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M H Stromer

Publications and source records attributed to M H Stromer.

12 recordsLinked to original sources
PubMed1978

Sythesis of tropomyosin in cultures of differentiating muscle cells.

The accumulation of tropomyosin in cultures of differentiating muscle cells was quantitatively measured. Tropomyosin was isolated from cultured cells during and after myoblast fusion; both alpha- and beta-subunits were present in myotube cultures. During fusion small amounts of tropomyosin were detectable, but, as fusion approached a maximum, tropomyosin accumulation began to increase. The increased synthesis of tropomyosin after the initiation of muscle cell fusion is consistent with the increased synthesis of other proteins characteristic of muscle, including myosin.

Animals↗
PubMed1977

N- and C-terminal amino acids of purified alpha-actinin.

Highly purified bovine cardiac alpha-actinin is obtained by successive chromatography on DEAE-cellulose and hydroxyapatite of a crude fraction obtained by salting out low ionic strength extracts of bovine cardiac muscle between 0 and 30% ammonium sulfate saturation. Hydroxyapatite chromatography removes a 43 000-dalton polypeptide chain that is difficult to remove by successive DEAE-cellulose columns. Removal of all 43 000-dalton material by hydroxyapatite chromatography is accompanied by disappearance of a very small 9 to 10 S boundary in analytical ultracentrifuge diagrams of DEAE-cellulose-purified 6.2S alpha-actinin. Approximately 95% of the protein in DEAE-cellulose and hydroxyapatite-purified alpha-actinin is the 100 000-dalton alpha-actinin polypeptide as estimated by SDS-polyacrylamide gel electrophoresis. Purified bovine cardiac, porcine skeletal, chicken gizzard, and chicken breast alpha-actinins all contain leucine as the C-terminal amino acid of both polypeptide chains in the alpha-actinin molecule. Bovine cardiac and porcine skeletal alpha-actinins contain arginine as the amino acid penultimate to C-terminal leucine. None of the four different alpha-actinins studied had a N-terminal amino group available for reaction with dansyl chloride, but all four alpha-actinins contained 1.6 to 1.8 acetate residues per molecule (200 000 daltons) of alpha-actinin. It seems likely that the N-terminal amino groups of both polypeptide chains in these four alpha-actinins are acetylated. A peptide having the composition N-Ac-Asp2-Glu4 was isolated from a proteolytic digest of bovine cardiac alpha-actinin. alpha-Actinin seems to be a conserved protein molecule found in many different motile systems.

Actinin↗
PubMed1976

Some properties of purified skeletal muscle alpha-actinin.

Highly purified alpha-actinin can be made by using the low ionic strength extraction procedure previously described (Arakawa N., Robson, R. M., and Goll, D. E. (1970) Biochim. Biophys. Acta 200, 284-295) and then subjecting the crude alpha-actinin fraction obtained with this extraction procedure to successive chromatography on DEAE-cellulose and hydroxyapatite. Hydrozyapatite chromatography specifically removes a protein having a subunit molecular weight of 42,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Hydroxyapatite-purified alpha-actinin sediments entirely as a 6.21 S boundary in the analytical ultracentrifuge with no trace of the small 9 to 10 S boundary seen in earlier alpha-actinin preparations purified by DEAE-cellulose chromatography. In 100 mM KCl, 20 mM Tris-acetate, pH 7.5, hydroxyapatite-purified alpha-actinin has a diffusion coefficient (D020,w) of 2.71 X 10(-7) cm2-s-1, an intrinsic viscosity of 20.6 ml-g-1, a molecular weight of 201,000 +/- 4,300 (plus or minus least squares standard error) as determined by sedimentation equilibrium, and a molecular weight of 210,000 as determined by sedimentation diffusion. In 6 M guanidine HCl, hydroxyapatite-purified alpha-actinin has a molecular weight of 106,000 +/- 6,300 as determined by sedimentation equilibrium and a molecular weight of 100,000 as determined by a calibrated 4% agarose gel permeation column. SDS-polyacrylamide gel electrophoresis gives a molecular weight of 96,000 to 100,000 for hydroxyapatite-purified alpha-actinin. Rod-shaped particles 44 X 390 to 400 A are seen in electron micrographs of negatively stained alpha-actinin. By assuming 45% hydration and a molecular weight of 206,000, dimensions of approximately 40 X 500 A can be calculated for the alpha-actinin molecule by using either s 020, w, D 020, w, intrinsic viscosity, or a calibrated 6% agarose gel permeation column. Hydroxyapatite-purified alpha-actinin has an alpha-helical content of 74% as measured by circular dichroism at 208 nm.

Actinin↗
PubMed1976

Effect of trypsin on rabbit skeletal muscle alpha-actinin.

5 min of tryptic digestion of purified rabbit skeletal alpha-actinin decreases by approximately 75% the ability of alpha-actinin to cross-link F-actin filaments as measured viscometrically at 27 degrees C, but has little effect on the sedimentation coefficient of alpha actinin at 20 degrees C or an alpha-actinin's ability to increase the Mg2+-modified ATPase activity and rate of turbidity increase of reconstituted actomyosin suspensions. Twenty to sixty min of trypsin treatment reduces the sedimentation coefficient of alpha-actinin and destroys much of alpha-actinin's ability to increase the MG2+-modified ATPase and rate of turbidity increase of reconstituted actomyosin suspensions. Therefore, the ability of alpha-actinin to increase the rate of in vitro measures of muscle contraction may not result directly from alpha-actinin's ability to cross-link F-actin filaments. Trypsin does not split alpha-actinin into large fragments as it does myosin. Previous studies have shown that 35 to 65% of total tryptic-susceptible peptide bonds in alpha-actinin are split after 60 min of incubation with trypsin and that 30% of these bonds split in 60 min are cleaved during the first 5 min in a rapid reaction. That splitting of this group of peptide bonds has little effect on the sedimentation coefficient of alpha-actinin indicates that these bonds are located in a region of the alpha-actinin molecule where noncovalent forces are strong enough to maintain conformation of the native alpha-actinin molecule even after these bonds have been split. This ostensible segregation of alpha-actinin's ability to cross-link F-actin filaments from its ability to increase rate of in vitro assays of contraction by tryptic digestion may suggest that alpha-actinin could have at least two different physiological roles: (1) to bind actin filaments to each other or to basal structures, and (2) to enhance the effectiveness of actin in supporting movement.

Actinin↗
PubMed1976

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme.

The purified Ca2+-activated protease (CAF) isolated from porcine skeletal muscle and capable of removing Z-disks from intact myofibrils is optimally active on either myofibril or casein substrates at pH 7.5 and in the presence of 1 mM Ca2+ and at least 2 mM 2-mercaptoethanol. No CAF activity is detected when 1 mM Mg2+, Mn2+, Ba2+, Co2+, Ni2+, and Fe2+ are added singly. When added with 1 mM Ca2+, Co2+, Cu2+, Ni2+, and Fe2+ inhibit, whereas Mg2+, Mn2+, and Ba2+ have no effect on CAF activity. CAF is irreversibly inhibited by iodoacetate but is unaffected by soybean trypsin inhibitor. S0/20,W=5.90 S, and sedimentation equilibrium molecular weight - 112 000 for purified CAF. Because purified CAF migrates as two polypeptide chains with molecular weights of 80 000 and 30 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CAF molecule must consist of one each of these two polypeptide chains. Approximate molecular dimensions of 38 X 220 A can be calculated for CAF from calibrated gel permeation column data or from S0/20,W and the molecular weight. Amino acid composition and physical properties of purified CAF distinguish it from the known catheptic enzymes and from other proteases found in blood or in granulocytes. Purified CAF removes Z-disks the 400-A periodicity associated with troponin in the I band and partly degrades M lines but causes no other ultrastructurally detectable effects when incubated with myofibrils. These results agree with the earlier finding that purified CAF degrades troponin, tropomyosin, and C-protein but has no effect on myosin, actin, or alpha-actinin, and suggest that CAF may have a physiological role in disassembly of intact myofibrils during metabolic turnover of myofibrillar proteins.

Amino Acids↗
PubMed1976

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Subcellular localization of the protease in porcine skeletal muscle.

A study was done to determine whether the Ca2+-activated muscle protease (CAF) that removes Z disks from myofibrils in the presence of Ca2+ is located in a sedimentable subcellular organelle. Porcine skeletal muscle cells were diced finely with a scalpel and were suspended in 0.25 M sucrose, 4 mM EDTA with a VIRTIS homogenizer. Filtration of the suspended muscle through four layers of cheesecloth removed most of the myofibrils and stromal protein. Nuclear (1,000 gavg for 15 min), mitochondrial-microsomal (50,000 gavg for 60 min), and supernatant fractions were assayed for succinic dehydrogenase, acid ribonuclease, cathepsin D, and CAF activities. Approximately 96% of total succinic dehydrogenase activity, 81% of cathepsin D activity, and 45% of acid ribonuclease activity, but only 14% of total CAF activity, were found in the nuclear and mitochondrial-microsomal fractions. Cathepsin D activity in the nuclear and mitochondrial-microsomal fractions was decreased if assays were done without prior treatment to rupture membranous structures; hence, our cell rupture and homogenization procedures preserved some intact lysosomal organelles. The results indicate that the small amount of CAF activity in the nuclear and mitochondrial-microsomal fractions was due to contamination by supernate and that CAF is not located in a membrane-bounded subcellular particle. Because CAF is active at the intracellular pH and temperature of living skeletal muscle cells and is in direct contact with the cytoplasm of muscle cells, its activity must be regulated by intracellular cellular Ca2+ concentration to prevent continuous and indiscriminate degradation of myofibrils.

Animals↗