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Cryospectrokinetic studies of zinc and cobalt carboxypeptidase A disclosed two intermediates in the hydrolysis of both peptides and depsipeptides and furnished all the rate and equilibrium constants for the reaction scheme E + S in equilibrium ES1 in equilibrium ES2---E + P [Auld, D. S., Galdes, A., Geoghegan, K. F., Holmquist, B., Martinelli, R. A., & Vallee, B. L. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5041-5045]. Since the ES2 intermediate is the predominate enzyme species present at steady state, its chemical nature is deducible from subzero chemical quench studies done after steady state is established. Extrapolation of the product concentration to zero time, [P0], measures the concentration of the enzyme species in which bond cleavage has occurred. For peptides, the [P0]values are zero, indicating that no product is generated prior to turnover and therefore the ES2 intermediate involves a complex between enzyme and intact peptide substrate. For depsipeptides, [P0] values are 1 mol of produce per mole of enzyme over the entire temperature range -20 to -50 degrees C, indicating cleavage of the ester bond occurs prior to the rate-limiting step so that ES2 is more properly denoted by EP1P2, where P1 and P2 are the substrates for the reverse reaction. The rate-limiting step for depsipeptides thus involves release of the products which may occur directly or through a mandatory conformational change followed by rapid product release.

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Xenopus laevis vitellogenin contains 2 g-atoms (g-at) of Zn and 3 g-at of Ca/dimer, transports zinc in plasma, and plays a role in its distribution within the oocyte [Montorzi et al. (1994) Biochem. Biophys. Res. Commun. 200, 1407-1413; Montorzi et al. (1995) Biochemistry 34, 10851-10858]. We here report the dynamics and time course of Zn65-labeled vitellogenin uptake by and distribution within stages II and IV oocytes, the fate of the metal in oocytes as they progress from stages II to VI, as well as in the first two cleavage blastomeres, the blastula, and subsequent stages of the developing embryo and tadpole. Zn65 bound to vitellogenin is taken up within less than 30 min by either stage II or IV oocytes incubated under in vitro culture conditions whereas free Zn65 is not. Once internalized, Zn65 remains within the cytosol of stage II, whereas in stage IV oocytes, it is transferred within 4 h of its entry from the cytosol into yolk platelets. Nearly all of the transferred Zn65 is found within yolk platelets and their precursors where it is associated with the vitellogenin cleavage product, lipovitellin. Its distribution within the oocyte organelles differs at each stage of oogenesis. In the early stages (III-IV) most of the oocyte zinc is located first in the small endocytosed vesicles and then in multivesicular bodies. When the zinc transfer process is finalized in the late stages of oogenesis (V-VI), > 90% of the total oocyte zinc is within yolk platelets while the remainder is in the cytosol. In embryos and tadpoles, the larger of these two pools remain sequestered in yolk platelets and is inaccessible to cytosolic apoproteins throughout the entire period of embryo formation. Its redistribution to the cytosol does not begin until several days after the tadpole has hatched. The smaller pool, on the other hand, is already present in the cytosol and is, therefore, postulated to constitute the sole source of zinc required for embryogenesis.

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