Crystal structure of thioltransferase at 2.2 A resolution.

We report here the first three-dimensional structure of a mammalian thioltransferase as determined by single crystal X-ray crystallography at 2.2 A resolution. The protein is known for its thiol-redox properties and dehydroascorbate reductase activity. Recombinant pig liver thioltransferase expressed in Escherichia coli was crystallized in its oxidized form by vapor diffusion technique. The structure was determined by multiple isomorphous replacement method using four heavy-atom derivatives. The protein folds into an alpha/beta structure with a four-stranded mixed beta-sheet in the core, flanked on either side by helices. The fold is similar to that found in other thiol-redox proteins, viz. E. coli thioredoxin and bacteriophage T4 glutaredoxin, and thus seems to be conserved in these functionally related proteins. The active site disulfide (Cys 22-Cys 25) is located on a protrusion on the molecular surface. Cys 22, which is known to have an abnormally low pKa of 3.8, is accessible from the exterior of the molecule. Pro 70, which is in close proximity to the disulfide bridge, assumes a conserved cis-peptide configuration. Mutational data available on the protein are in agreement with the three-dimensional structure.     

Changes in intracellular cations during the cell cycle in HeLa cells

Intracellular Na+, K+, and Mg2+ concentrations have been measured during the HeLa cell cycle and compared with changes in oxygen utilization and macromolecular synthesis. Cell water content remains relatively constant at 79 +/- 1% during the cell cycle. A biphasic change in intracellular Na+ occurs with low values as cells reach peak S phase and again in early G1. The decrease in S coincides with an increase in cell volume during increased macromolecular synthesis. The fall in intracellular Na+ during mitosis/early G1 coincides with decreased energy utilization as macromolecular synthesis decreases with a continued decrease in [Na+]i in G1 corresponding to a period of increasing cell volume and an increase in protein synthesis. Intracellular Na+ is relatively high during late S/G2 when phosphate incorporation into protein and phospholipid is maximal. Intracellular K+ concentrations largely parallel intracellular Na+ levels although the intracellular K+:Na+ ratio is significantly lower as the cell volume increases during late G2/mitosis. Additions of a Na+-pump inhibitor (strophanthidin) not only caused a rise in [Na+]i and fall in [K+]i but also inhibited protein synthesis. Conversely, addition of a protein synthesis inhibitor (cycloheximide) blocked amino acid incorporation and produces a fall in intracellular Na+ levels. These findings indicate that intracellular Na+ and K+ play an important role in regulating cell hydration during the cell cycle and that changes in Na+, K+-ATPase activity, synthesis and/or utilization of high energy phosphate compounds, fluid phase turnover (endocytosis), Na+:H+ exchange (pHi), Donnan forces, and ionic adsorption may all be involved.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

Chitinase is required for cell separation during growth of Saccharomyces cerevisiae

The Saccharomyces cerevisiae chitinase described by Correa et al. (Correa, J. U., Elango, N., Polacheck, I., and Cabib, E. (1982) J. Biol. Chem. 257, 1392-1397) has been cloned and sequenced. Analysis of the derived amino acid sequence suggests that the protein contains four domains: a signal sequence, a catalytic domain, a serine/threonine-rich region, and a carboxyl-terminal domain with high binding affinity for chitin. Most of the enzyme produced by cells is secreted into the growth medium and is extensively glycosylated with a series of short O-linked mannose oligosaccharides ranging in size from Man2 to Man5. Chitinase O-mannosylation was further examined in the temperature-sensitive secretion mutants sec18, sec7, and sec6. Oligosaccharides isolated from chitinase accumulating in cells at the nonpermissive temperature revealed Man1 and Man2 associated with the sec18 mutant. sec6 and sec7 accumulated Man2-Man5 with a higher proportion of Man5 relative to the secreted protein. A significant amount of chitinase is also found associated with the cell wall through binding of COOH-terminal domain to chitin. Disruption of the gene for the enzyme leads to a defect in cell separation but does not substantially alter the level of cellular chitin.     

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

Mechanisms involved in parasitic castration: in vitro effects of the trematode Prosorhynchus squamatus on the gametogenesis and the nutrient storage metabolism of the marine bivalve mollusc Mytilus edulis

The mechanisms involved in the parasitic castration of the marine mussel Mytilus edulis by the trematode parasite Prosorhynchus squamatus Odhner, 1905, have been investigated in vitro with two bioassays employing dissociated host tissues. There is no conclusive evidence that P. squamatus affects the secretion of two host neuroendocrine factors, viz., gonial mitosis-stimulating factor and glycogen mobilization hormone, involved in the gametogenesis/nutrient storage cycles of the mussel. In contrast, extracts of P. squamatus sporocysts and cercariae significantly stimulated glycogen mobilization in host glycogen cells and strongly inhibited host gonial mitosis. A gonial mitosis-inhibiting factor (GMIF) was found in the hemolymph of parasitized mussels. The existence of an endogenous GMIF in mantle tissue of uninfected mussels has been demonstrated. This factor appeared to be secreted into the hemolymph during the period of sexual maturity. Whether the parasite acts directly on the host gonia, or by provoking the liberation of this endogenous GMIF, has yet to be ascertained. It would appear, however, that the parasite acts directly on host glycogen cells.     

Detection of a soluble acrosome reaction-inducing factor, different from serum albumin, associated with the ovulated egg-cumulus complex.

Soluble extracts of the ovulated hamster egg-cumulus complex (ECC) were tested on capacitated sperm for activity in inducing the physiological acrosome reaction (AR). Evidence for occurrence of the physiological AR included enhanced sperm penetration of intact homologous zonae pellucidae as well as induction of AR in nonattached and in zona-bound sperm following a brief coincubation with test compound. Since hamster serum albumin, a major protein of hamster body fluids, also induces spontaneous ARs under certain conditions, it was used as one of the comparators for the acrosome reaction inducing factor (ARIF; Westrick et al., Biol Reprod 32 [Suppl 1]. 213, 1985) activity in the ECC. Sperm exposure to concentrations of the soluble ECC extract ranging from 0.04 to 0.2 mg protein/ml significantly increased penetration of salt-stored zonae by 36%, mean numbers of penetrating sperm by 90%, ARs in nonattached sperm by 65%, and ARs in zona-bound sperm by 102%. Hamster serum albumin added after completion of capacitation had no significant effect on these parameters. We conclude that 1) the ovulated ECC contains a soluble ARIF that augments zona-induced ARs and sperm penetration and 2) the ARIF is not serum albumin.