Expression of isoforms of internal Ca2+ pump in cardiac, smooth muscle and non-muscle tissues.

Smooth muscle and several non-muscle tissues contain mRNA for an alternative splice of the mRNA for the cardiac sarcoplasmic reticulum (SR) Ca,Mg-ATPase. Based on amino acid composition deduced from cDNA sequences the cardiac isoform (Ic) is 110 kDa while the smooth muscle and the non-muscle isoform (Is) is 115 kDa. This prediction in their molecular masses was tested at the protein level in rabbit stomach, aorta, uterus and vas deferens smooth muscles; stomach mucosa, brain, liver, kidney and heart. The major species of the acylphosphates formed in the presence of Ca2+ and electrophoresed in acid SDS-acrylamide gels were 5 kDa smaller for the heart (Ic) than those for all the other tissues (Is). The size difference was also confirmed in Western blots using a monoclonal antibody which binds to both Is and Ic. Thus consistent with the mRNA splices for the internal Ca2+ pumps previously reported to be present in these tissues, rabbit heart expresses predominantly the Ca2+ pump protein Ic and the various smooth muscle, mucosa, brain, liver and kidney express mainly the isoform Is.

Improved thin-layer chromatographic separation of 32P-postlabeled DNA adducts.

DNA adducts represent the putative initiating event in the chemical carcinogenesis process. 32P-Postlabeling is one of several assays which have been developed for the sensitive detection of DNA adducts. An integral part of the 32P-postlabeling assay is the separation of adducted nucleotides by multidirectional, multisolvent, anion-exchange polyethyleneimine-cellulose thin-layer chromatography. Standard since the introduction of this assay has been the use of high-salt, high-urea solvents for the resolution of adducts during the D3 and D4 phases of the chromatography. Urea solvents are able to separate adducts resulting from a number of chemicals, however, they are time-consuming, retain a lot of background noise, may push adducts into inadequately resolved diagonal radioactive zones, and may not separate adducts of similar structure. In this study we introduce the use of a dilute ammonium hydroxide solvent for D4 chromatography and compare it to other standard solvents such as lithium chloride-Tris.HCl-urea, sodium phosphate-Tris.HCl-urea, and isopropanol-4 M ammonium hydroxide for adduct separation, resolution, recovery, retention of background noise, and chromatography development time. We found that 0.2 M ammonium hydroxide worked well for the recovery, separation, and resolution of a wide array of adducts derived from highly lipophilic polycyclic aromatic hydrocarbons and aromatic amines. In addition, this solvent required much less time (< 1/4) as compared to the other solvents and more importantly allowed the separation of adducts which otherwise comigrated and were not visible when using the other three D4 solvents.(ABSTRACT TRUNCATED AT 250 WORDS)

Abundance of sarcoplasmic reticulum calcium pump isoforms in stomach and cardiac muscles.

Rabbit stomach smooth muscle contains mRNA for an internal Ca2+ pump identical in sequence to that reported for rabbit uterus [Lytton, Zarain-Herzberg, Periasamy & MacLennan (198) J. Biol. Chem. 264, 7059-7065]. This is an alternatively spliced form (Is) of the cardiac muscle sarcoplasmic reticulum Ca2+ pump (Ic). The splicing results in replacement of the last 4 amino acids (Ala-Ile-Leu-Glu) present in Ic by 49 amino acids and by a different 3'-non-coding region. Using cDNA probes against the conserved and the alternatively spliced regions, we determined that poly(A+) RNA isolated from rabbit stomach smooth muscle did not contain any transcripts for Ic. The poly(A+) RNA from cardiac muscle contained transcripts mostly for Ic, but also some for Is. The abundance of the Ca2(+)-pump transcripts as measured by the binding of a cDNA probe against the conserved region to poly(A+) RNA was 6-8 times higher in cardiac than in smooth muscle. The amount of the corresponding pump protein, measured using two antibodies, was 60-80 times higher in cardiac membranes than in smooth muscle membranes. Thus the protein-to-transcript level was approx. 10-fold higher in the cardiac muscle. We conclude that the regulation of the abundance of this protein occurs at steps leading to the formation of the mature mRNA for the two splices, which may differ in their translation efficiency.

The role of acetylation in benzidine metabolism and DNA adduct formation in dog and rat liver.

To determine whether benzidine is acetylated in dog, like rat, the metabolism of benzidine was assessed with dog and rat liver slices. Slices were incubated with 0.05 mM [3M]benzidine for 4 h. Media and cellular DNA were analyzed for acetylated benzidine metabolites and adducts. In rat, benzidine was rapidly converted to acetylated metabolites. At 1 h, benzidine, N-acetylbenzidine, and N,N'-diacetylbenzidine represented 5%, 23%, and 54%, respectively, of the total radioactivity in media. Within 2 h, 75% of the radioactivity was N,N'-diacetylbenzidine. In dog, 45% of the radioactivity was present in metabolites more polar than benzidine by 4 h. No N-acetylated metabolites were observed in dog liver slice media. To identify acetylated benzidine DNA adducts, N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine was prepared and identified by FAB MS. This nucleoside adduct was used to synthesize N-(deoxyguanosin-8-yl)-N-acetylbenzidine and N'-(deoxyguanosin-8-yl)-N-acetylbenzidine. Nucleoside adducts from slices incubated with [3H]benzidine were analyzed by HPLC. With this method of analysis, the 3H-material did not correlate with the synthetic adduct standards. To improve sensitivity and identify liver adducts, a 32P-postlabeling method was developed. 2'-Deoxyguanosine 3'-monophosphate adduct standards of acetylated benzidine were prepared. 32P-Postlabeling analysis demonstrated that rat liver contained only N'-(3'-monophosphodeoxyguanosine-8-yl)-N-acetylbenzidine after a 1- or 4-h exposure to benzidine. In contrast, no acetylated adducts were detected in dog. Results indicate that dog is a nonacetylator with respect to benzidine. The availability of acetylated benzidine nucleotide standards allowed unambiguous identification of N'-(3'-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine as the adduct present in rat liver slices. These nucleotide adduct standards will be useful in subsequent studies in animals and human.