Methionine-oxidized horse heart cytochromec. III. Ascorbate reduction and the methionine-80-sulfur-iron linkage

The ascorbate reduction of the CT-cytochromes—two chemically generated forms of horse heart cytochrome c, F^III and F^II, with both methionines, 80 and 65, as methionine sulfoxides, no iron-sulfur linkage, and potentiometric and physiological oxidoreduction properties distinct from those of the native protein and one another (J. Pande et al., 1987)—has been investigated using a stopped-flow technique. The reaction was monitored at 550 nm, and studies were conducted in 10 mM phosphate +0.17 M NaCl buffer,pH 7.4. Both CT-cytochromes are reduced by triphasic profiles, a faster and an intermediate ascorbate-dependent reaction and a slow, ascorbate-independent process. Both CT-cytochromes contain three molecular forms in slow equilibrium, two reducing directly by reaction with ascorbate and a third through conversion to one of the reducible forms. Like the reaction of the native protein, the ascorbate dependence of both the rapid and the intermediate process is nonlinear, approaching saturation values at high concentrations. The ascorbate profiles of the pseudo-first-order reduction constants are typical of the model for the reduction reaction of the unmodified protein, binding followed by a first-order reduction reaction (Myer et al., 1980; Myer and Kumar, 1984), but with distinct kinetic parameters, the first-order reduction constants and the protein-ascorbate stability constants. It has been concluded that the functional-conformational differences between the two CT-cytochromes are not operational to any significant extent in the reduction reaction with ascorbate. The methionine-80-sulfur-iron linkage of the protein is not a crucial requirement for the ascorbate reduction of the protein. The mechanism of the reaction in the main is also insensitive to the replacement of Met-80-S from heme coordination and/or the associated conformational-oxidoreduction properties of the protein. Of the two aspects of the reaction, the efficiency of the electron-transfer reaction and the stability of the ascorbate dianion-protein complex, the former is dependent on the integrity of the structural-conformational state of the molecule.     

Pemphigoid, pemphigus and desmoplakin as antigenic markers of differentiation in normal and tumorigenic mouse keratinocyte lines

Abstract The expression of differentiation stages in a murine epidermal cell transformation model has been investigated as a basis for studies of chemically-induced differentiation. Antibodies in sera of patients with the autoimmune diseases bullous pemphigoid and pemphigus vulgaris exhibit specific reactivity to antigenic determinants of basal and spinous cells, respectively, in sections of mouse and human epidermis. In addition, spinous cells in epidermis are reactive with a mouse monoclonal antibody to desmoplakin, a desmosomal component immunologically distinct from pemphigus. These antibodies were used to identify and attempt to quantify keratinocyte subpopulations in culture based on differentiation stage. Epidermal cell lines were cultured under conditions which favour proliferation (0.02 to 0.04 mm extracellular Ca²⁺, i.e. low Ca²⁺ conditions) or differentiation (0.1 mM to 1.4 mM Ca²⁺), as previously shown using primary cultures of mouse keratinocytes. Two independently-derived normal keratinocyte lines demonstrated Ca²⁺-dependent reactivity with pemphigoid and pemphigus antiserum, like that which has been observed in primary cultures. Furthermore, a Ca²⁺ and time-dependent reactivity with the three antisera was also observed in a papilloma cell line (derived from one of the normal cell lines after treatment in vitro with 7,12-dimethylbenz[α]anthracene). Papilloma cells cultured under conditions of low extracellular Ca²⁺ were comprised of three subpopulations: cells reactive only with pemphigoid anti-serum, cells reactive with pemphigoid and desmoplakin antibody (intracellular location), and cells reactive only with desmoplakin antibody. However, like the normal cell lines, papilloma cells underwent a transition to predominantly a spinous cell population (i.e. reactive with pemphigus and desmoplakin antibody) in response to extracellular Ca²⁺. A slower loss of pemphigoid antibody reactivity was noted in papilloma cells, consistent with an abnormal regulation of differentiation. The attempt to characterize these dynamic transitions from basal to spinous cell subpopulations in culture was considered to be prerequisite for the use of the model to investigate differentiation-inducing agents in carcinoma therapy.     

Structure of testicular angiotensin-converting enzyme. A segmental mosaic isozyme

The complete amino acid sequence of rabbit testicular angiotensin-converting enzyme has been deduced from the sequence of the corresponding cDNA clone. A protein of the expected molecular weight of 84,000 was translated in vitro from the mRNA encoded by this cDNA. All of the previously determined sequences of seven tryptic peptides from the enzyme are present in the deduced sequence, thus confirming the identity of the protein. From the deduced sequence it appears that the protein contains a signal peptide at the amino terminus and a hydrophobic anchoring domain near the carboxyl terminus. Northern analysis with oligonucleotide probes, whose sequences represented different regions of the cDNA, revealed not only the regions of extensive homology between the mRNAs encoding the testicular and the pulmonary isozymes but also a stretch of sequence near the 5' end unique to the testicular mRNA.     

Spectral studies of bovine dopamine beta-hydroxylase. Absence of covalently bound pyrroloquinoline quinone

Bovine dopamine beta-hydroxylase was examined spectroscopically for the presence of covalently bound pyrroloquinoline quinone (PQQ). Pure dopamine beta-hydroxylase had a featureless UV-visible spectrum above 300 nm. An equimolar solution of dopamine beta-hydroxylase and exogenously added PQQ (1 PQQ/active site) had a strong absorption maximum at 333 nm. Dialysis removed the added PQQ, indicating that dopamine beta-hydroxylase does not bind PQQ irreversibly. Reaction of dopamine beta-hydroxylase with 6 mM phenylhydrazine in the presence of 15 mM ascorbate caused 96% inactivation within 20 min and did not produce any spectrally detectable amounts of the phenylhydrazone adduct of PQQ, as reported by van der Meer et al. (van der Meer, R.A., Jongejan, J.A., and Duine, J.A. (1988) FEBS Lett. 231, 303-307). The peptide profile of phenylhydrazine inactivated dopamine beta-hydroxylase was monitored at 316 nm and did not reveal any peptides that might contain a PQQ-phenylhydrazone adduct. Thus, the absence of any spectrally detectable PQQ-phenylhydrazone adducts under these conditions demonstrates that the mechanism of phenylhydrazine inactivation does not involve covalent modification of PQQ at the active site of dopamine beta-hydroxylase and provides strong evidence that the native enzyme does not contain PQQ.     

Effector cell expression of NK1.1, a murine natural killer cell-specific molecule, and ability of mice to reject bone marrow allografts

The rejection of Hh-1 incompatible bone marrow cells in irradiated mice is mediated by NK cells and is genetically regulated. We tested the role of the NK-specific gene, NK1.1, in regulating the rejection of allogeneic bone marrow cell grafts. NK1.1+ mice, that are known to display strong resistance against Hh-1 incompatible grafts, were crossed to H-2/Hh-1 identical NK1.1-, poor responder mice, and the progeny were backcrossed to the poor responder parent. The segregating mice were individually typed for their expression of NK1.1 and the ability to resist Hh-1 incompatible bone marrow cells (BMC). A strong correlation was noted between expression of NK1.1 and rejection of H-2d/Hh-1d BMC. Our results support the idea that NK1.1 is one of the genes responsible for strong resistance to Hh-1d (determinant 2) but not for Hh-1j (determinant 3) BMC grafts. We suggest that the NK1.1 molecule functions as an accessory molecule in the cellular interactions involving the recognition of Hh-1 determinants.     

Nucleotide sequence and distinctive characteristics of the env gene of endogenous feline leukemia provirus.

Nucleotide sequence analysis of the env gene of two different endogenous feline leukemia virus (FeLV) loci, CFE-6 and CFE-16, of domestic cats revealed the following characteristics. (i) Both proviruses contain an open reading frame in the env region; (ii) whereas the full complement of the exogenous FeLV env is generally present in CFE-6 DNA, it is truncated in CFE-16 DNA such that the 5' half of the gp70 domain and the untranslated region 3' to the p15E domain have been fused by an internal deletion, resulting in loss of the C-terminal half of the gp70- and all of the p15E-coding sequences; (iii) endogenous env is highly homologous to large sequence domains conserved in all three exogenous FeLV subgroups (A, B, and C) but is similar to FeLV-B sequence domains in the variable regions detected in these viruses; and (iv) there are four other sequence domains, one residing at the C terminus of gp70 and three scattered in p15E, which are unique for the endogenous env, thereby distinguishing it from the FeLV-B gene.     

Synthesis and biological activity of a biotinylated vasopressin analog

To study vasopressin receptor-mediated endocytosis using electronmicroscopy methods and to develop avidin affinity columns for receptor purification, we synthesized and tested the biological properties of a biotinylated vasopressin (VP) analog [1-(2-mercapto) propionic acid] 8-[lysine-N6-biotin] VP (B-MLVP). B-MLVP was prepared by coupling biotin to the epsilon amine of the lysine residue in [1-(2-mercapto) propionic acid] 8-(lysine) VP (MLVP). The structure of HPLC purified B-MLVP was confirmed by fast atom bombardment mass spectrometry. B-MLVP effectively competed for arginine vasopressin (AVP) binding sites in canine renal plasma membranes on the surface of LLC-PK1 kidney cells. Dissociation constants of 15 nM and 202 nM were calculated from the results of competition binding assays conducted with membranes and cells, respectively. B-MLVP stimulated adenylate cyclase activity and elevated cellular 3',5',cyclic-AMP (cAMP) content in a manner similar to AVP, indicating it is an agonist of VP action in renal tissue. These observations indicate that B-MLVP is an agonist of VP action and may be used to study renal VP receptors by employing avidin coupled to various reporter groups.