Prolyl hydroxylase activity during sea urchin development

Preliminary

means of magnetic microspheres. Cell viability was good and virtually no cells were lost. Most of the cells in the T- and Bcell fractions could be recovered. So far, provided a good antiserum is available, the only technique that leads to cell subclasses of almost a 100% purity is the FACS [6, 71. However, as this device is very expensive and complicated in use, cheaper and simpler techniques are called for. With further improvement magnetic microspheres might be the best solution. As yet, however, this technique is not sufficiently sensitive to separate the different minor subclasses of cells present among other cells. In spite of these limitations, magnetic microspheres are useful for enriching populations in a given class of cells when a good antiserum is available. This work was supported by grants from the Finnish National Research Council for Natural Sciences, The Sigrid JusClius Foundation and the Nordisk Insulinfond. We wish to thank Dr Alan Rembaum for his kind help.

References

1. Ghetie,V, Mota,G & SjGquist, J, J immunol meth

21 (1978) 133. 2. B&urn, ‘A, Stand j clin invest 21, suppl. 97 (1968) 1. 3. Andersson, L C & Gahmberg, C G, Blood 52 (1978) 57. 4. Julius, M H, Simpson, E & Herzenberg, L A, Eur j immunoI3 (1973) 645. 5. Nordling, S, Andersson, L C & HLyry, P, Eur j immunol2 (1972) 405. 6. Banner, W A. Hulett. H R. Sweet. R G & Herzenberg, L A, R&J sci i&r 43 (1972) 404. 7. Herzenberg, LA, Sweet. R G & Herzenbere.w. LA. Sci Amer fi4 (1976) 108. 8. Molday, R S, Yen, S P S & Rembaum, A, Nature 268 (1977) 437. 9. Margel, S, Zisblatt, S & Rembaum, A, J immunol meth 28 (1979) 341. 10. Rembaum, A, Margel, S & Levy, J, J immunol meth 24 (1978) 239. Received May 6, 1980 Revised version received August 26, 1980 Accepted September 8, 1980

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467

Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/80/120467-04$02.0010

Prolyl hydroxylase activity during seaurchin development S. C. BENSON and A. SESSIONS, Department of Biological Sciences, California State University, Hayward, CA 94542, USA

Prolyl hydroxylase activity appears at the blastula stage of development in the sea urchin Strongylocentrotus purpuratus and increases over ‘I-fold by the prism larva stage. The enzyme requires ascorbate, ferrous ions, and a-ketoglutarate for maximum activity and is inhibited by a&-dipyridyl. The significance of prolyl hydroxylase activity in embryonic collagen metabolism and morphogenesis is discussed.

Summary.

The synthesis of collagen during sea urchin development has been studied by a number of investigators. Collagen synthesis begins during late cleavage and undergoes a several fold increase during gastrulation and pluteus formation [5, 6, 161. Several reports have implicated collagen in spicule formation [6, 161;however, the presence of abundant extracellular non-spicule collagen suggests that it may play a more general role in sea urchin morphogenesis [2, 171. Collagen is subject to a number of co- and post-translational modifications including hydroxylation of certain proline and lysine residues, glycosylation and cleavage of the procollagen polypeptide precursor [ 151. The formation of 4-hydroxyproline plays a significant role in collagen metabolism. Hydroxyproline residues contribute to the stability of the collagen triple helix [9, IS, 191 and evidence suggests that helix formation may be a prerequisite for secretion of procollagen [3, 11, 141. The enzyme prolyl hydroxylase (EC 1.14.11.2 proline, 2oxoglutarate dioxygenase) catalyses the synthesis of 4-hydroxyproline from certain prolyl residues in peptide linkage [l]. Despite the importance of prolyl hydroxylase in collagen metabolism we have been Exp Cell Res 130 (1980)

468

Preliminary

notes

Table 1. Cofactor requirements for 3H water re~ease~rom [3H]profyl substrate

Omission or addition”

[ZH]H,O” formed (dpm?

Minus~nzyme controt substracted @pm)

Rel. act. (%I

None Minus ascorbate Minus Fe*+ Minus cu-ketoglutarate Plus o,a’-dipyridyl Minus enzyme

5 363 2 162 1 680 I 563 865 607

4 756 1 5.75 I 073 956 258 0

100 32.6 22.5 20.1 5.4 0

n Incubations were performed as described in Materials and Methods except components were omitted as indicated or ff,ff‘-dipy~dyi was added to a final concentration of I.0 mM. Crude enzyme was extracted from 72 h larval stage embrvos and dialysed for 24 h against extraction buffer. b The results are averages of duplicate determinations.

able to find only one report of this enzyme in sea urchin embryos. In an abstract Ellis & Cain [4] report the presence of an activity capable of converting [‘“Cjproline to [‘*Clhydroxyproline. In the present report we describe the ontogeny and cofactor requirements of prolyl hydroxyIase from embryos of Strongylocentrotus purpuratus. Materials

and Methods

Gametes of Strongylocentrotus purwere obtained by intracoelomic injection of 0.5 M KCI. The eggs were washed, fertilized and cultured at 13°C at a concentration of 5~ 103/ml in Millipo~-~tered sea water (0.45 hm) containing 100 IU/mi penicillin G and SOrg/ml streptomycin sulfate.

Embryo puratus

Prolyl

culture.

~ydroxy~~~e

extraction

and assay.

Prolyl

hydroxylase activity was extracted from I liter of embryos at the desired developmentai stage. All operations were on ice or at 4°C. Embryos were concentrated by centrifugation at 900 g yielding a packed volume of 2.9-3.1 ml. Embryos were washed twice: first with 2 vol of 1.0 M dextrose, 0.01 M Tris-HCl, pH 7.6, and then with 2 vol of 0.01 M Tris-HCI, pH 7.6; 0.001 M dithiothreitol (DTT) (buffer A). Washed embryos (2.6-2.8 ml) were sonicated on ice in two volumes of buffer A by three bursts of 10 set each. T&on X-100 was added to a final concentration of 0.1% and the sonicate centrifuged for 20 min at 35 000 g. After cent~fugation the thin top layer of lipid was aspirated and the supernatant assayed immediately for prolyl hydroxylase activity or dialysed at 4°C for 24 h against buffer A. Proiyl hydroxylase activity was measured by a tritium release assay using [3,4-3H]~-proline labeled chick embryo unhydroxylated protocollagen as substrate [ 131.Incubation mixtures of 220 ~1 contained 150 ~1 enzyme extract (0.5 mg); 20 ~1 denatured protocolla8en substrate and 50 ~1 ot butter-cofactor mix yielding final concentrations of 40 mM Tris-HCI, pH 7.6; 1 mM sodium ascorbate, 1 mM o-ketoglutarate; 0.2 mM Exp Cell Res 130 (1980)

ferrous ammonium sulfate; 0.5 mM DTT; 2 mg/ml BSA and 0.4 mg/ml catalase. Assays were routinely run for 10 h at 25°C. When a,cY’dipyridyl was used it was added to a final concentration of I mM. Under these reaction conditions the hydroxylation of protocollagen is linear for at least 12-13 h (fig. IA) and embryonic prolyi hydroxylase is the limiting factor in the reaction (fig. 1B). Prolyl hydroxylase activity is expressed as disinte~tions per minute of 3H20 reieased in 10 h corrected for radioactivity released in the absence of enzyme. The presence of 2 mM chIoramphenico1 in the reaction mixture had no significant effect on tritium released at 10 h (results not shown). This minimizes the possibility that the tritium release was due to bacterial contamination. Protein was determined by the method of Lowry et al. [IO] using bovine serum albumin as standard.

Results and discussion

The cofactor requirements for prolyl hydroxylase activity previously documented in other organisms [l] could be demonstrated using 72 h prism larva embryo extract thus indicating the presence of prolyl hydroxylase (table 1). Using dialysed embryo extract there was a significant dependence on ascorbate, ferrous-ion and a-ketoglutarate. Activity was completely inhibited by addition of the iron chelator, &,a’-dipyridyl. The reason for the lack of complete dependency on added cofactors is unknown but may simply reflect very tight binding of endogenous cofactors by the crude extracts. Efforts are currently underway to further purify embryonic proiyl hy-

Preliminary notes

469

iooo-

0

4

8 12 16 HOURS AT 25’

20

.I5 .30 A.5 60 MG PROTEIN/lWJBATK)N

Fig. I. Protyl hydroxylase activity as a function of time and enzyme concent~tion. (A) Time course of 3H-

water release from [3H]protocollagen substrate in 0, the presence and A, absence of enzyme. (B) Release of 3H water from [3H]protocollagen substrate after 10 h in the presence of increasing concentration of enzyme. Minus enzyme control values were subtracted from each point. Incubations and analysis were carried out as described in Materials and Methods.

HRS OF-DEVELOPMENT

Fig. 2. Prolyl hydroxylase

activity during development. Prolyl hydroxylase was extracted at the indicated stages of development and assayed as described in Materials and Methods. The minus enzyme control value was subtracted from each point. Each point is the mean of two experiments.

number of adult systems. We have prektinary evidence (unpublished observations) that another enzyme involved in post-transdroxylase and re-examine the cofactor re- lational modification of collagen, lysyl oxiquirements. dase, also appears at blastula stage of deThe ontogeny of prolyl hydroxylase ac- velopment. Thus the possibility exists that tivity during sea urchin development is in sea urchin embryos, collagen and its asshown in fig. 2. Activity is absent in fer- sociated enzymes may represent a group of tilized eggs but is detectable at about 25 coordinately regulated genes. h of development which corresponds to The rapid rise in prolyl hydroxylase achatching blastula stage under our culture tivity and collagen synthesis during gastruconditions. Stages between fe~ilization and lation most likely represents the differentiahatchin blastula were not examined for tion of primary and secondary mesenchyme enzyme activity. Prolyl hydroxylase spe- cells. This differentiation is manifest by the cific activity increased almost 7-fold by formation of spicule matrix which may be midgastrula followed by a further slight collagenous in composition [6, 16]. In addicollagen fibrils become increase by the prism larva stage (75 h). tion, extr~ellul~ The initial appearance of prolyl hydroxydetectable at this time and may play a more lase activity at hatching blastula corre- general role in sea urchin morphogenesis such as the formation of cell attachment sponds to the first detectable hydroxyproline synthesis in sea urchin embryos [Sj. sites [7, 121 and basal lamina [8, 181. We The rapid rise in prolyl hydroxylase activity are currently investigating these possibihduring gastrulation correlates with the pre- ties. viously documented increases in collagen We would like to thank Drs R. Bahatnagar and 2. synthesis [5, 6, 161. Similar coordinate in- Hussein for providing the 3H-protocollagen used in the early phases of this work. This investigation was supcreases in collagen synthesis and prolyl ported in part by funds from the Research Committee, hydroxylase activity have been noted in a California State University, Hayward, CA, USA.

470

Preliminary

notes

References

Human diploid fibroblasts exhibit an age1. Cardinale, G J & Udenfriend, S, Adv enzymol or passage-dependent decrease in replica41 (1974) 245. tive capacity [l-5]. From kinetic analysis of 2. Crise-Benson, N & Benson, S C, Wilh Roux Arch Entwicklungsmech organ 186 (1979) 65. thymidine incorporation and from autora3. Dehm, P & Prockop, D J, Biochim biophys acta diographic analysis it is clear that this de240 (1971) 358. 4. Ellis; C H & Cain, G, Am zoo1 (1970) 318. crease results from the lengthening of the 5. Golob, R, Chetsanga, C J & Doty, P, Biochim average cell cycle transit time [24] and biophys acta 349 (1974) 135. 6. Gould, D & Benson, S C, Exp cell res 112 (1978) from a decreasing proportion of cells enter73. ing the S phase during a specific passage 7. Gustafson, T & Wolpert, L, Biol rev 42 (1%7) 442. 8. Hay, E, Am zoo1 13 (1973) 1085. [l, 4, 51. In addition, most in vivo studies 9. Jimenez, S A, Harsch, M & Rosenbloom, J, Biosuggest that this slowed or altered progresthem biophys res comm 52 (1973) 106. 10. Lowry, 0 H, Rosebrough, N J, Farr, A L & sion is not the result of an increased transRandall, R J, J biol them 193 (1951) 265. it time through the S phase, but instead 11. Margolis, R L & Lukens, L N, Arch biochem biophys 147 (1971) 612. is the result of slowed progression through, 12. McClay, D & Marchase, R, Dev biol71 (1979)289. 13. Peterkofsky, B & DiBlasio, R, Anal biochem 66 or permanent arrest in Gl [5-71. This in (1975) 279. turn suggests that the rate of DNA synthe14. Peterkofsky, B, Biochem biophys res comm 49 sis (in cells capable of entering the S phase) (1972) 1343. 15. Prockop, D J, Berg, R, Kivirikko, K I & Uitto, remains relatively constant. The DNA fiS, Biochemistry of collagen (ed G Ramachandran ber-autoradiographic data of Petes et al. & A Reddi) p. 163. Plenum, New York (1976). [8], however, indicate that in at least one 16. Pucci-Minafra. I. Casano. C & LaRosa. C., Cell diff 1 (1972) 157. 17. Spiegel, E & Spiegel, M, Exp cell res 123 (1979) human diploid fibroblast (MRCS) the rate of 434. DNA chain growth is slower in late passage 18. Tilney, L & Gibbins, J, J cell biol41 (1969) 227. than in early passage cultures. Because of 19. Ward, A R & Mason, P, J mol bio179 (1973) 431. these somewhat conflicting reports, we Received May 22, 1980 thought it worthwhile to determine if ageRevised version received August 1, 1980 or passage-associated changes could be deAccepted August 15, 1980 tected in WI38 or MRCS cultures rendered permeable to deoxyribonucleotides. An in Copyright 0 1980 by Academic Press, Inc. All tights of reproduction in any form reserved vitro assay was employed since analysis 0014-4827/80/120470-04$02.00/O of in vivo studies could be complicated if there were age- or passage-associated DNA synthesis in permeabilized WI38 changes in thymidine transport or in the and MRCS cells dNTP pools [9]. WI38 cells were included T. D. GRIFFITHS and J. G. CARPENTER, Desince most of the previous in vivo work partment of Radiation Biology and Biophysics, University of Rochester, School of Medicine and Denwhich indicated an absence of age cortistry, Rochester, NY 14642, USA related effects on DNA synthesis employed Summary. DNA synthesis was examined in cultures this cell line [5-71. of growing WI38 and MRCS cells made permeable to deoxyribonucleotides. Cells from late passage cultures showed a reduced rate of deoxythymidine triphosphate (dTTP) uptake as compared to cells from early- to mid-passage cultures. This reduction became evident earlier in WI38 cultures (passage 33) than in MRCS cultures (passage 41). Although this reduced rate of incorporation appeared to be primarily due to a reduced percentage of replicating (S phase) cells in later passage cultures, some effect on the rate of DNA synthesis in replicating cells was also evident. Exp Cell Res 130 (1980)

Materials

and Methods

Cultures of WI38 and MRCS cells were initially obtained through the Department of Microbiology, The University of Rochester School of Medicine and Dentistry. Cells were grown in MEM medium containing 10% fetal calf serum (FCS) and antibiotics. Cultures in our laboratory are tested for mycoplasmic contamination by the method of Schneider et al. [lo]. Cultures were salit, 1 : 2 at regular intervals and grown Printed

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