pH dependence of the tryptophan fluorescence in cytochrome c oxidase: further evidence for a redox-linked conformational change associated with CuA

When the low-potential metal centers of cytochrome c oxidase are reduced, the enzyme undergoes a conformational transition that shifts the fluorescence maximum of the emitting tryptophan residues from 329 to 345 nm. At pH 7.4, the change in this tryptophan fluorescence intensity is a nonlinear function of the electron equivalents added to the cyanide-inhibited enzyme. This nonlinear behavior is a result of the difference in redox potential between cytochrome a and CuA, which, at equilibrium, favors electron occupancy at cytochrome a. Studies on the cyanide-inhibited enzyme suggest that the conformational change is associated with reduction of CuA [Copeland, R. A., Smith, P. A., & Chan, S. I. (1987) Biochemistry 26, 7311-7316]. In this work we present tryptophan fluorescence data for the cyanide-inhibited enzyme at pH 8.9. Because of the pH dependence of the midpoint potential of cytochrome a in this form of the enzyme, the two low-potential centers become virtually isopotential at pH 8.9. The results obtained confirm our earlier conclusion that the observed conformational change is linked to the reduction of CuA only, rather than to the redox activity of both low-potential metal centers. We find that, in partially reduced cyanide-inhibited oxidase, raising the pH from 7.4 to 8.9 results in an intensification and red shift of the enzyme's tryptophan emission as the electron occupancy redistributes from cytochrome a to CuA. Moreover, when the fluorescence change is plotted as a function of the number of electrons added to the enzyme at pH 8.9, the data fit the nearly linear function expected for a conformational change triggered by reduction of CuA exclusively.     

Müllerian inclusions in peritoneal washings. Potential source of error in cytologic diagnosis

Müllerian inclusions in peritoneal washings from female patients may be mistaken for adenocarcinoma. Such findings were studied in the peritoneal washing cytology specimens from eight cases. The inclusions usually presented as tubular or papillary structures, often forming a single layer of epithelium surrounding psammoma bodies. The cells forming these structures often displayed some degree of atypia. Recognition of this entity in peritoneal fluids is important to avoid a misdiagnosis of disseminated cancer. A general outline is proposed for interpreting such findings in peritoneal washings, based on the cytomorphology of these structures as well as the microscopic features of the primary neoplasms.     

Envelope proteins of human T cell leukemia virus type I: characterization by antisera to synthetic peptides and identification of a natural epitope

Three peptides corresponding to selected regions of the env gene products of human T cell leukemia virus type I were synthesized by solid-phase Merrifield techniques. The sequence of peptide designated SP-65 was identical to the predicted C-terminal 12 residues of the transmembrane protein p21env, and peptide SP-74 was inferred from a region shown to be highly conserved among mammalian retroviruses. The third peptide, SP-70, was derived from a C-terminal region of the surface glycoprotein gp46. Antibodies to each peptide were raised in rabbits and were used to identify and further characterize the proteins coded by the env gene. Despite being present at very low levels in purified viral preparations, these proteins were chromatographed by reverse-phase high pressure liquid chromatography and were located by Western blot analysis of the column fractions. Anti-SP-70 recognized the surface glycoprotein (gp46) and also its C-terminal cleavage fragment (gp16). Anti-SP-65 and anti-SP-74 both reacted with the hydrophobic transmembrane protein (p21) and provided evidence that this protein does not undergo apparent C-terminal processing during viral maturation, unlike the trans-membrane protein of murine leukemia virus. As expected, anti-SP-74 also reacted with homologous proteins from other Type C and Type D viruses, confirming that peptide SP-74 corresponds to a broadly conserved region of retroviral transmembrane proteins. SP-70, which is predicted to be quite near the C terminus of the major surface glycoprotein, was also reactive with sera of HTLV-I-positive patients, indicating that this peptide corresponds to, or is part of, a native epitope recognized by the natural host.     

Chromosomal localization of Emv-16 and Emv-17, two closely linked ecotropic proviruses of RF/J mice.

Emv-16 and Emv-17, the two closely linked ecotropic proviral loci of RF/J mice, have been mapped to chromosome 1 between leaden, ln, and the mouse engrailed homeo-box locus, En-1, by using recombinant inbred strains and conventional backcross analysis.     

Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding

We have studied the sensitivity of sulfhydryl groups of a highly purified p21 protein of the v-rasH oncogene to a thiol-specific reagent, N-ethylmaleimide (NEM). Approximately 70% of GTP binding and autokinase activities of p21 were inactivated by NEM, and excessive amounts of GTP or GDP protected p21 activities. Thiol titration revealed the presence of one fast reactive cysteine residue, the susceptibility of which is modulated by GTP binding. A total of 4 and 6 residues, respectively, became titratable upon denaturation and reduction, suggesting the presence of a disulfide bond. This GTP-modulated sulfhydryl group was identified as Cys-80 in the following tryptic peptide sequence: NH2-Thr-Gly-Glu-Gly-Phe-Leu-Cys-Val-Phe-Ala-Ile-Asn-Asn-Thr-Lys-COOH. This is based on the comparative tryptic peptide mapping of [14C]NEM-modified p21 in the presence and absence of GTP. The GTP-modulated peptide co-chromatographed with a synthetic peptide of the predicted sequence. Amino acid analysis of the purified [14C]NEM-modified peptide from tryptic digests of p21 also confirmed its identity. This region of p21 shares an extensive sequence homology with various G-proteins and appears to be in the vicinity of the GTP-binding domain of these proteins.     

Fine structural study of gas secretion in the physoclistous swim bladder of Fundulus heteroclitus and Gadus callarias and in the euphysoclistous swim bladder of Opsanus tau

Summary The Haldane-Koch-Scholander-Kuhn-Steen theory of salting out countercurrent multiplication effect of the rete mirabile now accounts for release of most gases in the fish swim bladder. Evidence presented here indicates that final release is by microbubbles from the secretory epithelium. There is only one specific cell type in a highly vascularized epithelium. It is characterized by complex folds in the paravascular zone and by gas forming bodies which seem to form from plentiful Golgi material. The bodies are formed with dark amorphous matrix that becomes patterned (tubular or lamellar), finally froths and then is released to the gas surface. Residual material may form myelin-like layers on the lumenal surface. Active cells are also characterized by surface villi and subsurface, parallel cisternal spaces. Gas may be formed by cells not touching the gas surface and released through intercellular spaces. There are discontinuous desmosomes (maculae adhaerentes) near the gas surface and there are no tight junctions (zonulae occludentes). Gas release as bubbles would explain Wittenbergs observations that the gases found in swim bladders have ratios more closely related to their solubility coefficients in water than to ambient partial pressures. A surfactant may be present to lower the surface tension of the microbubbles. The carrier in the cytoplasm would have to be an iron-protein (or perhaps peroxidase) compound capable of binding molecular oxygen.Supported by grant-in-aid from the national Science Foundation (GB-676) and from the USPHS (General Medical Sciences Institute, GM-06836).     

An Association between Mitochondria and the Endoplasmic Reticulum in Cells of the Pseudobranch Gland of a Teleost

An elaborate and apparently unique specialization of the endoplasmic reticulum having the form of tubules and a precise orientation with respect to the mitochondria has been described for the specific cell of the pseudobranch gland. The tubules also are concentrated near the vascular border of the cell where they show continuity with the plasma membrane and open directly against the basement membrane. On the other side of the basement membrane, the endothelial cells of the sinusoid show openings or discontinuities characteristically associated with secretory cells. The pseudobranch gland is presumed to have carbonic anhydrase as one of its primary products, if not its only one, and the elaborate ultrastructure is thought to be associated with the special problems of secreting this enzyme.