Cytolytic pore-forming proteins and peptides: is there a common structural motif?

TIBS 16 - JUNE 1991 6 Endow, S. A., Henikoff, S. and Soler-Niedziela, L. (1990) Nature 345, 81-83 7 Yang, J. T., Laymon, R. A. and Goldstein, L. S. B. (1989) Cell 56, 879-889 8 Zhang, P., Knowles, B. A., Goldstein, L. S. B. and Hawley, R. S. (1990) Cell 62,1053-1062 9 Hagan, I. and Yanagida, M. (1990) Nature 347, 563-566 10 Otsuka, A. J., Jeyaprakash, A., AnnoverosGarcia, J., Tang, L., Rsk, G., Hartshome, T., Franco, R. and Bom, T. (1991) Neuron 6, 113-122 11 LeGuellec, R., Paris, J., Couturier, A., Roghi, C. and Philippe, M. Mol. Cell. Biol. (in press) 12 Hirokawa, N., Pfister, K. K., Yorifuji, H., Wagner, M. C., Brady, S. T. and Bloom, G. S. (1989) Cell 56, 867-878 13 Scholey, J. M., Heuser, J., Yang, J. T. and Goldstein, L. S. B. (1989) Nature 338, 355-357 14 McDonald, H. B. and Goldstein, L. S. B. (1990)

BIOLOGICAL MEMBRANES contain a phospholipid bilayer as their basic structural unit. Phospholipids are amphiphilic molecules, containing both hydrophobic and hydrophilic ends that aggregate spontaneously into a brayer where the polar lipid heads face the aqueous surface and the fatty acyl chains are sequestered within the hydrophobic interior. The bilayer represents a natural barrier to the flow of ions and other solutes, and transport of solutes across membranes is thus an essential process in living cells. Specific pumps and channels have evolved to allow the active translocation of solutes against solute concentration gradients or passive movement down the gradients, while more primitive channels are found in the outer membranes of Gramnegative bacteria and mitochondria, which function as molecular sieves that allow the passage of small solutes up to a defined exclusion limit. The highest transport rates are in fact accomplished by non-selective pores, which permit flux rates approaching those of free diffusionL Both vertebrate and invertebrate systems have taken advantage of the molecular structures involved in the latter type of pores for an entirely different purpose, having evolved largely non-specific pore-forming proteins (PFP) to lyse other cells. The wide range of cell types producing cytolytic PFP attests to the antiquity of this lethal strategy, for these toxins have now been isolated from sources as

Cell 61, 991-1000 15 Stewart, R. J., Pesavento, P. and Goldstein, L. S. B. (1990) J. Cell Biol. 111, 416a 16 Endow, S. A. and Hatsumi, M. Proo. Natl Acad. SCi. USA (in press) 17 Perrimon, N., Engstrom, L. and Mahowald, A. P. (1989) Genetics 121, 333-352 18 Euteneuer, U., Koonce, M. P., Pfister, K. K. and Schliwa, M. (1988) Nature 332,176-178 19 Walker, R. A., Salmon, E. D. and Endow, S. A. (1990) Nature 347, 780-782

20 McDonald, H. B., Stewart, R. J. and Goldstein, L. S. B. (1990) Cell 63,1159-1165 21 Vale, R. D. and Toyoshima, Y. Y. (1988) Cell 52, 459-469 22 Yang, J. T., Saxton, W. M., Stewart, R. J., Raft, E. C. and Goldstein, L. S. B. (1990) Science 249, 42-47 23 Block, S. M., Goldstein, L. S. B. and Schnapp, B. J. (1990) Nature 348, 348-352

24 Schnapp, B. J., Crise, B., Sheetz, M. P., Reese, T. S. and Khan, S. (1990) Proc. Natl Acad. Sci. USA 87, 10053-10057 25 Koshland, D. E., Mitchison, T. J. and Kirschner, M. W. (1988) Nature 331, 499-504 26 Steuer, E. R., Wordeman, L., Schroer, T. A. and Sheetz, M. P. (1990) Nature 345, 266-268 27 Pfarr, C. M., Coue, M., Grissom, P. M., Hays, T ~,., Porter, M. E. and Mclntosh, J. R. (1990) Nature 345, 263-265 28 Komma, D. J., Home, A. S. and Endow, S. A. (1991) EMBOJ. 10, 419-424 29 van Zeijl, M. J. A. H. and Marlin, K. S. (1990) Cell Regul. 1, 921-936 30 Kosik, K. S., Orecchio, L. D., Schnapp, B., Inouye, H. and Neve, R. L. (1990) J. Biol. Chem. 265, 3278-3283 3! Wright, B. D., Henson, J. H., Wedaman, K. P., Willy, P. J., Morand, J. N. and Scholey, J. M. J. Cell Biol. (in press)

Cytolytic po e-foFming p oteins and peptictes is theFe a common st uctuFal motif Pore-forming proteins or peptides (PFP) have now been isolated from a wide array of species ranging from humans to bacteria. A great number of these toxins lyse cells through a 'barrel-stave' mechanism, in which monomers of the toxin bind to and insert into the target membrane and then aggregate like barrel staves surrounding a central, water-filled pore. An evaluation of the secondary structures suggests that common secondary structures may be employed by most of these toxic PFP.

diverse as bacteria, the vertebrate immune system, sea anemones, amoeba, higher fungi and venoms from insects and snakes. A large number of pore-forming toxins are now known to create channels through a 'barrel-stave' mechanism2. Three discrete steps have been defined for this process: (I) water-soluble monomers bind to the membrane, (2) they insert into the membrane, and (3) they aggregate like barrel staves surrounding a central pore that increases in diameter through the progressive recruitment of additional monomers. The pore is formed by simple lateral oligomerization of the monomers such that the hydrophobic side of the protein is exposed to the membrane acyl chains and the hydrophilic sides line up the pore. As a result, stable transmemD. M. Oj©ius and J. DIE Young are at the brane pores are formed that allow the Laboratoryof CellularPhysiologyand Immunology,The RockefellerUniversity,1230 passive flux of ions and small molecules across the bilayer. Since many of the YorkAvenue,NewYork,NY10021, USA.

© 1991,ElsevierScience Publishers, (UK) 0376-5067/91/$02.00

homopolymeric channels formed by toxins are not large enough to allow the passage of cytoplasmic protein, this model predicts that the channels produce an ionic imbalance in the cell, which then results in colloid osmotic lysis. Some of the earliest evidence for this me,~ lism of pore formation was obj. ~d with the planar bilayer system using alamethicin, a small polypeptide produced by the fungus Trichoderma viride and rich in the unusual amino acid, a-aminoisobutyric acid (Aib). Analysis of the first measurements of alamethicin-induced conductance changes showed that the conductance increased proportionally to the sixth power of the alamethicin protein concentration, implying that the active unit may be a hexamer3. Subsequent studies of the concentration dependence of conductance yielded a ninth power dependence4, so that, while the number

22S

TIBS 16 - JUNE 1991

an amphiphilic [5-strand is predicted if the hydrophobicity varies with a period of two residues. However, as interactions between proteins and phospholipid surfaces are less specific than those between proteins and proteins, substitution of a single residue in a particular location by one of an apparOlo.1, ently opposite hydrophobicity may not represent an intolerable disruption in the toxin's amphiphilic structure. The skewed distribution of hydrophobic and hydrophilic residues that results in ahelices is evident if the n Alamethicin amino acid sequence is displayed on an Edmundson aRgure1 helical wheel. With small and Design of amphiphilic a-helices. The a-helical wheel representation of &toxin and alamethicin shows medium sized toxins (e.g. alathe hydrophobic residues (shaded) predominantly on one side and the hydrophilic residues on the methicin and 5-toxin, Fig. I), other side of the central axis. Aib, a-aminoisobutyric acid. the hydrophilic residues are readily found on one face of of monomers involved may be uncer- brahe proteins 6. However, it is now the helix, whereas the hydrophobic tain, it is clear that alamethicin clear that other forces are also residues segregate almost completely oligomerization takes place. This model involved. An example of alternate struc- on the opposite side of the helix. The has been substantiated for a group of tures can be found in porins, which cre- amphiphilic nature is characteristic of Aib-containing peptides related to ala- ate pores across the outer membrane of not only PFP of the barrel-stave class methicin (all of these being between 15 E. coli. Analysed only on the distri- but also of many putative transmemand 24 amino acids long), and, as will bution of hydrophobic residues in the brane helices of transmembrane probe discussed below, there is reason to amino acid sequence, this protein teins in general. believe that similar mechanisms of pore would not typically be assigned to the One may now consider a model for formation are used by a number of category of transmembrane proteins, the conformational changes involved in other toxins. since it lacks hydrophobic sequences pore formation by the barrel-stave class The pore diameters of these toxins long enough to span the membrane. of toxins. While the water-soluble can differ widely, in all cases, though, Nonetheless, the bulk of this largely monomer must have a surface that is the monomers must be long enough to hydrophilic protein exists within the predominantly hydrophilic, it may span the membrane, even if, for some lipid bilayer, and its secondary struc- assume either a random-coil structure toxins, the monomers could conceiv- ture appears to consist predominantly or a rigid a-helical or ~sheet structure ably oligomerize before inserting into of antiparallel ~-pleated sheets T. Each in solution. Binding of the monomer to the membrane. According to the 'pre- segment, consisting of 10-12 residues, the membrane would then initiate a aggregate' model 5, both aggregation runs roughly perpendicular to the plane conformational change in the monomer and incorporation of the PFP precedes of the membrane. Thus, rigid amphi- which exposes hydrophobic sites. the actual opening of the oligomeric philic secondary structures formed by Although the free monomer may channel. a-helices or ~sheets would be expected already be amphiphilic, a hydrophilic to be found in PFP. In fact, such struc- to amphiphilic transition is induced in Structures of barrel-stavePFP tures are thought to make up most the monomers that were originally in Insertion of the pore-forming toxins medium-sized peptides that interact random
TIBS 16 - JUNE 1991

channels while segregating the hydrophobic and hydrophilic surfaces of each monomeric constituent away and towards the central axis of the pore, respectively. A role for amphiphilic []-sheets or a-helices is clearly evident at this stage, as it is difficult to envision solely hydrophobic a-helices forming a water-filled channel. When in the []-sheet configuration, a minimum of eight amphiphilic [5-strands must be present to form a barrel ~°. This requirement could be satisfied by either eight separate monomers or fewer monomers containing multiple [~sheets. Either way, hydrogen bonds must be formed between neighboring []-sheets to produce a closed structure. From the point of view of the barrel-stave model, an attractive feature of amphiphilic ~sheets is that they have the tendency to self-associate". In addition to amphiphilicity, a minimal length requirement must also be fulfilled by the PFP. Thus, a minimum of 20 residues is needed to form a membrane-spanning a-helix and at least ten residues to form a membrane-spanning []-sheet12. Many of the general rules governing pore-formation have been gleaned from experimental work on the structure of synthetic peptides. In their elegant

study 13, DeGrado

and Lear synthesized isocompositional peptides of Leu (1,) and Lys (K) to investigate the effect of hydrophobic periodicity on peptide secondary structure. Based on their hydrophobic repeat periods, two pep-

tides, (LKKLLKL)2(I) and LKLKLKL(2), were predicted to assume amphiphilic a-helical and []-sheet configurations, respectively. This prediction was borne out by circular dichroism (CD) spectra measurements, which revealed that, depending on the peptide and salt concentrations, peptide 1 adopted either a random-coil or a-helical conformation. Similarly, peptide 2 could be found in an equilibrium between the random-coil and []-sheet configurations. For both peptides, the amphiphilic structure was stabilized by self-association at higher peptide concentrations or binding to extrinsic apolar surfaces ~3.The extent of association was much higher for peptide 2 (31 residues) than for peptide I, consistent with there being a high level of intermolecular hydrogen-bonding between the {]-sheets and mostly intramolecular hydrogen bonding in the a-helices. In an extension of this work, a 21residue peptide composed exclusively of Leu (L) and Ser (S) and designed to be a membrane-spanning amphiphilic

\/ RlPlre 2 Pore formation by the barrel-stave class of PIP. Dependingon the toxin, either a monomer with random-coil structure binds to the membrane, where the monomer acquires an amphiphilic conformation, or monomers already in an amphiphilic configuration bind to the membrane without undergoing major conformational change. The monomers subsequently insert into the membrane, and through lateral diffusion aggregate to form oligomeric pores of varying diameters.

a-helix, (LSSLI~L)3, was found to form well-defined ion channels in membranes 12. It was concluded that an a-helix must be approximately 20 residues in length to span the hydrocarbon portion of the membrane, it must be amphiphilic, and it should have a high enough degree of hydrophobicity to allow partitioning into the bilayer in a vertical orientation ~2. Although hydrophobicity is the primary driving force for peptide--peptide interactions, the two-dimensional nature of the membrane lattice also contributes to peptide oligomerization due to the presence in the membrane of other monomers at high concentrations and the preoriented state of the monomers. It has been estimated that, compared to the association of monomers tumbling freely in solution, the fact that oligomerization takes place in a restricted membrane volume enhances the likelihood of dimer formation by a factor of a million, and the enhancement is several orders of magnitude greater for formation of trimers and higher oligomers".

Specificexamplesof small PIP To illustrate the principles behind the structures of PFP, three representative examples of small PFP will be considered. This list is by no means exhaustive, as other pore-forming toxins have also been characterized. Staph)tococcal &toxin. The Gram-positive bacterium Staphylococcus aureus secretes several toxins, including four membrane-active agents, a-, ~-, y- and &toxin. Analysis by high-resolution ~H-NMRsuggests that &toxin, a peptide of 26 amino acid residues, forms a relatively stable a-helix Is. The distribution of the amino acid residues is such that, if displayed on an a-helical wheel, they

form an amphiphilic rod (Fig. I), and it has been proposed that the toxin forms barrel-stave pores through the aggregation of six monomers in the target membrane. CD studies with a 26residue analogue of 8-toxin revealed an a-helical configuration in both the free and lipid-bound states ~s. M~Inlns. These antimicrobial peptides are 23 amino acids long and have been isolated from the skin of Xenopus laevis. Primary structure analysis predicts that magainins form amphiphilic a-helices. Magainins have a random-coil conformation in aqueous solution, but assume a rod-like structure in the presence of organic co-solvent ~7. The concentration dependence of pore formation by magainins is suggestive of a multimeric structure, composed of at least four monomers. Cm:mplns. These 37-residue antibacterial toxins are produced by the humoral immune system of certain insects. Displayed on an a-helical wheel, the proteins assume an amphiphilic a-helical configuration. The lytic activities of six synthetic analogues of cecropin A were recently investigated ~s. A conserved Trp in position 2 of cecropins A, B and D could be substituted by another residue as long as the other residue was also hydrophobic. Thus, replacement of Trp2 with Glu but not Phe resulted in a sharp drop in antibacterial activity. However, an analog involving replacement of one hydrophilic residue with a hydrophilic residue of a different charge (Lys6 with Gill) gave variable antibacterial activities, which depended on the species of bacteria being tested TM. Hence, at least part of the specificity of cells being lysed could be due to electrostatic interactions between the peptide and the target membrane. 227

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specifically to sphingomyelin, since pretreatment of normally sensitive cells with sphingomyelinase renders the cells resistant. It is thought that the toxin oligomerizes into a functional unit consisting of a trimer z4. Antibodies against the S. helianthus toxin neutralize the lytic activity of an 18 kDa poreforming toxin from another sea anemone, S. kenti '-5, suggesting that the latter may lyse cells through a similar mechanism. Pedodn is one among several cytotoxins expressed by cytolytic lymphocytes from vertebrate immune systems'E This -70 kDa toxin binds to phospholipids Large proteins that form barrel-stave pores Although there is good evidence that on the target membrane and, in its the toxins described below create monomeric form, inserts into the memhomopolymeric pores through a barrel- brane, where it polymerizes into pores stave mechanism, the structural motifs with heterogeneous diameters of up to responsible for their lytic activity have 20 nm. Sequence analysis of the cDNA been largely uncharacterized. Thus, it corresponding to perforin revealed that has yet to be determined whether they there is appreciable homology between share a common structural motif, or perforin and the terminal components whether more complicated rules will be of the membrane attack complex of needed to describe their pore-forming complement, especially C9. Aer01ysln. The Gram~rlegative bacteria properties. Staphylococcal a-toxin is produced as a Aeromonas sobria produces a potent single-chain 34 kDa protein that inter- 54 kDa cytolysin known as aerolysin '-7. acts preferentially with phosphatidyl- This toxin probably binds non-specificholine from the target membrane. This cally to a glycoprotein or glycolipid, toxin is very hydrophilic, and the pres- whereupon it aggregates to form an ence of an amphiphilic surface suffices oligomeric pore. Like porin, aerolysin to induce its oligomerization into cylin- lacks a-helical structures, and its pridrical lesions consisting of hexamers z°. mary sequence is very hydrophilic and The CD spectrum of the hexamer in probably forms 15-pleated sheets. detergent micelles revealed that it conFinally, indirect evidence suggests tains a high proportion of 13-sheets, that both extracellular and intracellular which are assumed to reside within the parasites cause pathogenesls through lipid bilayer when pores are formed in the action of barrel-stave PFP2a. membranes 21. Interestingly, the toxin from the intraStrel~olysln 0. This 69 kDa protein is cellular parasite Trypanosoma cruzi has the prototype of a family of suifhydryi- recently been shown to be immunoactivated cytolysinsz2. It appears to logically related to the terminal compbind specifically to cholesterol, and lement component, C9 (Ref. 29). thus attacks all mammalian cells, but not bacteria, whose membranes lack A common structural motif? cholesterol. The pores are created While employing similar overall through lateral aggregation of choles- strategies for pore-formation, the barrelterol-toxin complexes on the surface of stave class of PIP can accommodate a the membrane 23. This toxin-toxin inter- wide group of proteins with widely action presumably causes a confor- differing properties. These PFP can vary mational change in the protein that greatly as to their receptor requireallows for membrane insertion of the ments, and both homogeneous and hetpreaggregated complex. Between 25 erogeneous PFP have been identified. and 80 monomers participate in form- Homogeneous structures are observed ing the streptolysin O lesions, so that when a constant number of monomers the pores are very heterogeneous and comprises each individual lesion, as is may be as large as 35 nm in diameter. likely the case for most staphylococcal Cnldadan toxins. Several species of sea (x-toxin pores 3°. Heterogeneous pores anemones elaborate a number of closely with varying diameters are expected related basic toxins of ,44, between when variable numbers of monomers 16000 and 20000. The toxin from constitute the pore, as is seen with ".'oichactis helianthus appears to bind streptolysin O and perforin 26,3°. Recently~9, it was found that the requirement for amphiphilic (x-helices can also be met by the D-enantiomers of naturally occurring cecropins or magainins. By CD, the synthetic D-isomers were shown to be mirror images of the corresponding L-isomers in several solvents, and both enantiomers were equally effective at producing ion channels and lysing bacteria. It was thus concluded that the peptides do not form stereospecific contacts with other protein receptors or the chiral components of the phospholipid membrane.

228

The amphiphilic a-helix/IS-sheet hypothesis for pore formation is on solid ground for peptides and small toxins of the barrel-stave class of PFP. if the same principles apply to the behavior of large PFP, then the existence of similar structural organization and immunological cross-reactivity would suggest that a bundle of amphiphilic ahelices or IS-sheets represents the only three-dimensional structure needed to create membrane pores, it is thus possible that pore-forming toxins have a conserved core region that is associated with the amphiphilic stretches making up the pore, and variable domains that could explain the functional diversity of the toxins. The variable domains could thus confer lipid or protein binding specificities to the monomer, and they could define the oligomeric size, which determines the pore diameter. The prevalence of the barrel-stave class of toxins throughout the biosphere could be explained if the toxins from invertebrate species evolved from a common ancestral gene that also gave rise to the vertebrate PFP. As the purification and sequencing of many of these PFP is currently underway, it will be fascinating to determine how far the structural homologies between the PFP extends to the level of primary sequences.

Acknowledgements We are grateful to R. B. Merrifield, R. M. Steinman and J. W. Taylor for critical reading of the manuscript, and to Z. A. Cohn for support and advice. References 1 Hille, B. (1984) Ionic Channelsof Excitable Membranes, SinauerAssociates 2 Ehrenstein,G. and Lecar,H. (1977) Q. Rev. Biophys. 10, 1-34 3 Mueller, P. and Rudin,D. O. (1968) Nature 217, 713-719 4 Eisenberg,M., Hall, J. E. and Mead, C. A. (1973) J. Memb. BioL 14,143-176 5 Gordon,L. G. M. and Haydon,D. A. (1976) Biochim. Biophys.Acta 436, 541-556 6 Engelman,D. M., Steitz, T. A. and Goldman,A. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 321-353 7 Kleffel, B., Garavito,R. M., Baumeister,W. and Rosenbusch,J. P. (1985)EMBOJ. 4,1589-1592 8 Kaiser,E. T. and Kedzy,F.J. (1983) Proc. Natl Acad. Sci. USA 80, 1137-1143 9 Bhakdi,S., Ruth, M., Sziegoleit,A. and TranumJensen,J. (1984) Infect. Immun. 46, 394-400 10 Lesk,A. M., "Branden,C-I.and Clothia,C. (1989) Proteins 5, 139-148 11 Osterman,D. G. and Kaiser,E. T. (1985) J. Cell. Biochem. 29, 57-72 12 Lear,J. D., Wasserman,Z. R. and DeGrado, W. F. (1988) Science 240, 1177-1181 13 DeGrado,W. F. and Lear,J. D. (1985) J. Am. Chem. Soc. 107, 7684-7689 14 Grasberger,B., Minton,A. P., DeLisi,C. and Metzger,H. (1986) Proc. Natl Acad. ScL USA

TIBS 16 - JUNE 1991 83, 6258-6262 15 Tappin, M. J., Pastore, A., Norton, R. S., Freer, J. H. and Campbell, I. D. (1988) Biochemistry 27, 1643-1647 16 Alouf, J. E., Dufourcq, J., Siffert, 0., Thiaudiere, E. and Geoffroy, C. (1989) Eur. J. Biochem. 183, 381-390 17 Marion, D., Zasloff, M. end Bax, A. (1988) FEBS Lett. 227, 21-26 18 Andreu, D., Merrifield, R. B., Steiner, H. and Boman, H. G. (1985) Biochemistry 24, 1683-1688 19 Wade, D., Boman, A., Wahlin, B., Drain, C. M., Andreu, D., Boman, H. G. and Merrifield, R. B.

A WIP.I.I. KNOWN generalization from the work of Otto Warburg is that high rates of glycolysis are observed in neoplastically transformed cells, even under highly aerobic conditions'. Warburg showed that pyruvate, most of which is synthesized in these cells from glucose provided in the serum or medium, is not taken up efficiently and oxidized by mitochondria, but is reduced to lactate. This observation has since been confirmed by many investigators in many systems 2, and put into a modern context by Racker3. Since cleaving vertebrate embryos have superficial similarities to tumor cells in that blastomeres divide rapidly without differentiating, it may be expected that they would also display high rates of aerobic glycolysis, and that changes in glycolytic rates might occur at critical points in development. The data described below indicate several similarities in metabolism between tumor cells and cleaving amphibian embryos, despite the absence of glycolytic activity in the embryos.

(1990) Proc. Natl Acad. Sci. USA 87, 4761-4765 20 Bhakdi, S., Fussle, R. and Tranum.Jensen, J. (1981) Proc. Natl Acad. Sci. USA 78, 5475-5479 21 Tobkes, N., Wallace, B. A. and Bayley, H. (1985) Biochemistry 24, 1915-1920 22 Geoffroy, C., Gaillard, J-L., Alouf, J. E. and Berche, P. (1987) Infect. Immun. 55, 1641-1646 23 Hugo, F., Reichwein, J., Awvand,M., Kramer, S. and Bhakdi, S. (1986) Infect. Immun. 54, 641-645 24 Michaels, D. W. (1979) Biochim. Biophys. Acta

555, 67-78 25 Bemheimer, A. W. and Lai, C. Y. (1985) Toxicon 23, 791-799 26 Ojcius, D. M. and Young, J. D-E (1990) Cancer Cells 2, 138-145 27 Chakraborty, T., Schmid, A., Notermans, S. and Benz, R. (1990) Infect. Immun. 58, 2127-2132 28 Ojcius, D. M. and Young, J. D-E (1990) ParasitoL Today6,163-165 29 Andrews, N. W., Abrams, C. K., Slatin, S. L. and Griffiths, G. (1990) Cell 61, 1277-1287 30 Bhakdi, S. and Tranum-Jensen, J. (1987) Rev. PhysioL Biochem. PharmacoL 107, 147-223

Xenopus embryos undergoing cleavage utilize amino acids as their main carbon source for metabolism. Glycolysis (from stored glycogen) begins near the onset of gastrulation. Thus, a major transition in the metabolism of the early embryo occurs before morphological differentiation. The enzymology that supports the carbon metabolism of the cleaving amphibian embryo resembles that of many mammalian tumor cells.

and can be easily injected with a variety of substances. Oocytes are arrested in prophase of the first meiotic division and can be stored for months or even years in the ovary as stable, non-dividing cells. Fully grown oocytes are induced by progesterone to reenter meiosis and undergo oocyte maturation, resulting in the formation of the fertilizable egg, which is arrested in metaphase of the second meiotic division (see Fig. I). Oocyte maturation in the clawed toad Xenopus laevis is accompanied by a Eady amphibian development Amphibian oocytes, eggs and number of well-defined biochemical embryos are convenient sources of ver- changes, including a decrease in the tebrate embryonic material, and can be cAMP concentration and the activation obtained in sufficient quantities for bio- of both $6 kinase and p34~c2/cyclin chemical experimentation. Oocytes are kinase (maturation-promoting factor) 4. fed by the mother's circulatory system, RNA synthesis ceases during matubut fertilized eggs are exuded into the ration and is not resumed until the blasmedium (pond water) and develop to tula stage s. The unfertilized egg is a the swimming tadpole stage by feeding non-stable, non-dividing cell, and will upon stored resources such as yolk and degenerate if not fertilized. The fertilized Xenopusegg undergoes glycogen (approximately 5% of the dry weight of amphibian eggs is glycogen). first cleavage in about 90 minutes. The Fully grown oocytes and eggs are large future dorsal-ventral axis of the embryo is not predetermined in the egg, but becomes established about halfway between fertilization and first M. B. Dworkin and E. Dwotkin-Rastlare at cleavage s. The fertilized egg thei~ the Emst-Boehringer-lnstitute,Dr divides synchronously 12 times in Boehri~gergasse5-11, A-1121Vienna, about six hours to form a 4000-cell Austria. © 1991, Elsevier Science Publishers, (UK) 0376-5067/91/$02.00

blastula. After the twelfth cleavage a transition occurs (the midblastula transition); cell division becomes asynchronous, cells become motile and RNA synthesis is resumed s. In Xenopus gastrulation commences about ten hours after fertilization and results in the formation of the three germ layers; the induction of mesoderm has already begun during cleavage and may involve growth-factor-mediated signaling7. Neurulation follows gastrulation over the next ten hours (Fig. 1). Transitions in carbon metabolism could be involved in the regulation of some of these events in the amphibian embryo.

Amphibianembryocleavage requires oxidative ph0sphorylati0n Studies with inhibitors have been used to measure the relative contributions of glycolysis and oxidative phosphorylation during amphibian development8.9.Exposure of Rana (frog) embryos to iodoacetic acid (a glycolysis inhibitor) does not inhibit cleavage, but affects gastrulation and completely blocks neurulation. Thus, glycolysis is not required for cleavage to proceed, b:,-t is required for subsequent morphogenesis. Exposure of cleaving Xenopus embryos to dinitrophenol or carbonyi cyanide phenylhydrazone (oxidative

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