It May, As Found for Hydroxylation of Proline
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VOL. 55, 1966 BIOCHEMISTRY: POPENOE ET AL. 393 E. H. Lincoln, and J. A. White, J. Am. Chem. Soc., 79, 1682 (1957); Moffett, R. B., B. D. Tiffany, B. D. Aspergen, and R. V. Heinzelman, J. Am. Chem. Soc., 79, 1687 (1957); Bahner, C. T., L. R. Barclay, G. Biggerstaff, D. E. Bilanco, G. W. Blanc, M. Close, and M. M. Isenberg, J. Am. Chem. Soc., 75, 4838 (1953); Wright, T. B., E. H. Lincoln, and R. V. Heinzelman, J. Am. Chem. Soc., 79, 1690 (1957). 6French, F. A., and B. L. Freedlander, Cancer Res., 18, 172 (1958); French, F. A., B. L. Freed- lander, A. Hosking, and J. French, Acta, Unio Intern. Contra Cancrum, 15, 614 (1960); French, F. A., and B. L. Freedlander, Cancer Res., 18, 1290 (1958). 7Friedemann, T. E., J. Biochem., 73, 33 (1927). 8 Klotzsch, H., in Methods of Enzymatic Analysis, ed. H. U. Bergmeyer (New York: Academic Press, 1965), p. 283. 9 Dr. S. E. Luria's strain B/1, 5, phage T, T5-resistant (MIT, Boston). 10 Roberts, R. B., P. H. Abelson, D. B. Cowie, E. T. Bolton, and B. J. Britten, Studies of Bio- synthesis of E. coli (Carnegie Institution of Washington, Publ. No. 607, 1957), p. 5. " Similar results were mentioned by First, A., J. Miller, A. Gross, and W. Cutting, Acta, Unio Intern. Contra Cancrum, 16, 625 (1960), that Kethoxal inhibited cell division of E. coli (strain K-12), and that glutamic acid antagonized inhibition. Glutamic acid similarly antagonized the action of Kethoxal-bisulphite complex in tumor-bearing animal experiments. 12Egyftd, L. G., to be published elsewhere. HYDROXYLYSINE FORMA TION FROM LYSINE DURING COLLAGEN BIOSYNTHESIS* BY EDWIN A. POPENOE, ROBERT B. ARONSON, AND DONALD D. VAN SLYKE MEDICAL RESEARCH CENTER, BROOKHAVEN NATIONAL LABORATORY, UPTON, NEW YORK Communicated December 22, 1965 Hydroxylysine as a constituent amino acid of a protein has been found only in collagen.' The obligatory source of collagen hydroxylysine is lysine. 2-4 Hydroxy- lysine itself, either fed or injected intraperitoneally, is not incorporated into collagen by rats,5 or presumably by other animals. Hydroxylation of lysine occurs in the early stages of collagen synthesis4' 6; it may, as found for hydroxylation of proline, occur in the precollagen peptide chain on the ribosomes.7 The present paper reports a continuation of a study of the biochemical mecha- nism by which a hydroxyl group is attached to the number 5 carbon of lysine. Con- ceivably the hydroxylation could proceed by (a) direct uptake of an atom of oxygen on carbon 5; (b) oxidation of carbon 5 to a ketone -CO- group followed by reduc- tion to -CH(OH)-; (c) dehydrogenation to form a 4-5 double bond followed by addition of the elements of water; (d) dehydrogenation to form a 5-6 double bond followed by addition of the elements of water. We have previously reported the results of studies using lysine-4-5-H3 as a pre- cursor of collagen hydroxylysine.8 In these studies we found that only one of the four labeled H atoms of lysine-4-5-H3 was lost in the conversion to hydroxylysine. One hydrogen atom on carbon 5 was replaced by -OH; therefore none of the other three labeled H atoms could be involved in the hydroxylation in such a way that they became equilibrated with the H of the medium. The results eliminated 5- keto-lysine and 4,5-dehydrolysine (mechanisms b and c) as possible intermediates in the hydroxylation of lysine. Downloaded by guest on September 25, 2021 394 BIOCHEMISTRY: POPENOE ET AL. PROC. N. A. S. Ill the present experiments we have used lysine-6-H3 as a precursor of collagen hydroxylysine, and have found that the conversion to hydroxylysine occurs without the loss of any of the tritium from carbon 6. Mechanism d thus appears to be eliminated. Materials and Methods.-Alpha-aminoadipic acid was purchased from California Corp. for Bio- chemical Research. D-Glucose-l-H3, uniformly labeled i-lysine-C14, and standards of C14- and H3-toluene were obtained from New England Nuclear Corp. 2,5-Diphenyloxazole (PPO) and 1,4-bis-2-(4-methyl-5-phenyloxazolyl) benzene (POPOP) scintillators were from Packard Instru- ment Co. New England Nuclear Corp. prepared Dilysine-6-H3 chemically by catalytic hydrogenation of 5-cyano-2-amino-pentanoic acid (personal communication, Dr. Thomas F. Sullivan, New England Nuclear Corp.). The radiochemical purity of the lysine was confirmed by paper chro- matography. Biosynthetic preparation of L-lysine-6-H3: The 6-tritiated lysine was prepared by the action of baker's yeast (Saccharomyces cerevisiae) on alpha-aminoadipic acid in the presence of D-glucose-1-H3 by a procedure derived from the work of Broquist9 and his collaborators. A two-step reaction converts -COOH of the adipic acid to -CH2NH2. In both the initial and final steps, reduction is mediated by DPNH, although in crude systems TPNH can be substituted for DPNH. For our preparation, into each of two 50-ml beakers were placed 2.5 me (0.8 mg) of D-gluCose-l-H3, 3.4 mg of alpha-aminoadipic acid, 0.5 ml of phosphate buffer (1 M KH2PO4 adjusted to pH 3.5 with H3PO4), and a suspension of 125 mg (moist) of baker's yeast. The total volume in each beaker was 5 ml. The beakers were shaken vigorously in a water bath at 300 for 3 hr. The cells were then collected by centrifugation and heated at 1150 in sealed tubes with 0.2 N HCl (4 ml) for 10 min. Lysine was isolated from the 0.2 N HCl extract by chromatography on Amberlite IR-120 ion exchange resin as described below for isolation of the amino acids from the metabolic experiments. Paper chromatography (butanol, acetic acid, water, 5:1:4, upper layer) showed that the product was contaminated by a very small amount of slower-moving, ninhydrin-positive material. However, all detectable radioactivity, as measured on a paper strip scanner, migrated with the lysine. The yield of radioactive lysine was 10 liC. Determination of radioactivity: All counting was done in a Tri-Carb liquid scintillation spec- trometer, model 4322. The instrument permits simultaneous counting of C14 and H3 in the same sample with good accuracy if the ratio H3/C"4 does not deviate too much from the optimal range. Activities of the two isotopes, when they were determined in the same sample, were calculated by the discriminator-ratio method described by Kabara et al.10 Counting efficiencies were calculated from the count rates of standards of C'4 and H3 toluene. In determining the H3 activities of the products of degradation of the lysine-6-H3 preparations (Tables 1 and 2), weighed samples were dissolved in 0.1 ml of water to which was added 5 ml of a 2:1 (v/v) mixture of ethylene glycol monomethyl ether and ethanolamine, and 10 ml of toluene containing 15 gm of PPO and 50 mg of POPOP per liter. In determining the H3/C"4 ratios of lysine and hydroxylysine from the hydroxylation experi- ments (Tables 3 and 4), the amino acids, after ion-exchange separation and desalting, were brought to 1 ml vol, and aliquots of 0.1 ml were handled in the same way described above, except that the scintillation mixture contained 5 ml of ethylene glycol monomethyl ether instead of 5 ml of the 2: 1 mixture of ethylene glycol monomethyl ether and ethanolamine. Degradation of the lysine-6-H3 preparations to confirm the location of the tritium: A sample of the chemically prepared Dilysine-6-H3 was mixed with nonradioactive Di-lysine, and a sample of the biosynthetic zlysine-6-H3 was mixed with nonradioactive ilysine. Each mixture was crystallized to constant specific activity and degraded by the procedures of Strassman and Wein- house.'1 In each degradation, one sample of lysine was oxidized directly with hot acidic per- manganate to glutaric acid. Another sample was converted by more gentle permanganate oxida- tion to delta-aminovaleric acid. This was then. converted by a Schmidt reaction to 1,4-diamino- butane followed by alkaline permanganate oxidation to succinic acid. The reactions were carried out exactly as described by Strassman and Weinhouse," except that the succinic acid was puri- fied by crystallization from water rather than by way of the silver salt. Melting points of the final products agreed satisfactorily with known values. The purity of the delta-aminovaleric acid was Downloaded by guest on September 25, 2021 VOL. 55, 1966 BIOCHEMISTRY: POPENOE ET AL. 395 further checked on the amino acid analyzer and the purity of the two dicarboxylic acids by thii layer chromatography.12 Incorporation of labeled lysine into the collagen of rat sponge biopsy tissues: The procedures fol- lowed were those of Kao and Botucek,13 and have been described in detail previously.8 In the present experiments, either the chemically prepared D-lysine-6-H' or the biosynthetic ilysine-6-H3 was mixed with an appropriate amount of uniformly labeled L-lysine-C4 and incu- bated with the sliced sponge biopsy tissue for 12 hr in Krebs-Henseleit solution under an atmos- phere of 95% 02 and 5% Co2. After the incubation, from four to six sponges were pooled for each experiment. Collagen was extracted from the tissues with hot trichloroacetic acid14 and hydro- lyzed. Lysine and hydroxylysine were isolated by chromatography on a 20 X 0.9-cm column of Amberlite IR-120 ion exchange resin with pH 4.26, 0.38 N buffer" at 300. The fractions contain- ing lysine or hydroxylysine were pooled and desalted on a column of Dowex-50 of similar size, as in a previous report.8 Results.-Location of tritium in the lysine-6-H3 preparations: The results of the degradations are given in Tables 1 and 2.