VOL. 54, 1965 BIOCHEMISTRY: BEKHOR ET AL. 615

24 Felsenfeld, G., and A. Rich, Biochim. Biophys. Acta, 26, 457 (1957). 5 Ludlum, D. B., R. C. Warner, and A. J. Wahba, Science, 145, 397 (1964). 26 Szer, W., and D. Shugar, Acta Biochim. Polom., 7, 491 (1960). 27 Nirenberg, M. W., and P. Leder, Science, 145, 1399 (1964).

THE OF MICROSOMAL-BOUND COLLAGEN PRECURSORS IN RABBIT EMBRYO SKIN IN VITRO* BY ISAAC J. BEKHOR,t ZIAEDDIN M1OHSENI, 1\IARCEL E. NIMNI,t AND LUCIEN A. BAVETTA§

DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF SOUTHERN CALIFORNIA SCHOOL OF DENTISTRY, LOS ANGELES Communicated by J. P. Guilford, June 21, 1965 Previously,1 it was reported from this laboratory that the specific activity of microsomal-bound in rabbit embryo skin increased linearly over several hours when incubated with -C1'. In addition to this finding, it was also reported that when actinomycin D was added to the incubation media, it exerted an immediate inhibitory effect on the formation of microsomal-bound hydroxyproline. One of the difficulties in the reported studies was the una-vaila- bility of a suitable method for simultaneous quantitative separation and isotopic analysis of proline and hydroxyproline. In the following experiments we have utilized an analyzer in combination with a liquid scintillation spectrom- eter to investigate further the nature of the collagen-synthesizing system. Addi- tional evidence is presented showing that actinomycin D inhibits the formation of microsomal-bound hydroxyproline as well as that of hydroxylysine, while incorpora- tion of a labeled amino acid into noncollagenous is not significantly altered. Collagen precursors refer to microsomal hydroxylysine- or hydroxyproline-contain- ing materials that are extractable with hot 5 per cent TCA. Materials and Methods.-The preparation of the skin, its method of incubation, and the prepara- tion of the microsomal fractions were carried out as described in the previous communication.' In addition to L-proline-C'4 (u.l., specific activity, 180 mc/miNI), L--C'4 (U.l., specific ac- tivity, 220 mc/mMI) and DL--2,3-H3 (specific activity, 5 c/mMI) were used. The labeled amino acids were obtained from New England Nuclear. Skin samples weighing approxi- mately 0.5 gm were preincubated for 2 hr with or without 25 ,og/ml actinomycin D (a gift from Merck, Sharp and Dohme), unless otherwise stated. Labeled amino acids were added at this time, and incubation continued for 4-5 hr. Isolation of proline-Cl4 and hydroxyproline-C'4 following the preparation of their nitroso deriva- tives: Hydroxyproline- or hydroxylysine-bound was extracted from the microsomes with hot 5% TCA as previously described,l and the free amino acids were released by hydrolysis.I The nitroso imino acids were prepared following the method described by MIyhill and Jacksoii.2 The nitrous acid was prepared by the addition of 3 ml of 40% aqueous NaNO2 to 10 ml of concen- trated HC1 in an ice bath. The hydrolysates were decolorized with activated charcoal, and the solution was filtered and evaporated to dryness. About 1 ml of nitrous acid per mg of amino acids was added to the hydrolyzate, and heated to 140'C in a vacuum oven until a faint yellow color was obtained. The acid mixture was evaporated to dryness. The residue was extracted with ether, and the nitroso imino acids were dissolved in 1.0 ml citrate buffer 0.2 N, pH 2.2. They were then separated on a 69 X 0.9-cm column containing Beckman spherical resin type AA-15, operated at 550C and using a pH 3.28, 0.2 N citrate buffer. The Beckman amino acid analyzer Downloaded by guest on September 29, 2021 616 BIOCHEMISTRY: BEKHOR ET AL. PROC. N. A. S.

0.10 N-Prelime

N-lydroxy- ' ~prelime N- Argimise

40 i0 126 166 200 241 mi. EFFLUENT FIG. 1.-Profile of the separation of N-proline, N-hydroxyproline, and N- on the Beckman amino acid analyzer model 120B, at 550C, using pH 3.28, 0.2 N citrate buffer.

model 120B was used to automate this procedure. Standards of 100 ,ug of each nitroso imino acid were used. The separation is shown in Figure 1. In addition to N-hydroxyproline and N- proline, a third peak was obtained and was identified to be a nitroso derivative of arginine. For the detection of radioactivity the effluent was passed through a 1.0-ml flow cell in a Nuclear- Chicago scintillation spectrometer, at a flow rate of 1.0 ml per 70 sec. After counting, the sample was passed through the reaction bath, to which ninhydrin was fed at one half the buffer rate. Counting was carried out at 50% efficiency at a setting which gave a background count of 3-7 cpm, thus affording a very sensitive system for the detection of small amounts of radioactivity in the effluent. Specific activity represents cpm/hmole of amino acid. A recovery of 95-100 per cent was observed both in iAmoles of N-imino acid and in cpm placed on the column. Isolation of other amino acids and the measurement of their radioactivity: Lysine and hydroxy- lysine were separated on a short column (15 X 0.9 cm), packed with Beckman spherical resin type AA-27, operated at 30'C, using a pH 5.28, 0.35 N citrate buffer. Counting of radioactivity was carried out as above. Studies with tryptophan: Following the incubation with tryptophan-H3, the microsomes were isolated, washed, and collagenous materials were removed with hot 5% TCA.1 The TCA-pre- cipitated protein was dissolved in 1.0 ml of hyamine, then placed in 15 ml of toluene containing 100 mg POPOP and 3 gm PPO per liter for counting. The measurement of free proline and hydroxyproline in skin: The incubated tissue was washed in saline, homogenized in 5 ml of 1% picric acid, and the precipitate was discarded. Picric acid was removed by passing through a column containing Dowex-2X-8 resin. The effluent was evaporated to dryness and the nitroso derivatives of proline and hydroxyproline were prepared as previously indicated. Separation and counting were carried out as described above. The isolation of total tissue collagen: When the incubation was terminated, the tissue was re- moved, washed in saline, and homogenized in 5 ml of 5% TCA. The precipitate was washed as described previously,' and collagenous material was extracted with hot 5% TCA. The super- natant was dialyzed against distilled water for about 20 hr. The nondialyzable material was evaporated to dryness, and the amino acids were rendered free upon hydrolysis.' The nitroso derivatives of proline and hydroxyproline were prepared, separated, and counted as above. Results.-The biosynthesis of microsomal-bound collagen precursors: In the pre- vious communication' it was reported that the uptake of proline and its incorpora- tion into bound hydroxyproline in the skin microsomes increased linearly with time of incubation. Thias isn contrast to the typical behavior of a tissue slice,' a homogenate,4 or a cell-free system5 where equilibration between the specific activity of an amino acid in the intracellular pool, or in the medium, and on the microsomes occurs quite rapidly. This delay of reaching the stage of equilibrium seems to be a peculiarity of the tissue under investigation. As shown in Figure 2, the specific activity of total free proline and hydroxyproline in this tissue does not reach a Downloaded by guest on September 29, 2021 VOL. 54, 1965 BIOCHEMISTRY: BEKHOR ET AL. 617

12 L /-,~t F"S 2 FIG. 2.-The change in the specific activity / of total skin free proline-C'4 and hydroxypro- _ line-CH4 with time of incubation. A skin _ a ; / _ 2 sample of about 0.5 gm was preincubated for ! 2 hr before the addition of 1 jsc of proline-C'4 to the medium, and incubation was continued ,1 -- for an additional 5 hr. The technique of analysis was described in Methods.

2 4 2 4 Isrt plateau until after 3 hr of incubation. The incorporation into the skin microsomes of lysine and its conversion to bound hydroxylysine are shown in Figure 3. The in- crease in the specific activity of microsomal-bound lysine and hydroxylysine is evident over the period of incubation. In order to understand the sequence of events that accompanies this process, the variation in the specific activity ratios of lysine to hydroxylysine or of proline to hydroxyproline was followed as a function of incubation time. Analysis of the change in the ratios is shown in Figure 4. The data for lysine was calculated from Figure 3, and for proline was calculated from an. independent experiment. The initial increase in the specific activity ratio in Fig- ure 4 suggests that the incorporation of either lysine or proline into microsomal

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9- 2 1 2 3 4 II fl~~~~~~~~~~~~~~~~~~~Nours 1 2 3 4 ~lifS FIG. 4.-The change in the specific activity ratio of lysine to hydroxylysine and of proline FIG. 3.-Time course incorporation of radio- to hydroxyproline from a 2-hr preincubated activity into lysine and into hydroxylysine skin. The label, 2 Muc of lysine-C'4 or 1 juc of from hot 5% TCA-soluble microsomal protein. proline-C'4, was added at the end of the pre- The skin was preincubated for 2 hr, at which incubation time. The specific activity ratio of time 2 Muc of lysine-C"4 was added to the lysine to hydroxylysine was calculated from medium. Separation was carried out as de- Fig. 3, and of proline to hydroxyproline from scribed in Methods. an independent experiment. Downloaded by guest on September 29, 2021 618 BIOCHEMISTRY: BEKHOR ET AL. PROC. N. A. S.

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1 2. 3 4 FIG. 5.-The effect of 25 jig/ml of actinomy- Neer$ cin 1) on the formation of microsomal-bound hydroxyproline-C 14. The control was studied FIG. 6.-The change in the specific activity as in Fig. 3. For actinomycin, skin samples ratio of lysine to hydroxylysine as affected by were preincubated with actinomycin D for 2 hr 25 ,ug/ml of actinomycin D. Studies with the before the addition of 1 lie of proline-C'4 to the antibiotic were carried out as in Fig. 5, except media. that 2 jie of lysine-C14 were used. polypeptides exceeds the rate of formation of bound hydroxylysine or hydroxypro- line. This increase may represent a lag phase in the synthesis of bound hydroxy- amino acids. The rate of incorporation and the rate of hydroxylation become constant after 2 hr of incubation. No change in the ratio can be observed between the second and third hour of incubation. Thus, proline or lysine are incorporated into the microsomes and are hydroxylated at a constant rate with a constant in- crease in their specific activities. Following this "constant" period, the ratio be- gins to drop, probably because the incorporated labeled amino acid is approaching a constant specific activity due to equilibration with the intracellular pool while that of the hydroxy-amino acids is increasing. The effect of actinomycin D on the formation of microsomal-bound collagen pre- cursors: In the earlier communication' it was suggested that collagen messenger RNA was unstable and hence the formation of bound hydroxyproline was inhibited by the antibiotic. Further evidence that actinomycin D inhibits the formation of collagen is presented in the following experiments. This is shown by the effect of the antibiotic on the specific activity of newly formed microsomal-bound hydroxyproline (Fig. 5), on the specific activity ratio of micro- somal lysine to hydroxylysine (Fig. 6), and on total tissue collagen hydroxyproline (Fig. 7). When the specific activity ratio plot for the actinomycin D-treated tissue is compared with the control (Fig. 4), a significant drop in the formation of bound hydroxylysine is implied. A linear increase in the ratio is extended over a period of 3.5 hr, and reaches much higher values than the control. The ratio of incorpora- tion to that of hydroxylation of lysine does not reach a constant level as occurred with the control (Fig. 4), indicating that the formation of hydroxylysine is lagging throughout this period of incubation. The data summarized in Figure 7 seems to reflect a total inhibition of collagen biosynthesis within 2-3 hr, with an over-all in- Downloaded by guest on September 29, 2021 VOL. 54, 1965 BIOCHEMISTRY: BEKHOR ET AL. 619

10 1

FIG. 7-The inhibition of the biosynthesis of total skin collagen with actinomycin D as isft measured by the change in the specific activity of hydroxyproline-C 14. The control was stud- Z / ied as in Fig. 3, using 1 .tc of proline-C'4. In- cubation with actinomycin was done as in Fig. 5, using 1 ,ucof proline-C'4. The isolation of -/ o the hot TCA-soluble protein is described in 5"_;/ Illn*|!el_O'A**,0*____ _ Methods.

1 2 3 4 S

hibition of 57 per cent following the first hour of incubation. This inhibitory effect was also reflected by the proline-C14 incorporation. Table 1 shows the values ob- tained when the proline-hydroxyproline ratios as well as the ratios of their specific activities were determined in the TCA-extractable fractions from both control and actinomycin-treated skins. These values obtained for the chemically determined proline-hydroxyproline ratios are in close agreement with those reported in the literature for skin of newborn rabbits.' The ratios of the specific activities of all specimens remained quite constant, in spite of the fact that actinomycin-treated tissues synthesized significantly less new collagen molecules. The lack of an inhibitory effect on the biosynthesis of noncollagenous proteins was studied with tryptophan-H3. Tryptophan, which is absent in collagen, has been used as an index for examining the amount of noncollagenous protein that is extracted with collagen from carrageenan granuloma.7 The results, shown in Figure 8, suggest that actinomycin D does not significantly inhibit the incorporation of tryptophan into microsomal protein over the experimental period. Discussion.-In the present experiments, using embryonic skin, it has been ob- served that the microsomal fraction exhibits a unique behavior in the sense that its specific activity is constantly increasing as a function of time. This is quite different from what occurs in other systems such as liver slices, homogenates, or cell-free preparations where the specific activity of microsomal-bound amino acid and that of the amino acid pool reach a maximum rather rapidly. Liver micro- somes, following an injection of a labeled amino acid into the animal, also reach a constant specific activity in a short time.8 Other in vivo studies utilizing the chick embryo have shown that the microsomes reach maximal specific activity at about 1 hr withrespect to proline, and about 2 hr with respect to lydroxyproline.9 The highest

TABLE 1 RATIOS OF PROLINE TO HYDROXYPROLINE IN TOTAL COLLAGEN FROM CONTROL AND ACTINOMYCIN D-TREATED TISSUE Time ,- Ratio (pmole) - Specific Activity Ratio (hr) Control Act. D Control Act. D 1 1.33 1.16 1.76 1.73 2 1.37 1.28 2.56 2.62 3 1.02 1.21 2.04 2.10 4 1.39 1.27 1.76 2.20 5 1.43 1.30 2.04 2.10 Avg. 1.31 1.24 2.03 2.15 Downloaded by guest on September 29, 2021 620 BIOCHEMISTRY: BEKHOR ET AL. PROC. N. A. S.

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/e / D$I 1 2 3 4 5 Nears FIG. 8.-The uninhibitory effect of actinomycin D on the incorporation of tryptophan-H3 into microsomal proteins. The control was preincubated for 2 hr in the absence of the labeled amino acid; 2.5 ,Ac of tryptophan-H3 were then added. For actinomycin, the skin was preincubated with 25 ;&g/ml of the antibiotic for 2 hr, at which time 2.5 4c of tryp- tophan-H3 were added. incorporation in liver homogenates is reached in 15 min.4 The time required to reach a maximal constant specific activity on the microsomes may be dependent not only on the rate of protein synthesis but also on the availability of the labeled amino acid to the site of protein synthesis. Studies on the intracellular accumula- tion of labeled amino acids in liver slices have shown that the tracer amino acid ap- proaches its highest specific activity within a period of 30 min.10 Studies using granuloma tissues in vitro have shown that the total free tissue labeled proline ap- proaches a constant value in about 10 min.3 The intracellular concentration of proline could not be estimated because of the large proportion of extracellular space in that tissue. The present studies show (Fig. 2A) that the specific activity of total free proline reaches a plateau in 3 hr. No reliable estimate of the specific activity of intracellular proline can be made at this time; however, it may be assumed that the specific activity of intracellular proline is increasing proportionally. The in- crease in the specific activity of free hydroxyproline (Fig. 2B) parallels that of total free proline in the skin; and free hydroxyproline represents intracellular activity. Many a time it has been shown that proline or lysine are the only source of free or bound hydroxyproline or hydroxylysine.11-"3 Examining the specific activities of microsomal-bound hydroxyproline, free hydroxyproline, and total collagen hy- droxyproline, it seems that free hydroxyproline may originate not only from total collagen hydroxyproline but also from the possible breakdown of microsomal polypeptides containing hydroxyproline. This is because free hydroxyproline has a higher specific activity than total collagen hydroxyproline and lower specific activity than microsomal hydroxyproline. The specific activity ratio (Fig. 4) represents a satisfactory way of understanding the sequence of events in the formation of microsomal-bound collagen precursors. The increase in the specific activity ratio from 30 to 120 min serves as evidence for a lag phase in the formation of hydroxylysine and hydroxyproline. The plateau Downloaded by guest on September 29, 2021 VOL. 54, 1965 BIOCHEMISTRY: BEKHOR ET AL. 621

signifies a condition where both collagen precursor formation and noncollagenous protein synthesis have reached a constant rate. The drop in the ratio indicates that the increase in the specific activity of hydroxy-amino acids becomes greater than that of the incorporated labeled proline or lysine. In conclusion, microsomal col- lagen-precursor formation may follow this proposed sequence: (1) a lag phase due to delay in hydroxylation; (2) a plateau condition where increase in incorporation and in hydroxylation becomes constant; and (3) a regression phase where the spe- cific activity of the collagenous hydroxy-amino acids is attempting to reach that of the precursor amino acids. The studies with actinomycin D show a definite inhibition in the formation of bound collagenous hydroxy-amino acids (Figs. 5-7). The data from Figure 6 suggests that the inhibition of collagen synthesis is on the microsomal level because the ratio of microsomal lysine to hydroxylysine only levels off at the time when maximal specific activity is approached. The data from Figure 7 unequivocally shows that total collagen synthesis is arrested after 3 hr of incubation. The conclusion that collagen messenger RNA may be unstable' again seems to be im- plied by these results; however, inhibition at another site cannot be ruled out. This conclusion appears to be in contrast to the reported study on the lifetime of collagen and noncollagen messenger RNA,14 using 3T6 mouse fibroblasts, an isolated cell culture, where studies were carried out on total cell protein. As has been sug- gested by these authors, the presence of coexistent messengers of different half lives in animal tissues may be due to mixed cell population. However, the signifi- cance of the present experiments lies in the fact that whole embryonic skin is being investigated in terms of collagen synthesis as well as the synthesis of other proteins. In addition, the experiments of Bloom et al.'4 should be carried out under various concentrations of actinomycin D, as levels which will produce total inhibition of proline-C14 incorporation will undoubtedly result in inhibition of hydroxyproline formation. Therefore, their interpretation that collagen messenger RNA has the same half life as that of noncollagen mRNA cannot be based on these findings alone. Our data suggests that collagen biosynthesis is more sensitive to actinomy- cin D than that of noncollagenous proteins in the rabbit embryo skin. Summary.-The biosynthesis of collagen on skin microsomes was followed by examining the specific activity ratio of either lysine to hydroxylysine or proline to hydroxyproline at this site. A lag in the formation of bound collagenous hydroxy- amino acids was evident. It was also found that both free proline and hydroxypro- line reached a constant specific activity only after 3 hr of incubation with labeled proline. Further evidence is also presented that actinomycin D exerts an inhibi- tory effect on the biosynthesis of collagen precursors, but has no effect on non- collagenous protein synthesis, as judged by the incorporation of tryptophan under these experimental conditions. The authors wish to thank Mr. Charles Lyons for his technical assistance. * This investigation was supported by a research grant from the National Institute for Dental Research. t Supported by training grant #5-T1-DE-94-02 from the National Institutes of Health, USPHS. In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at the University of Southern California, Los Angeles. t Postdoctoral trainee grant #5-T1-DE-94-02 from the National Institutes of Health, USPHS. Downloaded by guest on September 29, 2021 622 BIOCHEMISTRY: TRILLING AND APOSHIAN PROC. N. A. S.

§ Recipient of Career Development Award #1-K6-DE-6083 from the National Institutes of Health, USPHS. l Bekhor, I. J., and L. A. Bavetta, these PROCEEDINGS, 53, 613 (1965). 2 Iyhill, D., and D. S. Jackson, Anal. Biochem., 6, 193 (1963). 3 Green, N. M., and D. A. Lowther, Biochem. J., 71, 55 (1959). 4McCorquodale, D. J., E. G. Veach, and G. C. Mueller, Biochim. Biophys. Acta, 46, 335 (1961). 6 Peterkofsky, B., and S. Udenfriend, J. Biol. Chem., 238, 3966 (1963). 6 Gonzalez Cadavid, N., and A. C. Paladini, Biochem. J., 92, 436 (1964). 1Meister, A., N. Stone, and J. A. Manning, Advan. Chem. Ser., 44, 67 (1964). 8 Keller, E. B., P. C. Zamecnik, and R. B. Loftfield, J. Histochem. Cytochem., 2, 378 (1954). 9 Prockop, D. J., B. Peterkofsky, and S. Udenfriend, J. Biol. Chem., 237, 1581 (1962). '° Gulidotti, G. G., G. Ragnotti, and C. B. Rossi, Ital. J. Biochem., XIII, 145 (1964). 11 Stetten, -M. R., and R. Schoenheimer, J. Biol. Chem., 153, 113 (1943). 12 Sinex, MI. F., and D. D. Van Slyke, J. Biol. Chem., 216, 245 (1955). 13 Piez, K. A., and R. C. Likins, J. Biol. Chem., 229, 101 (1957). 4 Bloom, S., B. Goldberg, and H. Green, Biochem. Biophys. Res. Commrn., 19, 317 (1965).

A DEOXYRIBONUCLEASE FOUND AFTER INFECTION OF BACILLUS SUBTILIS WITH PHAGE SP3* BY DAVID M. TRILLINGt AND H. VASKEN APOSHIAN

DEPARTMENT OF MICROBIOLOGY, SCHOOL OF MEDICINE, TUFTS UNIVERSITY, BOSTON, MASSACHUSETTS Communicated by S. E. Luria, June 28, 1965 SP3 is a bacteriophage capable of infecting transformable strains of Bacillus subtilis.1' 2 The DNA of this phage contains thymine (unpublished observation). In order to understand the events which lead to the synthesis of mature phage, we have been investigating a number of viruses3 which infect B. subtilis. After in- fection of B. subtilis SB19 with phage SP3, we have observed a 50-fold increase of a deoxyribonuclease (DNase) that degrades heat-denatured DNA to acid-soluble components. Evidence is presented that this increase is the result of the synthesis of a new protein after phage SP3 infection. Materials and Methods-Materials: B. subtilis SB19 and phage SP3 were obtained from I)r. W. R. Romig. Calf thymus DNA, crystalline pancreatic ribonuclease, and crystalline pancreatic deoxyribonuclease were purchased from Worthington Biochemical Corp.; mitomycin C was purchased from Nutritional Biochemical Corp.; bovine plasma albumin from Armour Corp.; yeast sRNA type III was purchased from Sigma Chemical Co.; chloramphenicol was purchased from Parke, Davis and Co. Methods: The medium used for growth of B. subtilis SB19 and for phage propagation was the TY broth described by Romig and Brodetsky4 except that CaCl2 was omitted and the MnCl2 concentration was increased 10-fold. Lysates of phage SP3 were obtained by growing B. subtilis SB19 at 370 with vigorous aeration to a cell titer of approximately 1 X 108/ml. One phage per two cells was added and aeration was continued until the optical density of the culture decreased to approximately 10% of the value at the time of infection. The culture was then centrifuged at 14,000 X g for 10 min and the supernatant (approximately 1 X 1010 phages/ml) was saved. The phages were concentrated and partially purified by centrifuging 40-ml aliquots of the super- natant, at 40,000 X g for 1 hr. The supernatants were discarded and each pellet was washed with 2 ml of phage diluant (0.01 M potassium phosphate buffer, pH 7.1; 0.01 M MgSO4; 10 ,ug/ml bovine plasma albumin) and then resuspended by standing in 4 ml of phage diluant overnight at Downloaded by guest on September 29, 2021