The solid-state catalytic isotope exchange of hydrogen in dalargin

A [³H]Dalargin preparation with a molar radioactivity of 52 Ci/mmol was obtained by the high temperature solid-state catalytic isotope exchange (HSCIE) of tritium for hydrogen at 150°C. This tritium-labeled peptide was shown to completely retain its biological activity in the test of binding to opioid receptors from rat brain. The dissociation constant of the Dalargin-opioid receptor complex was found to be 4.3 nM. The dependences of the chemical yield and the molar radioactivity on the reaction time and temperature of HSCIE were determined. The activation energy of the HSCIE reaction for the peptide was calculated to be 32 kcal/mol. The amino acid analysis showed that tritium is distributed between all the amino acid residues of [³H]Dalargin at the HSCIE reaction, with the temperature growth significantly increasing the total tritium incorporation and, especially, enhancing the radioactivity incorporation into aromatic residues.     

Study of solid-phase catalytic isotopic exchange of hydrogen in alpha-conotoxin G1 under the effect of spillover-tritium

Tritium-labeled alpha-conotoxin G1 with a molar radioactivity of 35 Ci/mmol and full biological activity (according to the binding to nicotinic acetylcholine receptor) was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE). The tritium distribution in the molecule of alpha-conotoxin G1 was revealed by 3H NMR spectroscopy. Tritium was found in all amino acid residues except for the Asn4-Pro5-Ala6 fragment. The data on the comparative reactivity of C-H bonds, the ab initio quantum-chemical calculation of the hydrogen exchange reaction, and the information on the spatial structures of alpha-conotoxin G1 in solution and in crystal state allowed us to establish that the reactivity of H atoms may be increased by their interaction with the electron donor O and N atoms at the transition state of the HSCIE reaction. A decrease in the rate of the HSCIE reaction could be caused by both a poor spatial accessibility of C-H bonds and a limited mobility of the peptide fragment containing these bonds.     

The solid-state catalytic isotope exchange of hydrogen in α-conotoxin G1 by the tritium spillover

Tritium-labeled α-conotoxin G1 with a molar radioactivity of 35 Ci/mmol and full biological activity (according to the binding to nicotinic acetylcholine receptor) was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE). The tritium distribution in the molecule of α-conotoxin G1 was revealed by³H NMR spectroscopy. Tritium was found in all amino acid residues except for the Asn4-Pro5-Ala6 fragment. The data on the comparative reactivity of C-H bonds, theab initio quantum-chemical calculation of the hydrogen exchange reaction, and the information on the spatial structures of α-conotoxin G1 in solution and in crystal state allowed us to establish that the reactivity of H atoms may be increased by their interaction with the electron donor O and N atoms at the transition state of the HSCIE reaction. A decrease in the rate of the HSCIE reaction could be caused by both a poor spatial accessibility of C-H bonds and a limited mobility of the peptide fragment containing these bonds.     

The solid-state catalytic synthesis of tritium labeled amino acids,peptides and proteins

Summary New catalytic reaction between a solid bioorganic compound and activated spillover tritium (ST), based on High-temperature Solid-state Catalytic Isotopic Exchange (HSCIE) was examined. The HSCIE mechanism and determination of the reactivity of hydrogen atoms in amino acids, peptides and proteins was investigated. Quantum mechanical calculations of the reactivity of hydrogen atoms in amino acids in the HSCIE reaction were done. The carbon atom with a greater proton affinity undergoes a greater exchange of hydrogen for tritium in HSCIE. The electrofilic nature of spillover hydrogen in the reaction of HSCIE was revealed. The isotope exchange between ST and the hydrogen of the solid organic compound proceeds with a high degree of configuration retention at the carbon atoms. The HSCIE reaction enables to synthesize tritium labeled proteins with a specific activity of 20–30 mCi/mg and kept biological activity.Presented at the 3rd International Congress on Amino Acids, Peptides and Analogues. Vienna, August, 23–27, 1993     

Inactivation of monoamine oxidase by allylamine does not result in flavin attachment

[1-3H]Allylamine was synthesized by sodium boro[3H]hydride reduction of acrolein followed by direct conversion of the [1-3H]allyl alcohol to N-allylphthalimide with triphenylphosphine, diethylazodicarboxylate, and phthalimide. The protecting group was removed with hydrazine. Inactivation of beef liver mitochondrial monoamine oxidase with [1-3H]allylamine led to incorporation of 1-6 eq of inactivator/active site depending upon the length of incubation time. Inactivation and radioactivity incorporation coincided; however, after 1 eq of tritium was incorporated and 5% enzyme activity remained, additional radioactivity continued to become incorporated into the enzyme. The optical spectrum of the FAD coenzyme changed during inactivation from that of oxidized to reduced flavin. Following dialysis of the inactivated enzyme, the spectrum remained reduced, but denaturation in urea rapidly resulted in reoxidation of the flavin. Under these same denaturing conditions, 96% of the radioactivity associated with the enzyme remained bound, therefore indicating that allylamine attachment is not to the flavin coenzyme but rather to an active site amino acid residue. The adduct also was stable to base and, to a lesser degree, acid treatment. Although allylamine and N-cyclopropylbenzylamine appear to be oxidized by monoamine oxidase to give 3-(amino acid residue) propanal adducts, two different amino acids seem to be involved because of a difference in stability of the adducts. The mechanisms for inactivation of monoamine oxidase by allylamine and reactivation by benzylamine are discussed in relation to previously reported results.     

Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.     

The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.     

Stereospecific abstraction of epsilon-pro-R-hydrogen of L-lysine by L-lysine epsilon-dehydrogenase from Agrobacterium tumefaciens

The stereochemical aspects of the L-lysine epsilon-dehydrogenase reaction were examined with (6R)-L-[6-3H]lysine and (6S)-DL-[6-3H]lysine. When (6S)-DL-[6-3H]lysine was used as a substrate, the tritium was found in the product, delta 1-piperideine-6-carboxylate. In contrast, the radioactivity from (6R)-L-[6-3H]lysine was not retained in the product. Thus, the pro-R hydrogen at the prochiral C-6 carbon of L-lysine is specifically abstracted by the enzyme: the enzyme behaves stereochemically as an amino acid D-dehydrogenase.     

Histidine 235 of human sex hormone-binding globulin is the covalent site of attachment of the nucleophilic steroid derivative, 17 beta-bromoacetoxydihydrotestosterone

This article deals with the elucidation of the steroid-binding site of human sex hormone-binding globulin (SHBG). 17 beta-Bromoacetoxydihydrotesterone (BA-DHT) reacted with highly purified SHBG in a time-dependent and irreversible fashion. The interaction could be totally inhibited by the simultaneous addition of an excess of dihydrotesterone. At the completion of the reaction, the molar ratio of BA-DHT to SHBG was approximately unity. SHBG was affinity labeled with [14C]BA-DHT and submitted to acid hydrolysis. The released amino acids were evaluated on high performance liquid chromatography, and virtually all of the 14C was identified as 3-[14C]carboxymethylhistidine. Furthermore, [14C]BA-DHT-labeled SHBG was digested with trypsin, followed by isolation of the released tryptic peptides by reverse-phase high performance liquid chromatography. The 14C was localized to a single tryptic peptide. It contained 2' histidyl residues, corresponding to residues 235 and 251 in the known amino acid sequence of SHBG. Although most of the 3-[14C]carboxymethylhistidine, or its phenylthiohydantoin derivative, was trapped on the filter of the amino acid sequenator, sufficient radioactivity emerged to identify histidyl residue 235 as the labeled amino acid.     

Evenly tritium-labeled peptides and their in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50-150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium-labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a tg scale of biological samples both in vitro and in vivo.     

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.     

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)     

Enrichment for auxotrophic and heat-sensitive mutants of Neurospora crassa by tritium suicide.

Tritium suicide is shown to be an effective technique for mutant enrichment in Neurospora crassa. When mutagenized conidia were labelled to a high specific radioactivity either with a tritiated amino acid mixture or with [5-3H]uridine at a non-permissive temperature and stored at 4 degrees C to accumulate decays, there was a 13-15 fold enrichment for temperature-sensitive mutants relative to the original mutagenized cultures. For a wild type culture of Neurospora crassa labelled with [5-3H]uridine at 35 degrees C the probability of cell killing per tritium decay was calculated to be 3.64 X 10(-5).     

The effect of three-dimensional structure on the solid state isotope exchange of hydrogen in polypeptides with spillover hydrogen

The effect of the three-dimensional structure of polypeptides and proteins on their ability to undergo isotopic exchange under the action of spillover hydrogen (SH) in the high temperature solid state catalytic isotope exchange reaction (HSCIE) was theoretically and experimentally studied. The HSCIE reaction in the beta-galactosidase protein from Thermoanaerobacter ethanolicus (83kDa) was studied. The influence of the beta-galactosidase structure on isotopic exchange as peptide fragments with spillover tritium was studied. The most accessible peptide fragment, which does not contribute to alpha-helix and beta-strand formations (KEMQKE215-220), had the largest relative reactivity. The one located in the contact area between the subunits (YLRDSE417-422) showed the smallest relative reactivity. The relative reactivities of these peptides differ more than 150 times. Data collected during a study devoted to the HSCIE reaction of the beta-galactosidase protein indicate that the HSCIE reaction might be employed for acquiring information about their three-dimensional structure and protein-protein interactions. The results of ab initio calculations have shown that alpha-helix formation in polypeptides decreases the reactivity in HSCIE. Hydrogen exchange in the alpha-helical fragment Trp1-Leu8 of zervamycin IIB was also analyzed using theoretical methods. It was shown by ab initio quantum-chemical calculations that the high degree of substitution of C(alpha)H for tritium in Gln3 might be associated with the participation of electron donor O and N atoms in transition state stabilization in the HSCIE reaction.     

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.     

Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-³H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [³H]thymidine experiments and compared the degree of nonspecific labeling by [³H]adenine with that derived from [³H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.     

THE INCORPORATION OF RADIOACTIVE AMINO ACIDS INTO BRAIN SUBCELLULAR PROTEINS DURING TRAINING

—Total proteins, free amino acids, tritiated water and subcellular proteins of mouse brain were examined for changes in radioactivity during operant conditioning after subcutaneous administration of labelled amino acids. The conditioning was based on appetitive learning, using sweetened milk as a reward. During training and incorporation for 20-30 min, both [³H]leucine and [1-¹⁴C]leucine underwent a significant increase in catabolism, resulting in a decreased radioactivity in the free amino acids. [2-²H]Methionine underwent a rapid loss of isotope, so that 90% of the radioactivity was in the form of tritiated water at the end of training, and this phenomenon masked any possible effect of training. The brain uptake of [³⁵S]methionine increased during the training, resulting in an increased radioactivity in the proteins. Uptake of [³H]lysine increased slightly during training only after 1 h incorporation and not after 20 or 30 min, as judged from a time course of radioactivity in the free amino acids. Incorporation into nuclear proteins increased selectively during 20 min, and into nuclear and cytosol proteins after 60 min incorporations. It is concluded that changes in the observed rate of incorporation of a precursor into brain subcellular proteins under the influence of behaviour might be the result of changes in precursor catabolism or uptake, or both, and that each amino acid behaves in a different way. Even the same amino acid gives different results depending on the isotope and its position in the amino acid.     

Determination of the effect of acetylation of specific lysine residues in human growth hormone on its affinity for somatogenic receptors by an affinity selection technique

A technique is described to study the effect of acetylation of individual lysine residues in peptide hormones on the affinity for their receptors, and is illustrated for the case of human growth hormone (hGH) binding to somatogenic receptors. The hGH was partially acetylated with high specific activity [3H]-acetic anhydride and the product ([3H]-Ac-hGH) was incubated with solubilised affinity-purified somatogenic receptors (from male rat liver) in the presence and absence of excess unlabelled hGH. The receptor-bound and unbound labelled hormone were separated by gel filtration and subjected to HPLC tryptic peptide mapping after the addition of cold carrier Ac-hGH. Peaks of [3H] radioactivity were assigned to peptides corresponding to the acetylation of specific lysine residues in the hGH sequence by amino acid analysis and sequencing. Comparison of the relative intensities of corresponding [3H] peaks in the peptide maps of added receptor, bound and unbound [3H]-Ac-hGH, enabled the relative receptor-binding potencies of different acetylated hGH species to be determined. Acetylation of lysine 168 or 172 in hGH greatly decreases its receptor-binding affinity, acetylation of lysine 115 probably causes a minor decrease, whereas acetylation of lysines 38, 70, and the N-terminal amino group have no appreciable effect. Acetylation of lysine 140 causes a significant increase in receptor-binding affinity.     

Identification of a tyrosine residue in rat guanidinoacetate methyltransferase that is photolabeled with S-adenosyl-L-methionine.

Exposure of rat guanidinoacetate methyltransferase to ultraviolet light in the presence of S-adenosyl-L-[methyl-3H]methionine ([methyl-3H]AdoMet) results in covalent linking of radioactivity to the enzyme protein. The incorporation of radioactivity shows no lag and is linear with respect to time up to 1 h. The photolabeling is saturable with [methyl-3H]AdoMet, and the binding constant of the enzyme for AdoMet determined in this experiment is similar to that obtained by equilibrium dialysis. Low concentrations of competitive inhibitors S-adenosyl-L-homocysteine and sinefungin effectively prevent the photoinduced labeling by AdoMet. Although guanidinoacetate methyltransferase is irreversibly inactivated upon ultraviolet irradiation in the absence of AdoMet, the enzyme inactivated by 1-h exposure to ultraviolet irradiation has been shown to bind AdoMet with an affinity identical to that of the native enzyme. These results indicate that photolabeling occurs at the active site. Following proteolysis of the [methyl-3H]-AdoMet-labeled enzyme with chymotrypsin, a radioactive peptide is isolated having a sequence Asp-Thr-X-Pro-Leu-Ser-Glu-Glu-Thr-Trp. The peptide corresponds to residues 134-143, with X being modified Tyr-136. The same peptide is photolabeled when [carboxy-14C]AdoMet is used. High-performance liquid chromatography of this peptide after acid hydrolysis and phenyl isothiocyanate derivatization suggests that the entire molecule of AdoMet is attached to Tyr-136.     

Sialic acid, galactose and fucose on the surface of Ehrlich ascites tumor cells grown in glucose-free medium in the presence of uridine

1) The content and accessibility of terminal sialic acid and galactose residues as well as the incorporation of [3H]fucose into glycoconjugates were determined in 48-h cultures of Ehrlich ascites tumor cells in a glucose-free medium supplemented with uridine, a compound which can fulfil the necessary functions of glucose. 2) Sialic-acid residues accessible to sialidase cleavage were reduced from 695 +/- 80 nmol/10(9) cells (controls) to 284 +/- 22 nmol/10(9) cells (43% of controls). In situ labeling using periodate oxidation followed by sodium borotritiide reduction revealed a tritium incorporation of 47 +/- 11% that of controls (= 4.1 x 10(5) cpm/mg protein). 3) Labeling of galactose residues of 80-90% of that of controls was achieved after treatment of the cells with galactose oxidase/sodium borotritiide. A nearly six-fold enhancement of tritium incorporation into galactose of control cells was observed after sialidase/galactose oxidase treatment and sodium borotritiide reduction (1.5----8.8 x 10(5) cpm/mg protein); only a 3.6-fold increase (1.2 x 10(5)----4.3 x 10(5) cpm/mg protein) was found with glucose-free cultured cells. It is concluded that the galactose content of the cell surface is reduced to about 50% of controls. 4) The incorporation of tritium into acid-insoluble precipitate after 24 h incubation with [3H]fucose and the activity of the acid-soluble fraction were enhanced by about 85% as compared to controls. The pattern of inhibition by tunicamycin of [3H]fucose uptake and incorporation was the same in glucose-containing standard medium and in glucose-free uridine medium.(ABSTRACT TRUNCATED AT 250 WORDS)