Surgical treatment of mammary carcinomas in dogs with or without postoperative chemotherapy.

This retrospective study identified prognostic factors associated with survival; and compared survival data in 94 canine mammary carcinoma (MCA) dogs treated with surgery (n = 58), or surgery and adjunct chemotherapy (n = 36), and a subset of dogs with poor prognostic factors. On multivariate analysis independent predictors of median survival time (MST) were clinical stage, lymphatic invasion (LI; present 179 days; none 1098 days), ulceration (present 118 days; none 443 days) and surgical margins (incomplete 70 days; complete 872 days). Complete surgical margins were associated with MST in dogs with stages 1-3 MCA (incomplete 68 days; complete 1098 days) and dogs with LI (incomplete 70 days; complete 347 days). There was no statistically significant improvement in MST in dogs with advanced disease (stage 4 or LI) treated with adjunctive chemotherapy (chemotherapy 228 days; none 194 days); although five dogs with complete surgical margins that received mitoxantrone and carboplatin had a mean survival of 1139 days.

Catalytic activity of an isolated domain of Na,K-ATPase expressed in Escherichia coli.

Fusion proteins of glutathione-S-transferase and fragments from the large cytoplasmic domain of the sheep Na,K-ATPase alpha1-subunit were expressed in Escherichia coli. The Na,K-ATPase sequences begin at Ala345 and terminate at either Arg600 (DP600f), Thr610 (DP610f), Gly731 (DP731f), or Glu779 (DP779f). After affinity purification on glutathione-Sepharose, the fusion proteins were labeled with [alpha-32P]-2-N3-ATP, and incorporation of the radiolabel into the fusion proteins was measured by scintillation counting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kd values of 220-290 microM for 2-N3-ATP binding to the fusion proteins were obtained from the photolabeling experiments. Approximately 1 mol of 2-N3-ATP was calculated to be incorporated per mole of fusion protein after correction for photochemical incorporation efficiency. Labeling of all of the fusion proteins by 25 microM 2-N3-ATP was reduced in the presence of MgATP, Na2ATP, MgCl2, 2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and p-nitrophenylphosphate, and Ki values of 2-11 mM for Na2ATP, 0.2-5 mM for MgCl2, 0.1-5 mM for MgATP, and 20-300 microM for p-nitrophenylphosphate were calculated for these ligands. All of the fusion proteins catalyze the hydrolysis of p-nitrophenylphosphate. The reaction requires MgCl2 and is inhibited by inorganic phosphate, which is similar to the hydrolysis of p-nitrophenylphosphate by native Na,K-ATPase. Based on these observations, it appears that the soluble fragments from the large cytoplasmic domain of Na,K-ATPase expressed in bacterial cells are folded in an E2-like conformation and are likely to retain much of the native structure.

The RXRalpha gene functions in a non-cell-autonomous manner during mouse cardiac morphogenesis.

Germline mutation in mice of the retinoic acid receptor gene RXRalpha results in a proliferative failure of cardiomyocytes, which leads to an underdeveloped ventricular chamber and midgestation lethality. Mutation of the cell cycle regulator N-myc gene also leads to an apparently identical phenotype. In this study, we demonstrate by chimera analysis that the cardiomyocyte phenotype in RXRalpha-/- embryos is a non-cell-autonomous phenotype. In chimeric embryos made with embryonic stem cells lacking RXRalpha, cardiomyocytes deficient in RXRalpha develop normally and contribute to the ventricular chamber wall in a normal manner. Because the ventricular hypoplastic phenotype reemerges in highly chimeric embryos, we conclude that RXRalpha functions in a non-myocyte lineage of the heart to induce cardiomyocyte proliferation and accumulation, in a manner that is quantitatively sensitive. We further show that RXRalpha is not epistatic to N-myc, and that RXRalpha and N-myc regulate convergent obligate pathways of cardiomyocyte maturation.

Photoaffinity labeling of the active site of the Na+/K(+)-ATPase with 4-azido-2-nitrophenyl phosphate.

Na+/K(+)-ATPase will hydrolyze small acylphosphates such as p-nitrophenyl phosphate (pNPP) in addition to ATP and can derive sufficient energy from the hydrolysis of these small molecules to catalyze active ion transport. In this report, 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive analog of pNPP, was used as a probe of the substrate binding site of dog renal Na+/K(+)-ATPase. ANPP was slowly hydrolyzed by Na+/K(+)-ATPase with a Vmax of 0.19 mumol mg-1 min-1 and with an apparent Km of 1.0 mM. The Km for hydrolysis of pNPP was 1.7 mM. ANPP competitively inhibited the hydrolysis of pNPP with a Ki of 0.37 mM. Both the ATPase and pNPPase activity of the Na+/K(+)-ATPase were irreversibly inhibited after photolysis of the enzyme and ANPP with UV light, although neither activity was completely inhibited by up to 200 microM ANPP. Inhibition of activity was prevented by including 0.2 mM ATP in the reaction or by excluding Mg2+ from the photolysis buffer. Photolysis with [32P]ANPP labeled only the alpha subunit of the Na+/K(+)-ATPase, and the amount of labeling was substantially reduced by 0.2 mM ATP or in the absence of Mg2+. The stoichiometry of labeling extrapolated to a maximum of about 1.2 nmol/mg of protein at 100% inhibition of Mg(2+)-dependent activity. Limited proteolytic digestion showed labeling sites on nonoverlapping tryptic peptides derived from the alpha subunit of Na+/K(+)-ATPase, and two radiolabeled peptides were purified from an exhaustive tryptic digest of [32P]ANPP-labeled Na+/K(+)-ATPase. One peptide contained amino acids Met-379 to Lys-406, and the second contained amino acids Ala-655 to Lys-676. Amino acids corresponding to Asn-398 and Pro-668 were missing from the sequences and may represent residues derivatized by ANPP from within the substrate binding site of Na+/K(+)-ATPase.

Identification of an amino acid in the ATP binding site of Na+/K(+)-ATPase after photochemical labeling with 8-azido-ATP.

[alpha-32P]-8-N3-ATP, [2-3H]-8-N3-ATP, and non-radioactive 8-N3-ATP have been used as photoaffinity probes of the ATP binding site of dog kidney Na+/K(+)-ATPase. 8-N3-ATP has previously been shown to bind to Na+/K(+)-ATPase with high affinity, to be a substrate for Na+/K(+)-ATPase, and to inactivate the enzyme upon ultraviolet irradiation [Scheiner-Bobis, G., & Schoner, W. (1985) Eur. J. Biochem. 152, 739-746]. 8-N3-ATP competitively inhibits the high-affinity binding of [2,8-3H]-ATP to Na+/K(+)-ATPase with a Ki of 3.4 microM, which is comparable to the reported KD of 3.1 microM for the binding of 8-N3-ATP to the enzyme. The extent of inhibition of ATP hydrolysis by 8-N3-ATP was linearly correlated with the stoichiometry of covalent incorporation of 8-N3-ATP into Na+/K(+)-ATPase up to about 50% inhibition of activity; however, the linkage between the protein and 8-N3-ATP was unstable, and the maximum incorporation of 8-N3-ATP was less than the nucleotide binding capacity of the protein. After photolysis with ultraviolet light, 8-N3-ATP was specifically incorporated into the carboxy-terminal 58-kDa fragment of the alpha-subunit of Na+/K(+)-ATPase generated by limited trypsin digestion in the presence of KCl, and the beta-subunit was not labeled. 8-N3-ATP-labeled Na+/K(+)-ATPase was digested with trypsin, and a single peak containing the nucleotide was identified after HPLC fractionation of the digest.(ABSTRACT TRUNCATED AT 250 WORDS)

Photochemical labeling and inhibition of Na,K-ATPase by 2-Azido-ATP. Identification of an amino acid located within the ATP binding site.

2-Azido-ATP (2-N3-ATP) was investigated as a reagent for the identification of amino acids located within the catalytic ATP binding site of Na,K-ATPase. The enzymatic activity of Na,K-ATPase was inhibited up to 50% by 2-N3-ATP (K0.5 = 5-10 microM) after irradiation with ultra-violet light, and inhibition was prevented by 0.2 mM ATP. The binding of ATP to Na,K-ATPase (KD = 0.1 microM) was inhibited competitively by 2-N3-ATP. [alpha-32P]2-N3-ATP labels the alpha subunit of Na,K-ATPase, and the stoichiometry of covalent ATP-protectable incorporation of the probe into the protein is approximately equal to the stoichiometry of high-affinity binding of ATP to the Na,K-ATPase. 2-N3-ATP is also hydrolyzed by Na,K-ATPase as a substrate. From these data, it is concluded that 2-N3-ATP photochemically labels the Na,K-ATPase from within the catalytic ATP site on the protein. Trypsin digestion of Na,K-ATPase after photochemical labeling with [alpha-32P]2-N3-ATP generated a large 30-kDa fragment containing the radiolabeled nucleotide. This fragment was resistant to further cleavage by trypsin, but it could be digested further after denaturation in urea. High pressure liquid chromatography separation of tryptic peptides from the 30-kDa fragment and subsequent amino acid sequence analysis of the radiolabeled peptides identified the region between His496 and Arg510 of the Na,K-ATPase alpha subunit as the region labeled by [alpha-32P]2-N3-ATP. Gly502 was absent from all sequences of the radiolabeled peptides from this region, consistent with the derivatization of this amino acid by 2-N3-ATP and localization of Gly502 within the ATP binding site.

Inhibition of ion pump ATPase activity by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP): assessment of BzATP as an active site-directed probe.

The interaction of 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) with the renal (Na+ + K+)-ATPase, the sarcoplasmic reticulum Ca-transport ATPase, and the gastric (H+ + K+)-ATPase has been investigated in order to determine whether BzATP is a suitable probe for the labeling and identification of a peptide from the ATP binding sites of these ion pumps. After ultraviolet irradiation BzATP inhibited the enzymatic hydrolysis of ATP by each of the ion pumps, and also was covalently incorporated into the 100 000 dalton polypeptides of each protein. The presence of excess ATP in the reaction solution did not prevent either the inactivation of ATPase activity or the labeling of the catalytic polypeptides by BzATP. Prior modification of the ATPases with fluorescein-5'-isothiocyanate (FITC), however, prevented much of the labeling of the 100 000 dalton polypeptides by BzATP. BzATP competitively inhibited the high-affinity binding of ATP to the ion pumps, but ATP did not block the high-affinity binding of BzATP by the enzymes. BzATP binds to the membrane-bound ATPases at a high-affinity site with a Kd of 0.8-1.2 microM and a Bmax of 2-3 nmol/mg, and also binds to at least one low-affinity, high-capacity site on the membranes. HPLC separation of the soluble peptides from a tryptic digest of BzATP-labeled (Na+ + K+)-ATPase revealed the presence of several labeled peptides, none of which was protected by either ATP or FITC. Although BzATP can displace ATP from a high-affinity binding site on the ion pumps, it appears, therefore, that inactivation of enzymatic activity is the result of reactions between BzATP and the proteins at locations outside this site. Thus, it is concluded from these experiments that BzATP is not likely to be a useful probe for the ATP binding sites on the ion transport ATPases.

The amino acid sequence of a fluorescein-labeled peptide from the active site of (Na,K)-ATPase.

(Na,K)-ATPase in an active-transport protein that couples the energy obtained from the hydrolysis of ATP to the transport of Na+ and K+ across animal cell membranes. In order to investigate the enzymatic mechanism of this activity, a peptide derived from the ATP-binding site of (Na,K)-ATPase has been purified and its amino acid sequence has been determined. The peptide was identified by the covalent incorporation of a fluorescent probe, fluorescein 5'-isothiocyanate, into the active site before trypsin digestion of the protein. The labeling of (Na,K)-ATPase by fluorescein 5'-isothiocyanate was associated with the irreversible inhibition of enzymatic activity, and both the labeling of the tryptic peptide and inhibition of activity were prevented when the reaction was performed in the presence of ATP. An apparent KD of 5.7 microM was calculated when the reaction between (Na,K)-ATPase and fluorescein 5'-isothiocyanate was performed under pseudo first-order conditions. The amino acid sequence of the active-site peptide, His-Leu-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Arg, is similar to the sequence of a fluorescein-labeled peptide derived from the active site of the sarcoplasmic reticulum Ca2+-transport ATPase (Mitchinson, C., Wilderspin, A. F., Trinaman, B. J., and Green, N. M. (1982) FEBS Lett. 146, 87-92).