Pyrrolo[1,4]benzodiazepine antibiotics. Biosynthesis of the antitumor antibiotic sibiromycin by Streptosporangium sibiricum

The biosynthesis of the antitumor antibiotic sibiromycin by Streptosporangium sibiricum requires the construction of four units: the amino sugar from glucose; the anthranilate ring from DL-tryptophan probably via kynurenine; the aromatic methyl group from methionine; the propylidene proline from L-tyrosine with the loss of two aromatic carbons and addition of a C-1 from methionine. Retention of tritium from DL-[5-3H]tryptophan in sibiromycin suggest an NIH shift during hydroxylation of an intermediate.     

Thiamin biosynthesis in Saccharomyces cerevisiae. Origin of carbon-2 of the thiazole moiety.

Radioactivity from [2-14C]glycine enters C-2 of the thiazole moiety of thiamin and no other site, in Saccharomyces cerevisiae (strains A.T.C.C. 24903 and 39916, H.J. Bunker). Radioactivity from L-[Me-14C]methionine or from DL-[2-14C]tyrosine does not enter thiamin.     

Incorporation of Label from 13C-, 2H-, and 15N-Labeled Methionine Molecules during the Biosynthesis of 2[prime]-Deoxymugineic Acid in Roots of Wheat

The biosynthetic pathway of 2[prime]-deoxymugineic acid, a key phytosiderophore, was investigated by feeding 13C-, 2H-, and 15N-labeled methionine, the first precursor, to the roots of hydroponically cultured wheat (Triticum aestivum L. cv Minori). The incorporation of label from each methionine species was observed during their conversion to 2[prime]-deoxymugineic acid, using 2H-, 15N-, and 13C-nuclear magnetic resonance (NMR). L-[1-13C]Methionine (99% 13C) was efficiently incorporated, resulting in 13C enrichment of the three carboxyl groups of 2[prime]-deoxymugineic acid. Use of D,L-[15N]methionine (95% 15N) resulted in 15N enrichment of 2[prime]-deoxymugineic acid at the azetidine ring nitrogen and the secondary amino nitrogen. When D,L-[2,3,3,-2H3-S-methyl-2H3]methionine (98.2% 2H) was fed to the roots, 2H-NMR results indicated that only six deuterium atoms were incorporated, and that the deuterium atom from the C-2 position of each methionine was almost completely lost. [2,2,3,3-2H4]1-Aminocyclopropane-1-carboxylic acid (98% 2H) was not incorporated into 2[prime]-deoxymugineic acid. These data and our previous findings demonstrated that only the deuterium atom from the C-2 position of L-methionine was lost, and that other atoms were completely incorporated when three molecules of methionine were converted to 2[prime]-deoxymugineic acid. These observations are consistent with the conversion of L-methionine to azetidine-2-carboxylic acid, suggesting that L-methionine is first converted to azetidine-2-carboxylic acid during biosynthesis leading to 2[prime]-deoxymugineic acid. Based on these results, a hypothetical pathway from L-methionine to 2[prime]-deoxymugineic acid was postulated.     

Properties of tyrosine hydroxylation in living mouse neuroblastoma clone N1E-115

Tyrosine hydroxylation was studied in intact cells of mouse neuroblastoma clone N1E-115 which have high levels of tyrosine 3-monooxygenase (EC 1.14.16.2) and which have been fully characterized for tyrosine transport. Measurement of [3H]OH formed from L-[3,5(-3)H]tyrosine in the medium was the method of assay and [3H]OH formed was stoichiometric with the formation of L-[3H]3,4-dihydroxyphenylalanine. Tyrosine hydroxylation was dependent on time of incubation, cell number, and the concentration of [3H]tyrosine in the medium. From velocity vs. [3H]tyrosine concentration experiments, two apparent Km values were obtained: Km1 = 10 +/- 2 microM; Km2 = 140 +/- 10 microM. Substrate inhibition occurred with tyrosine concentrations between 20 and 50 microM. The reaction was twice as fast at pH 5.5 as at pH 7.4. alpha,alpha'-Dipyridyl (1 mM) caused major inhibition (75%) when [3H]tyrosine concentration was 10 microM. L-3-Iodotyrosine was a competitive inhibitor with Ki = 0.3 microM. Dopamine was a non-competitive inhibitor with Ki = 500 microM. 1-Norepinephrine had no effect. These results show that the hydroxylation of tyrosine by living N1E-115 cells has many of the properties of the reaction catalyzed by purified tyrosine 3-monooxygenase from normal tissue.     

The contribution of phenylalanine to tyrosine metabolism in vivo. Studies in the post-absorptive and phenylalanine-loaded rat.

1. Rates of appearance and oxidation of plasma L-leucine, L-phenylalanine and L-tyrosine, as well as conversion of plasma phenylalanine into plasma tyrosine, were determined in 90-120 g rats after overnight starvation and while receiving 115-120 mumol of L-phenylalanine/h. 2. In the post-absorptive state, plasma tyrosine and phenylalanine appearances were similar, despite the fact that 22% of plasma tyrosine appearance could be attributed to the hydroxylation of phenylalanine. 3. A constant infusion of 115-120 mumol of L-phenylalanine/h did not significantly alter plasma leucine kinetics, but increased appearance of plasma phenylalanine and tyrosine. The percentage of phenylalanine and tyrosine appearance that was oxidized increased from 12.1% and 24.4% to 37.3% and 48.0% respectively. In phenylalanine-loaded rats, 72% of plasma tyrosine appearance could be attributed to the conversion of phenylalanine. 4. Whole-body tyrosine oxidation measured from a continuous infusion of either L-[14C]tyrosine or L-[14C]phenylalanine differed by 165%. 5. It can be concluded that, in the post-absorptive state, phenylalanine hydroxylation makes a substantial contribution to the plasma appearance of tyrosine and is significantly increased when phenylalanine is administered. The disposal of excess infused phenylalanine is a result of a greater percentage of plasma phenylalanine being converted into tyrosine and a greater proportion of tyrosine being further oxidized. However, apparent tyrosine oxidation rates estimated from plasma tyrosine specific radioactivities and appearance of expired 14CO2 during administration of [14C]tyrosine are underestimates of true rates, in part because tyrosine generated from phenylalanine hydroxylation is catabolized without freely equilibrating with the plasma compartment.     

Radiometric analysis of oxidative reactions in aromatization by placental microsomes. Presence of differential isotope effects

In order to study the initial as well as the final steps in the aromatization of androgens to estrogens, high-specific activity [19-C3H3]androstenedione and testosterone were synthesized. Incubations of [19-C3H3]androstenedione with human placental microsomes resulted in the generation of [3H]water, as a result of the dual hydroxylation at C-19, and [3H]formic acid reflecting final aromatization. After an initial lag in the production of [3H]formic acid, the two radiolabeled products were formed linearly with time at a ratio of 2 to 1 under subsaturating conditions and 2.2 to 1 when saturating levels of substrate were present. Incubation of a mixture of [19-C3H3]- and [4-14C]androstenedione with human placental microsomes yielded 19-hydroxy- and 19-oxoandrostenedione, respectively, products of one and two hydroxylations at C-19. The isotope ratios of these derivatives revealed the presence of a tritium isotope effect in the first but not in the second hydroxylation at that site. When [19-C3H2]- and [4-14C]19-hydroxyandrostenedione were used as the substrate, the isotope ratio of the isolated 19-oxoandrostenedione showed no evidence of any isotope effect in its formation. Thus, the second hydroxylation at C-19 exhibits no isotope effect irrespective of whether androstenedione or 19-hydroxyandrostenedione are the substrates, and therefore, a concerted process and catalytic commitment are not responsible for the difference in isotope effects between the first and second C-19 hydroxylation by the placental aromatase complex. Radiometric kinetic analysis employing [19-C3H3]- and [1 beta,2 beta-3H]androstenedione as the comparative substrates provided evidence that the isotope effect is exerted solely through the Vmax component of the reaction. The distinction between the successive hydroxylations at C-19 in the aromatization sequence suggests, but does not prove, that different mechanisms, and hence different catalytic sites, may be involved in these steps.     

Studies on the biosynthesis of sulfolipids in the Diatom Nitzschia alba.

Labeling of sulfolipids in Nitzschia alba was studied after growth of the cells in media containing L-[35S]cystine, L-[35S], L-[35S]cysteine, L-[35S]-methionine or a mixture of L-[Me-3H]methionine and L-[35S]methionine, [35S]Cysteine or [35S]cystine labeled the deoxyceramide sulfonate and the sulfonium analog, phosphatidylsulfocholine (and its lyso derivative) but not the sterol sulfate nor the sulfoquinovosyl diglyceride; [35S]methionine labeled only the phosphatidylsulfocholine and its lyso derivative. With the [35S]- and [Me-3H]methionine mixture (3H/35S ratio 1.0) the phosphatidylsulfocholine had a 3H/35 S ratio of 1.5 indicating that both sulfonium methyl groups were derived from methionine. Probable biosynthetic pathways for these novel sulfolipids are discussed.     

Biosynthesis of an unusual amino acyl moiety contained in bestatin

The biosynthetic pathway of an unusual amino acyl [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl (AHP)] moiety which is contained in bestatin has been studied by testing the incorporation of potential precursors. L-[U-14C]-Phenylalanine, L-[U-14C]leucine, and [U-14C]acetic acid were efficiently incorporated into bestatin, but the radioactivity of L-[1-14C]phenylalanine, [1-14C]glyoxylic acid, and [14C]oxalic acid were not incorporated. Incorporation of acetic acid into 1- and 2-carbon of the AHP moiety was confirmed by incorporation of [13C]acetic acid. Thus, the AHP moiety was shown to be biosynthesized from L-phenylalanine and two carbon atoms of acetic acid, accompanied by decarboxylation of the phenylalanine.     

Conversion of 5-S-methyl-5-thio-D-ribose to methionine in Klebsiella pneumoniae. Stable isotope incorporation studies of the terminal enzymatic reactions in the pathway

Extracts of Klebsiella pneumoniae convert 5-S-methyl-5-thio-D-ribose (methylthioribose) to methionine and formate. To probe the terminal steps of this biotransformation, [1-13C]methylthioribose has been synthesized and its metabolism examined. When supplemented with Mg2+, ATP, L-glutamine, and dioxygen, cell-free extracts of K. pneumoniae converted 50% of the [1-13C]methylthioribose to [13C]formate. The formation of [13C]formate was established by 13C and 1H NMR spectroscopy studies of the purified formate, and by 13C and 1H NMR spectroscopy and mass spectrometry studies of its p-phenylphenacyl derivative. By contrast, no incorporation of label from [1-13C]methylthioribose into the biosynthesized methionine was detected by either mass spectrometry or 13C and 1H NMR spectroscopy. The most reasonable interpretation of these results is that C-1 of methylthioribose is converted directly to formate concomitant with the conversion of carbon atoms 2-5 to methionine. The penultimate step in the conversion of methylthioribose to methionine and formate is an oxidative carbon-carbon bond cleavage reaction in which an equivalent of dioxygen is consumed. To investigate the fate of the dioxygen utilized in this reaction, the metabolism of [1-13C]methylthioribose in the presence of 18O2 was also examined. Mass spectrometry revealed the biosynthesis of substantial amounts of both [18O1]methionine and [13C, 18O1]formate under these conditions. These results suggest that the oxidative transformation in the conversion of methylthioribose to methionine and formate may be catalyzed by a novel intramolecular dioxygenase. A mechanism for this dioxygenase is proposed.     

Biosynthesis of methanopterin

The biosynthetic pathway for the generation of the methylated pterin in methanopterins was determined for the methanogenic bacteria Methanococcus volta and Methanobacterium formicicum. Extracts of M. volta were found to readily cleave L-7,8-dihydroneopterin to 7,8-dihydro-6-(hydroxymethyl)pterin, which was confirmed to be a precursor of the pterin portion of the methanopterin. [methylene-2H]-6-(Hydroxymethyl)pterin was incorporated into methanopterin by growing cells of M. volta to an extent of 30%. Both the C-11 and C-12 methyl groups of methanopterin originate from [methyl-2H3]methionine, as confirmed by the incorporation of two C2H3 groups into 6-ethyl-7-methylpterin, a pterin-containing fragment derived from methanopterin. Cells grown in the presence of [methylene-2H]-6-(hydroxymethyl)pterin, [ethyl-2H4]-6-[1 (RS)-hydroxyethyl]pterin, [methyl-2H3]-6- (hydroxymethyl)-7-methylpterin, [ethyl-2H4, methyl-2H3]-6-[1 (RS)-hydroxyethyl]-7-methylpterin, and [1-ethyl-3H]-6-[1 (RS)-hydroxyethyl]-7-methylpterin showed that only the non-7-methylated pterins were incorporated into methanopterin. Cells extracts of M. formicicum readily condensed synthetic [methylene-3H]-7,8-H2-6-(hydroxymethyl)pterin-PP with methaniline to generate demethylated methanopterin, which is then methylated to methanopterin by the cell extract in the presence of S-adenosylmethionine. These observations indicate that the pterin portion of methanopterin is biosynthetically derived from 7,8-H2-6-(hydroxymethyl)pterin, which is coupled to methaniline by a pathway analogous to the biosynthesis of folic acid. This pathway for the biosynthesis of methanopterin represents the first example of the modification of the specificity of a coenzyme through a methylation reaction.     

Insights into the catalytic mechanisms of phenylalanine and tryptophan hydroxylase from kinetic isotope effects on aromatic hydroxylation

Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.     

Assay of the possible organization of particle-bound enzymes with squalene synthetase and squalene oxidocyclase systems

Lanosterol was biosynthesized in pig liver homogenate from [4,8,12-(14)C(3)]farnesyl pyrophosphate and [4S-4-(3)H]NADPH through the intermediary formation of squalene labelled asymmetrically with (3)H. The biosynthetic lanosterol, freed from labelled 24,25-dihydrolanosterol, which was also synthesized, was converted into 24,25-dihydrolanosteryl acetate and subjected to chemical degradations to locate the position(s) of the (3)H label in the molecule. The ratio of (3)H at C-11 to that at C-12 was found to be 1.28. Although a certain inequality of labelling was thus indicated, experimental uncertainties did not permit the conclusion that the asymmetrically labelled squalene might have been cyclized preferentially from one end.     

Biosynthesis of tritium-labeled S-adenosyl-L-methionine in yeast cells

Biosynthetic preparation of S-adenosyl-L-[methyl-3H]methionine from L-[methyl-3H]methionine by cultivation of diploid yeast Saccharomyces cerevisiae (methionine-auxotrophic) in a cultural medium with the high concentration of L-methionine is described. The radiochemical purity was over 95%. Biological activity of the preparations has been shown in transmethylation reactions in the presence of the yeast homocysteine-methyltransferase.     

Biosynthesis of thiamine. Incorporation experiments with 14C-labelled substrates and with [15N]glycine in Saccharomyces cerevisiae

1. Yeast was grown in a minimal synthetic medium together with a range of (14)C-labelled substrates under standardized conditions. After isolation, the purified thiamine was cleaved by sulphite and the pyrimidine and thiazole moieties were purified and assayed for radioactivity. 2. In order of decreasing incorporation, [(14)C]formate, [3-(14)C]serine, [2-(14)C]glycine and [2-(14)C]acetate supplied label for the pyrimidine, and [2-(14)C]glycine, [3-(14)C]serine, [1-(14)C]glycine, [(14)C]formate and [2-(14)C]acetate for the thiazole. Incorporation of label into the fragments from several other (14)C-labelled substrates, including [Me-(14)C]- and [3,4-(14)C(2)]-methionine, was insignificant. 3. [3-(14)C]Serine was shown not to contribute label to C-2 of the thiazole ring. 4. Significant incorporation of nitrogen from [(15)N]glycine into the thiazole moiety, but not into the pyrimidine moiety, was established. 5. It appears that C-2 and N-3 of the thiazole ring are formed from C-2 and the nitrogen atom of glycine, but the entire methionine molecule does not appear to be implicated.     

Two Related Biosynthetic Pathways of Mugineic Acids in Gramineous Plants

The biosynthesis of mugineic acids was studied by feeding 2H- or 13C-labeled compounds to water-cultured roots in several gramineous plants. The fate of labeled compounds was monitored by using 2H- and 13C-nuclear magnetic resonance. On investigating the proton changes during biosynthesis by feeding D,L-[3,3,4,4-d4]-methionine (98.6% 2H), 2H-labeled 2[prime]-deoxymugineic, mugineic, and 3-epihydroxymugineic acids were isolated from root washings of wheat (Triticum aestivum L. cv Minori), barley (Hordeum vulgare L. cv Minorimugi), and beer barley (Hordeum vulgare L. cv AM Nijo Tochigi), respectively. The 2H-nuclear magnetic resonance study indicated that 12 deuteriums were incorporated into the labeled 2[prime]-deoxymugineic acid, suggesting that three molecules of L-[3,3,4,4-d4]methionine were combined. In comparison, one of the deuteriums at C-2[prime] position in the mugineic acid, and one each of the deuteriums at C-2[prime] and C-3 positions in the 3-epihydroxymugineic acid, were lost. However, all other deuteriums were incorporated in a manner similar to that of the labeled 2[prime]-deoxymugineic acid. When [1,4[prime],4"-13C3]2[prime]-deoxymugineic acid (20% 13C) was fed to oat roots (Avena sativa L. cv Amuri II), avenic acid A, which was 13C enriched at the corresponding positions, was obtained. These results revealed that L-methionine was the precursor for all these mugineic acids and that cleavage of the azetidine ring or hydroxylation of the 2[prime]-deoxymugineic acid produced two related biosynthetic pathways in different gramineous plant species: L-methionine -> 2[prime]-deoxymugineic acid -> avenic acid A in oat; and L-methionine -> 2[prime]-deoxymugineic acid -> mugineic acid -> 3-epihydroxymugineic acid in barley and beer barley.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain.

The incorporation of [3H]phenylalanine, [3H]tyrosine, and [3H]tryptophan into protein and amino acyl-tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl-tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl-tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl-tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both Km and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than Km.     

Biosynthesis of securinine,the main alkaloid of Securinega suffruticosa

Biosynthesis of securinine was studied by incorporation experiments in Securinega suffruticosa. Among presumed precursors tested, lysine, cadaverine, and tyrosine showed the highest incorporation into securinine. Degradation experiments revealed that cadaverine-[1,5-¹⁴C] labelled specifically the piperidine ring of securinine and the radioactivity from dl-tyrosine-[2-¹⁴C] was introduced into the C-11 lactone carbonyl. Experiments with L-tyrosine-[U-¹⁴C] and L-tyrosine-[3′,5′-³H; U-¹⁴C] prove that the remaining C₆Sz.sbnd;C₂ moiety is derived from the aromatic ring and the C-2 and C-3 or tyrosine.     

Use of 13C in biosynthetic studies. The labelling pattern in tenellin enriched from isotope-labelled acetate, methionine, and phenylalanine

The biogenetic origin of the carbon atoms in tenellin has been established by adding 13C-enriched compounds to cultures of Beauveria bassiana, and determining the isotopic distribution in the metabolite by 13C nuclear magnetic resonance spectrometry. Tenellin is formed by condensation of an acetate-derived polyketide chain with a phenylpropanoid unit that may be phenylalanine. Alternate carbon atoms of the polyketide chain were labelled with sodium [1(-13C)]- and [2-(13C]-acetate; sodium [1,2-(13C)]acetate was incorporated as intact two-carbon units, the presence of which in tenellin was apparent from coupling between adjacent 13C-enriched carbons. Substituent methyl groups of the polyketide-derived alkenyl chain were labelled with L-[Me-13C]methionine. The labelling patterns from DL-[carboxy-13C]phenylalanine and DL-[alpha-13C]phenylalanine indicated a rearrangement of the propanoid component at some stage in the synthesis. The mass spectrum of tenellin from cultures administered L-[15N]phenylalanine showed isotopic enrichment similar to that obtained with 13C- or 14C-labelled phenylalanine. During incorporation of L-[carboxy-14C, beta-3H]phenylalanine 96% of the tritium label was lost, discounting the possibility of a 1,2-hydride shift during biosynthesis of the metabolite.     

The cell surface receptor for immunoglobulin E. Effect of tunicamycin on molecular properties of receptor from rat basophilic leukemia cells

Cell surface receptors for immunoglobulin E were isolated by repetitive affinity chromatography from rat basophilic leukemia cells biosynthetically labeled with L-[35S]methionine and D-[3H]mannose. Native immunoglobulin E receptor appeared as a very broad band in the 45,000 to 62,000 Mr region in sodium dodecyl sulfate polyacrylamide gels. However, from cells cultured in the presence of tunicamycin, a relatively narrow band with an apparent Mr of 38,000 was isolated. The 38,000 Mr band rebound to immunoglobulin E-Sepharose, was immunoprecipitated with antibodies to immunoglobulin E receptor, shared tryptic peptides with native receptor, and was labeled with L-[35S]methionine but not D-[3H]mannose, and thus appears to be immunoglobulin E receptor lacking N-linked oligosaccharides. It is demonstrated that N-linked oligosaccharides account for much of the apparent heterogeneity of native receptor in sodium dodecyl sulfate polyacrylamide gels and in two-dimensional gel electrophoresis. A receptor-associated protein with apparent Mr = 30,000, prominently labeled with L-[35S]methionine but not with D-[3H]mannose, did not have altered molecular properties when isolated from tunicamycin-cultured cells, and did not share tryptic peptides with receptor.     

Biosynthesis of methylphosphomannosyl residues in the oligosaccharides of Dictyostelium discoideum glycoproteins. Evidence that the methyl group is derived from methionine

The phosphorylated oligosaccharides of Dictyostelium discoideum contain methylphosphomannosyl residues which are stable to mild-acid and base hydrolysis (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we present evidence that these methyl groups are derived from [methyl-3H]methionine, in vivo and [methyl-3H]S-adenosylmethionine in vitro. About 18% of the macromolecules secreted from vegetative cells labeled with [methyl-3H]methionine are released by digestion with preparations of endoglycosidase/peptide N-glycosidase F. The majority of the released molecules are sulfated, anionic high mannose-type oligosaccharides. Strong acid hydrolysis of the [3H]methyl-labeled molecules yields [3H]methanol with kinetics of release similar to those found for the generation of Man-6-P from chemically synthesized methylphosphomannose methylglycoside. Treatment of the [3H]methyl-labeled molecules with a phosphodiesterase from Aspergillus niger which is known to cleave this phosphodiester also releases [3H]methanol from a portion of the oligosaccharides. In vitro incorporation of [methyl-3H]S-adenosylmethionine into endogenous acceptors found in membrane preparations shows that the [3H]methyl group of the methylphosphomannose residues can be derived from this molecule.