Nuclear proteins in the heart of the cardiomyopathic Syrian hamster. Fractionation of phenol-soluble nonhistone proteins by two-dimensional polyacrylamide gel electrophoresis.

Abstract

SUMMARY Nonhistone nuclear proteins (NHNP) were isolated from the hearts of hamsters in the myolytic, hypertrophic, and failing phases of cardiomyopathy and from paired controls. These proteins were solubilized in phenol and fractionated by polyacrylamide gel electrophoresis. One-dimensional gel electrophoresis, using either isoelectrofocusing or sodium dodecyl sulfate, showed quantitative differences between the dystrophic hearts and the controls. A high resolution of NHNP was achieved by two-dimensional gel electrophoresis, revealing both quantitative and limited qualitative differences between the two groups. Proteins focusing from pH 5.0 to 5.6 with molecular weights of 55,000 (P 5 ) and 100,000 (P,) were strikingly increased in the cardiomyopathy. A protein (P 2 ) with an isoelectric point of 5.1 and some NHNP from dystrophic hearts in the entire region from pH 7.0 to 9.0 with molecular weights ranging from 35,000 (P 7 ) to 68,000 (P 4 ) were markedly reduced or absent. Differences in NHNP could be detected during the myolytic phase of cardiomyopathy but were most striking during the terminal phase of the disease. There were no detectable differences between the profiles of proteins derived from whole heart homogenates of dystrophic hamsters and controls. There were no significant differences at the failing phase between NHNP isolated from a purified preparation of myocardial cells and those isolated from whole heart. Therefore, the differences in NHNP appear to be reflections of alterations in nuclear composition of the dystrophic myocardial cell. Some of these observations may represent changes secondary to heart disease. However, if NHNP interacting with DNA play a major role in genetic expression, some of the manifestations of hamster cardiomyopathy could be due to a different constitution of NHNP in the dystrophic heart.

Nuclear proteins in the heart of the cardiomyopathic Syrian hamster. Phosphorylation of phenol-soluble nonhistone proteins.

“…They were characterized by isoelectric points of 5.1 (A,) and 4.9 (A 2 ) and molecular weights of 60,000 and 25,000, respectively. We demonstrated in the preceding paper 26 that there were striking differences in NHNP between the control and the dystrophic hearts. Protein (A,) appears to correspond to protein (P 2 ) in our previous report.…”

“…In an earlier study, we demonstrated alterations in NHNP composition of myocardium from patients with muscular subaortic stenosis as compared to those with infundibular hypertrophy or controls. 25 In a previous report, 26 we showed that there are striking differences in NHNP between the hearts of cardiomyopathic hamsters and controls. This report deals with the covalent modification of heart NHNP at different stages of the development of the cardiomyopathy.…”

“…26 Radioactive phosphorus acid ( 32 P-carrier free) was purchased from New England Nuclear. Each hamster was given 2.5 mCi 32 P, intraperitoneally, and was killed by decapitation 100 minutes after injection.…”

Electrophoretic profiles of nonhistone nuclear proteins of human hearts with muscular subaortic stenosis.

“…The electrophoretic patterns of both IH and normal were strikingly similar and clearly distinct from that of MSS. Furthermore, we have shown that the NHNP profile obtained from purified cardiac muscle cells of failing cardiomyopathic hamsters did not differ significantly from that obtained from unpurified whole heart nuclei (Liew and Sole, 1978a).…”

“…The pellet was resuspended once with Medium A, recentrifuged, then suspended in 30 ml of 2.2 M sucrose containing 1 mM MgCl 2 ,10 mM Tris-HCl (pH 8.0), and 0.1 mM PMSF and underlaid with 5 ml of the same solution. This suspension was centrifuged at 25,000 rpm for 1 hour in a Beckman SW 27 rotor as described previously (Taylor and Liew, 1976;Liew and Sole, 1978a). The pellet then was suspended in Medium A and centrifuged at 2000 rpm in the IEC centrifuge prior to fractionation of the nuclear proteins.…”