were prepared, applied to Sephadex G-50, and 3-carboxymethylhistidine-bearing fractions identified. These peptides were further purified by cation exchange chromatography, gel filtration, and paper electrophoresis. The purified, 3-carboxymethylhistidine-bearing peptides labeled by the two steroids had identical electrophoretic mobilities at pH 6.5, 3.5, and 1.9. The amino acid sequence of the radioactive peptide alkylated by 3-bromo[2'-14C]acetoxyesterone was determined as: Leu-Ala-3-[14C]CmHis-Ser-Lys. The smaller quantity of peptide obtained from the inactivation with 12 beta-bromo[2'-14C]acetoxy-4-estrene-3,17-dione precluded the determination of its complete sequence. However, the first 3 residues were found to be Leu-Ala-3-[14C]CmHis and the amino acid composition showed that serine and lysine were also present. It is concluded that the steroid-binding site of human placental estradiol 17 beta-dehydrogenase contains a histidine residue which proximates the upper A-ring region of the steroid as it undergoes the reversible binding step.     

The putative 15 S precursor of globin mRNA contains a poly (A) sequence

[3H] Uridine or [3H] adenosine pulse-labelled nuclear RNA was isolated from chicken immature red blood cells and separated on denaturing formamide sucrose gradients. RNA of each gradient fraction was hybridized with unlabelled globin DNA complementary to mRNA (cDNA) and subsequently digested by RNAase A and RNAase T1. The experiments revealed two RNA species with globin coding sequences sedimenting 9 S and approx. 15 S, the latter probably representing a precursor of 9 S globin mRNA. A poly (A) sequence was demonstrated in this RNA by two different approaches. Nuclear RNA pulse-labelled with [3H] uridine was fractionated by chromatography on poly (U)-Sepharose. Part of the 15 S precursor was found in the poly(A)-containing RNA. In the second approach 15 S RNA pulse-labelled with [3H]adenosine was hybridized with globin cDNA, incubated with RNAase A and RNAase T1 and subjected to chromatography on hydroxyapatite. The hybrids were isolated and after separation of the strands degraded with DNAase I, RNAase A and RNAase T1. By this procedure poly(A) sequences of approximately 100 nucleotides could be isolated from the 15 S RNA with globin coding sequences. The poly(A) sequence was completely degraded by RNAase T2.     

Affinity labeling of the membrane protein-binding component of human polymorphonuclear leukocyte receptors for leukotriene B4.

A radiolabeled N-(3-aminopropyl)-leukotriene B4 amide ([3H]LTB4-APA) analog of the potent leukocyte chemotactic factor leukotriene B4 (LTB4) binds to receptors for LTB4 in plasma membrane-enriched preparations from human blood polymorphonuclear leukocytes (PMNL) and intact PMNL with respective mean dissociation constants of 2.3 nM and 69 nM at 4 degrees C. The [3H]LTB4-APA bound to plasma membrane-enriched preparations from PMNL was covalently cross-linked to membrane proteins with disuccinimidyl suberate. Solubilization and resolution by SDS-PAGE of proteins from [3H]LTB4-APA-labeled PMNL membranes revealed predominant labeling of a 60-kDa protein. Labeling of the PMNL membrane protein was inhibited by LTB4 and its analogs at concentrations similar to those inhibiting the binding of [3H]LTB4 to its receptor, with an identical rank order of potency of LTB4 greater than 20-hydroxy-LTB4 greater than LTB4-APA = 5(S),12(R)-dihydroxy-eicosa-14-cis-6,8,10-trans-tetraenoic acid much greater than LTD4 = LTC4. GTP suppressed the labeling of the 60-kDa PMNL membrane protein to an extent consistent with the decrease in receptor affinity for LTB4 induced by GTP. The stereospecificity of the affinity cross-linking reaction and the regulation by GTP support the identification of an approximately 60-kDa protein as the binding component of the PMNL receptor for LTB4.     

N-nitroso-N-2-fluorenylacetamide: a new direct-acting mutagen and teratogen

Reaction of N-2-fluorenylacetamide (2-FAA, CAS No. 53-96-3) with nitrous fume (N2O3) in glacial acetic acid at 0 degree C yields N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA), 3-nitro-N-2-fluorenylacetamide, N-nitroso-3-nitro-N-2-fluorenylacetamide and other compounds. N-NO-2-FAA is the major product (80%) and fairly stable at low temperature (-20 degrees C), but extremely labile at ambient temperature. The chemical structure of N-NO-2-FAA is characterized by spectrometric analysis of its naphthol coupling derivatives. This new compound is highly mutagenic to Salmonella typhimurium TA97, TA98, TA100 and TA1538 and requires no microsomal metabolic activation. The mutagenicity of N-NO-2-FAA in TA98 is higher than that of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, CAS No. 70-25-7) and N-acetoxy-N-2-fluorenylacetamide (N-AcO-2-FAA). The teratogenic potential of N-NO-2-FAA was studied with white Leghorn chick embryos given a single dose of 1-100 micrograms/egg on day 6 of incubation. A high incidence of flaccid paralysis of the legs and a low incidence of feather, claw and bill malformations were found in the treated group; no such malformed embryos were found in the control group. The teratogenicity of N-NO-2-FAA was found to be weaker than that of MNNG, but comparable to that of N-methyl-N-nitrosourea (CAS No. 684-93-5). N-NO-2-FAA is a strong electrophile and reacts readily with histidine, lysine, cysteine, glutathione, tryptophan, adenosine, cytidine at neutral pH. In contrast to N-AcO-2-FAA, N-NO-2-FAA does not react significantly with guanosine and thymidine. It seems that N-NO-2-FAA is a strong direct-acting mutagen and probably a new prototype of synthetic carcinogen.     

Two isoforms of eIF-5A in chick embryo. Isolation, activity, and comparison of sequences of the hypusine-containing proteins.

Eukaryotic translation initiation factor 5A (eIF-5A) (older terminology, eIF-4D) is unique in that it contains the unusual amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Hypusine is formed by a post-translational event in which a specific lysine residue is modified by a structural contribution from spermidine. Metabolic labeling of chick embryo fibroblasts with [3H]spermidine or [3H]lysine gives rise to two distinct proteins, designated I (approximately 20 kDa and pI 5.6) and II (approximately 18 kDa and pI 5.35), that contain [3H]hypusine. Upon incubation with [3H]lysine the labeling of the two proteins followed a similar time course and showed approximately the same ratio over the 6-h incubation period. [3H]Hypusine-containing proteins from cells which had been cultured with [3H]spermidine were employed as tracers for isolation of hypusine-containing proteins from whole chick embryos. Four such proteins were obtained. Two of these proteins, I and II, correspond to the two native proteins synthesized in chick embryo fibroblasts; the other two forms, Ia and IIa, displayed properties suggesting that they were derived from the native proteins, I and II, respectively, during purification. The amino acid compositions and the tryptic peptide maps of the 20-kDa protein (I) and the 18 kDa protein (II) suggest that they are closely related but distinct proteins. In fact, amino acid sequence analysis of the two major proteins revealed differences in the polypeptide backbone of the two proteins. In spite of structural differences, the two native forms (I and II), as well as the two altered forms (Ia and IIa), were effective in stimulating methionyl-puromycin synthesis, providing evidence that they are indeed functional isoforms of eIF-5A.     

Mass spectrometric analysis of catechol-histidine adducts from insect cuticle

Adducts of catechols and histidine, which are produced by reactions of 1,2-quinones and p-quinone methides with histidyl residues in proteins incorporated into the insect exoskeleton, were characterized using electrospray ionization mass spectrometry (ESMS), tandem electrospray mass spectrometry (ESMS-MS, collision-induced dissociation), and ion trap mass spectrometry (ITMS). Compounds examined included adducts obtained from acid hydrolysates of Manduca sexta (tobacco hornworm) pupal cuticle exuviae and products obtained from model reactions under defined conditions. The ESMS and ITMS spectra of 6-(N-3')-histidyldopamine [6-(N-3')-His-DA, pi isomer] isolated from M. sexta cuticle were dominated by a [M + H]+ ion at m/z 308, rather than the expected m/z 307. High-resolution fast atom bombardment MS yielded an empirical formula of C14H18N3O5, which was consistent with this compound being 6-(N-1')-histidyl-2-(3, 4-dihydroxyphenyl)ethanol [6-(N-1')-His-DOPET] instead of a DA adduct. Similar results were obtained when histidyl-catechol compounds linked at C-7 of the catechol were examined; the (N-1') isomer was confirmed as a DA adduct, and the (N-3') isomer identified as an (N-1')-DOPET derivative. Direct MS analysis of unfractionated cuticle hydrolysate revealed intense parent and product ions characteristic of 6- and 7-linked adducts of histidine and DOPET. Mass spectrometric analysis of model adducts synthesized by electrochemical oxidative coupling of N-acetyldopamine (NADA) quinone and N-acetylhistidine (NAcH) identified the point of attachment in the two isomers. A prominent product ion corresponding to loss of CO2 from [M + H]+ of 2-NAcH-NADA confirmed this as being the (N-3') isomer. Loss of (H2O + CO) from 6-NAcH-NADA suggested that this adduct was the (N-1') isomer. The results support the hypothesis that insect cuticle sclerotization involves the formation of C-N cross-links between histidine residues in cuticular proteins, and both ring and side-chain carbons of three catechols: NADA, N-beta-alanyldopamine, and DOPET.     

Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol.

The cellular receptor for human urokinase-type plasminogen activator (u-PAR) is shown by several independent criteria to be a true member of a family of integral membrane proteins, anchored to the plasma membrane exclusively by a COOH-terminal glycosyl-phosphatidylinositol moiety. 1) Amino acid analysis of u-PAR after micropurification by affinity chromatography and N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 2-3 mol of ethanolamine/mol protein. 2) Membrane-bound u-PAR is efficiently released from the surface of human U937 cells by trace amounts of purified bacterial phosphatidylinositol-specific phospholipase C. This soluble form of u-PAR retains the binding specificity toward both u-PA and its amino-terminal fragment holding the receptor-binding domain. 3) Treatment of purified u-PAR with phosphatidylinositol-specific phospholipase C or mild alkali completely alters the hydrophobic properties of the receptor as judged by temperature-induced detergent-phase separation and charge-shift electrophoresis. 4) Biosynthetic labeling of u-PAR was obtained with [3H]ethanolamine and myo-[3H]inositol. 5) Finally, comparison of amino acid compositions derived from cDNA sequence and amino acid analysis shows that a polypeptide of medium hydrophobicity is excised from the COOH terminus of the nascent u-PAR. A similar proteolytic processing has been reported for other proteins that are linked to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor.     

Use of p-aminobenzoic acid and tritiated cyanoborohydride for the detection of pyruvoyl residues in proteins

A procedure for the detection of covalently bound pyruvic acid in purified proteins or in crude extracts is described. The dialyzed sample is first treated with sodium cyanoborohydride to reduce any Schiff bases present and then incubated with p-aminobenzoic acid and sodium [3H]cyanoborohydride. Derivatized proteins are visualized by fluorography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel slices containing the labeled proteins are hydrolyzed, and, after removal of polyacrylic acid, the hydrolysate is subjected to ion-exchange high-performance liquid chromatography. The presence of pyruvic acid is established by the detection of a tritiated, 280-nm absorbing compound with a retention time corresponding to that of synthetic N-(p-carboxyphenyl)alanine. The procedure is capable of detecting protein-bound pyruvic acid in the picomolar range and is easily modified to screen for other covalently bound keto acids.     

Interaction of benzo[alpha]pyrene diol-epoxide with nuclei and isolated chromatin.

Chicken erythrocyte chromatin and nuclei were labeled with benzo[alpha]-pyrene (B[alpha]P) diol-epoxide (anti) and digested with micrococcal nuclease to mono- and dinucleosomes. Analysis of the distribution of the carcinogen showed that the internucleosomal region bound 3-4 times more carcinogen per unit DNA than did nucleosomes. The enhanced binding of the 'ultimate' carcinogen to the internucleosomal region was similar when isolated chromatin or nuclei were used for in vitro labeling. Furthermore, isolation of the histone core proteins, H2A, H2B, H3 and H4, revealed that only 15% of the carcinogen was associated with the histones and that the majority of the carcinogen was bound to chromosomal DNA. Fluorography of purified nucleosomal histones showed that the covalent association of the carcinogen was mainly with histones H3 and H2B.     

Revised mechanism for inactivation of mitochondrial monoamine oxidase by N-cyclopropylbenzylamine

A mechanism previously proposed for inactivation of monoamine oxidase (MAO) by N-cyclopropylbenzylamine (N-CBA) [Silverman, R. B., & Hoffman, S. J. (1980) J. Am. Chem. Soc. 102, 884-886] is revised. Inactivation of MAO by N-[1-3H]CBA results in incorporation of about 3 equiv of tritium into the enzyme and release of [3H]acrolein. Treatment of inactivated enzyme with benzylamine, a reactivator for N-CBA-inactivated MAO, releases only 1 equiv of tritium as [3H]acrolein concomitant with reactivation of the enzyme. Even after MAO is inactivated by N-[1-3H]CBA, the reaction continues. At pH 7.2, a linear release of [3H]acrolein is observed for 70 h, which produces 55 equiv of [3H]acrolein while 2.3 equiv of tritium is incorporated into the enzyme. At pH 9, only 3.5 equiv of [3H]acrolein is detected in solution after 96 h, but 40 equiv of tritium is incorporated into the enzyme, presumably as a result of greater ionization of protein nucleophiles at the higher pH. N-[1-3H]Cyclopropyl-alpha-methylbenzylamine (N-C alpha MBA) produces the same adduct as N-CBA but gives only 1-1.35 equiv of tritium bound after inactivation of the enzyme. Denaturation of labeled enzyme results in reoxidation of the flavin without release of tritium, indicating attachment is not to the flavin but rather to an amino acid residue. Enzyme inactivated with N-[1-3H]C alpha MBA is reactivated by benzylamine with the release of 1 equiv of [3H]acrolein, which must have come from an adduct attached to an active site amino acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial phosphate transport: Correlation between alkylation of membrane proteins with N-[3H]ethylmaleimide and inhibition of transport

N-ethylmaleimide inhibits the mitochondrial phosphate carrier. Mitochondria were titrated with N-[³H]ethylmaleimide, dissolved in dodecylsulfate-mercaptoethanol, and their proteins separated on dodecylsulfate-polyacrylamide gels. While the phosphate transport is essentially insensitive to low concentrations of N-ethylmaleimide, the six primary N-ethylmaleimide reactive inner membrane proteins are labeled in direct proportion to the amount of inhibitor added. The reaction of N-[³H]ethylmaleimide with proteins I and III is independent of the preincubation of the mitochondria with p-mercuribenzoic acid, a membrane impermeable inhibitor of the transport. Comparing the alkylation of proteins II, IV, V and VI with the inhibition of phosphate transport, it is found that only proteins IV (45,000 daltons) and V (32,000 daltons) are maximally labeled at the same N-[³H]ethylmaleimide concentration that maximally inhibits the transport.     

Thiol-based inhibitors of mammalian collagenase. Substituted amide and peptide derivatives of the leucine analogue, 2-[(R,S)-mercaptomethyl]-4-methylpentanoic acid

To define the inhibitory requirements of mammalian collagenase, several N-substituted amide and peptide derivatives of the mercaptomethyl analogue of leucine, 2-[(R,S)mercaptomethyl]-4-methylpentanoic acid (H psi[SCH2]-DL-leucine), were synthesized and tested as inhibitors of pig synovial collagenase with soluble type I collagen as substrate. H psi[SCH2]-DL-leucine (IC50 = 320 microM) was about 10 times more potent than the beta-mercaptomethyl compound, N-acetylcysteine. The amide of H psi[SCH2]-DL-leucine was six times more potent than the parent thiol acid. Aliphatic N-substituted amides were less potent than the unsubstituted amide, whereas the N-benzyl amide was slightly more potent. Dipeptides, particularly those with an aromatic group at P2', were up to 20-fold more potent, while tripeptides with an aromatic L-amino acid at P2' and Ala-NH2 at P3' were up to 2200 times more potent than H psi[SCH2]-DL-leucine. The resolved diastereomers of H psi[SCH2]-DL-Leu-Phe-Ala-NH2 inhibited by 50% at 0.3 and 0.04 microM, respectively. The most potent inhibitor synthesized, an isomer of H psi[SCH2]-DL-Leu-L-3-(2'-naphthyl)alanyl-Ala-NH2, exhibited an IC50 of 0.014 microM, a value about 300 times less than similar thiol-based analogues of the P'-cleavage sequence of type I collagen, H psi[SCH2]-DL-Leu-Ala-Gly-Gln-. These structure-function studies establish within the present series of compounds that the most effective inhibitors of mammalian collagenase are not closely related to the P2'-P3' elements of the cleavage site of the natural substrate but rather have an aromatic group at the P2' position and Ala-NH2 at the P3' position.     

The biosynthesis of brain gangliosides. Separation of membranes with different ratios of ganglioside sialylating activity to gangliosides.

Brain subcellular fractions were analysed for ganglioside-sialylating activity by measuring the incorporation of N-[3H]acetylneuraminic acid from CMP-N-[3H]acetylneuraminic acid into endogenous ganglioside acceptors (endogenous incorporation) and into exogenous lactosyceramide (haematoside synthetase activity). The ratios of endogenous incorporation to gangliosides and of haematoside synthetase to gangliosides for the synaptosomal and mitochondrial fractions from a washed crude mitochondrial fraction were lower than those obtained for other membrane fractions. The differences appear to reflect intrinsic characteristics of each membrane fraction. The results of labelling in vitro and the time course of labelling of gangliosides of the different subcellular fractions in vivo after injection of N-[3H]acetylmannosamine are consistent with the possibility of a subcellular site for synthesis of gangliosides different from that of ganglioside deposition.     

Palmitate uptake by neonatal rat myocytes and hepatocytes. Role of extracellular protein.

The role of extracellular binding proteins in the rate of [3H]palmitate uptake by neonatal cardiac myocytes and hepatocytes was investigated using a model-independent approach. Binding proteins used in this study included alpha1-acid glycoprotein [isoelectric point (pI) approximately 2.7], conalbumin (pI approximately 6.4), lysozyme (pI approximately 11.0), albumin (pI approximately 4.9), and albumin which had been modified to yield proteins with pI values of 3.5, 4.7, 7.5 and 8.6. All uptake studies were conducted at similar unbound ligand fractions. There was a linear relationship between the rate of neonatal hepatocyte [3H]palmitate clearance and protein pI (r2 = 0.98). In contrast, there was an overall poor relationship between neonatal cardiac myocyte [3H]palmitate-clearance rate and protein pI (r2 = 0.48). However, the relationship improved when the data on [3H]palmitate-clearance were analyzed using only the modified albumins. The study indicates that an ionic interaction between extracellular proteins and the hepatocyte surface enhances the overall uptake of [3H]palmitate. This interaction may be limited to albumin for neonatal cardiac myocytes.     

Covalent binding of benzo[a]pyrene metabolites to DNA, RNA and chromatin proteins in the AKR mouse embryo cell-line

A specific fraction from the nuclei of the AKR mouse embryo cell-line (fraction I) displayed a much greater localization of radioactivity compared to fraction II and III when the chemical carcinogen, [3H]benzo[a]pyrene (B[a]P) was incubated with the cells for 24 h. The radioactivity in fraction I consisted of both covalently and non-covalently bound metabolites. Isolation of the DNA, RNA and protein of fraction I revealed that 94% of the covalently bound radioactivity was to protein, 5% to RNA and 1% to DNA. Analysis of the fraction I proteins by SDS gel electrophoresis revealed that there was more radioactivity covalently bound to the larger proteins than to smaller proteins. Isoelectric focusing (IEF) of the purified proteins displayed two peaks of radioactivity, one at a pH of 5 and the other at 11. The former proteins bound more radioactivity per mass of protein than the latter proteins. Analysis of fraction I histones on acid urea polyacrylamide gels showed that the radioactivity coincided with histones H3 and H2B and low levels of radioactivity associated with histones H1, H2A and H4. Two significant peaks of radioactivity closely migrated near but did not co-migrate with histone H1. The distribution of the bound radioactivity is probably a reflection of the availability of the proteins to the reactive carcinogen metabolites. The possible binding of B[a]P metabolites to phosphorylated histones and to the high mobility of group (HMG) proteins 1 and 2 is discussed.     

Reactions of the carcinogen N-acetoxy-4-acetamidostilbene with nucleosides.

The carcinogen N-acetoxy-4-acetamidostilbene (N-AcO-AAS) yields multiple products in reactions with guanosine, adenosine or cytidine in aqueous acetone. The major product from the reaction with cytidine is a deamination product, 1-(4-acetamidophenyl)-1-(3-uridyl)-2-hydrosy-2-phenylethane. Three minor products were unstable and were characterized only by their UV spectra and pK values. Adenosine yielded two major products, one of them 1-(4-acetamidophenyl)-1-(N6-adenoxyl)-2-hydroxy-2-phenylethane, and the second 3-(beta-D-ribosyl)-7-phenyl-8-(4-acetamidophenyl)-7,8 dihydroimidazo [2,1-i] purine. The major adduct with guanosine is 1-(4-acetamidophenyl)-1-(1-guanosyl)-2-hydroxy-2-phenylethane. One minor adduct also appears to be a guanosine-N-1 derivative, while two other minor adducts yield 1-(4-acetamidophenyl)-2-phenyl-1, 2-ethanediol on acid hydrolysis, and thus appear to be O6-derivatives. None of the guanine adducts isolated had the properties of N-7, C-8 or N2 adducts. In this respect, N-Aco-AAS appears to behave more like a classical alkylating agent than like previously studied N-acetoxy-N-arylacetamides, although the target organs of 4-acetamidostilbene are the same as those of other N-arylacetamides.     

Expression of external-surface membrane proteins in differentiated and undifferentiated mouse neuroblastoma cells.

Murine neuroblastoma cultures were labeled externally with the cationic reagent N,N,N-[3H]-trimethylamino-beta-alanyl-N-hydroxy-succinimide ester ([3H]Me3N-beta Ala-NSuc) or with 125I/lactoperoxidase. The cells were labeled in the logarithmic and confluent growth phases as well as in a highly differentiated state following treatment with 2% dimethylsulfoxide. The labeled exterior membrane proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Major changes in the exterior membrane proteins were observed during maturation and differentiation of the cells. Most of these changes were clonal-specific, while others were common to several clones. Two proteins of Mr 55,000 and 65,000 were labeled by both 125I/lactoperoxidase and Me3N-[3H]-beta Ala-NSuc. The level of labeling was dependent on the clonal lines used and the state of the cell maturation. A group of proteins displaying a molecular weight between 150,000 and 200,000 was found to be related to the transition of a culture from logarithmic to confluent growth phases. An additional protein, with an apparent molecular weight of 95,000, was common to differentiated cells of the two inducible clones used. In general the maturation of logarithmic phase cells into confluent cells resulted in a less complex electrophoretic distribution of the pattern of labeling. After dimethyl-sulfoxide treatment, further reduction in the complexity of the externally labeled proteins was observed.     

Active site chlorination of D-amino acid oxidase by N-chloro-D-leucine.

N-Chloro-D-leucine is an irreversible inhibitor or D-amino acid oxidase on a time scale of seconds. Studies with N-[36C]chloro-D-leucine, N-chloro-D-[1-14C]leucine and N-chloro-D-[4,5-3H]leucine show that the modified enzyme has been chlorinated at a site, or sites, on the apoenzyme. The 36Cl measurements agree with titrations of catalytic activity in showing that two chlorine equivalents are incorporated per active site flavin. Kinetically, the interaction with N-chloro-D-leucine behaves in a manner which is consistent with consecutive chlorinations of an amino acid residue, or residues, in the active site region by the first 2 molecules of N-chloro-D-leucine to be processed by the enzyme. The effect of chlorination of the enzyme on the steady state parameters for oxidation of D-alanine is entirely explained by a single perturbation, namely, a 1000-fold reduction in the specific rate of flavin reduction as measured directly by rapid reaction techniques.     

Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase

S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide. In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-[ethyl-2-3H]maleimide/subunit. Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold. Two N-[ethyl-2-3H]maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography. The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence. These are the first residues to be implicated in the activity and/or structure of the enzyme. N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer. Accumulation of the dimer parallels the loss of the enzyme activity. When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-[ethyl-2-3H] maleimide was present predominantly in the dimer. Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M. Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M. Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol.