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Biomedical subjects

R M Robson

Publications and source records attributed to R M Robson.

16 recordsLinked to original sources
PubMed1992

Assembly of contractile and cytoskeletal elements in developing smooth muscle cells.

Specific developmental changes in smooth muscle were studied in gizzards obtained from 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 20-day chick embryos and from 1- and 7-day posthatch chicks. Myoblasts were actively replicating in tissue from 6-day embryos. Cytoplasmic dense bodies (CDBs) first appeared at Embryonic Day 8 (E8) and were recognized as patches of increased electron density that consisted of actin filaments (AFs), intermediate filaments (IFs), and cross-connecting filaments (CCFs). Although the assembly of CDBs was not synchronized within a cell, the number, size, and electron density of CDBs increased as age increased. Membrane-associated dense bodies (MADBs) also could be recognized at E8. The number and size of MADBs increased as age increased, especially after E16. Filaments with the diameter of thick filaments first appeared at E12. Smooth muscle cells were able to divide as late as E20. The axial intermediate filament bundle (IFB) could first be identified in 1-day posthatch cells and became larger and more prominent in 7-day posthatch cells. Immunogold labeling of 1- and 7-day posthatch cells with anti-desmin showed that the IFB contained desmin IFs. The developmental events during this 23-day period were classified into seven stages, based primarily on the appearance and the growth of contractile and cytoskeletal elements. These stages are myoblast proliferation, dense body appearance, thick filament appearance, dense body growth, muscle cell replication, IFB appearance, and appearance of adult type cells. Smooth muscle cells in each stage express similar developmental characteristics. The mechanism of assembly of myofilaments and cytoskeletal elements in smooth muscle in vivo indicates that myofilaments (AFs and thick filaments) and filament attachment sites (CDBs and MADBs) are assembled before the axial IFB, a major cytoskeletal element.

Animals↗
PubMed1992

Effect of porcine stress syndrome on the solubility and degradation of myofibrillar/cytoskeletal proteins.

This study examined the effect of stress classification (stress-positive, stress-carrier, stress-negative) of pigs on selected properties of postmortem muscle, including protein solubility and degradation of proteins such as titin. Longissimus muscle samples were removed 45 min postslaughter, divided into samples, and stored at 0 to 2 degrees C for analysis at 0, 1, 3, 5, and 7 d postmortem. Whole-muscle samples (homogenates) and purified myofibrils were prepared from each sample for analysis by SDS-PAGE. A portion of each muscle sample also was extracted 1) with a low-ionic-strength solution to obtain a sarcoplasmic protein fraction and 2) with two different high-ionic-strength solutions to obtain a myofibrillar/cytoskeletal protein fraction for measurement of protein solubility and for analysis of extracts by SDS-PAGE. No significant differences were observed between muscle from stress-negative and stress-carrier animals in this study. Sarcoplasmic (P less than .05) and myofibrillar/cytoskeletal (P less than .01) protein solubility was lower in muscle samples from stress-positive animals than in muscle samples from stress-carrier and stress-negative animals at all postmortem times studied. The high molecular weight protein titin was degraded more slowly postmortem in muscle from stress-positive than in muscle from stress-negative animals, as observed by SDS-PAGE analysis of whole-muscle samples (homogenates) an myofibrils. The combination of lowered protein solubility and reduced rate of postmortem degradation of structural proteins such as titin may explain, at least in part, the reduced quality and protein functionality of muscle from stress-positive pigs.

Animals↗
PubMed1979

Interaction of alpha-actinin, filamin and tropomyosin with F-actin.

The abilities of alpha-actinin, filamin and tropomyosin to bind F-actin were examined by cosedimentation experiments. Results indicated that smooth muscle alpha-actinin and filamin can bind to actin filaments simultaneously with little evidence of competition. In contrast, tropomyosin exhibits marked competition with either filamin or alpha-actinin for sites on actin filaments.

Actinin↗
PubMed1978

Filamin-actin interaction. Dissociation of binding from gelation by Ca2+-activated proteolysis.

Chicken gizzard filamin has been digested with purified Ca2+-activated protease. The subunits of (Mr = 250,000) of the protein are cleaved asymmetrically into two fragments, heavy merofilamin, Mr = 240,000, and light merofilamin, Mr = 9,500. Digestion is complete at substrate to enzyme ratios of 100:1 and requires Ca2+ concentrations in excess of 0.3 mM. Heavy merofilamin binds to F-actin as evidenced by cosedimentation with F-actin, by direct observation under the electron microscope, and by its ability to inhibit actin activation of heavy meromyosin ATPase. Heavy merofilamin does not form a gel when mixed with actin, except at very low concentrations of KCl. Thus, actin binding and gelation are separable activities of filamin. We speculate that Ca2+-stimulated proteolysis may play a role in the regulation of actin-filamin interactions.

Actins↗
PubMed1978

Laser Raman light-scattering observations of conformational changes in myosin induced by inorganic salts.

The Raman spectra of aqueous solutions of myosin and mixtures of myosin in solutions of the salts CaCl(2), MgCl(2), and LiBr have been taken. The spectrum of the solvent background has been subtracted by means of a computer, leaving only the Raman peaks of the protein. From an analysis of the Raman bands in the regions at 900, 940, 1,240-1,300, and 1,650-1,670 cm(-1), it seems likely that CaCl(2) effects an alpha-to beta-transition in myosin, probably owing to the interaction of the Ca(2+) ion, LiBr appears to denature the protein leading to increased random coil structure, and MgCl(2) appears to have an effect intermediate between the two other salts. These results are reported for concentrations as low as 10(-5) M of CaCl(2) and MgCl(2).This investigation indicates the usefulness of the Raman light-scattering technique for the study of protein conformational changes.

Calcium Chloride↗
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. Purification from porcine muscle.

Ca2+-activated Z-disk-removing activity in the P0-40 crude muscle extracts described by Busch et al. (Busch, W. A., Stromer, M. H., Goll, D. E., and Suzuki, A. (1972), J. Cell Biol. 52, 367) was purified from porcine skeletal muscle extracts by using five column chromatographic procedures in succession: (1) 6% agarose; (2) DEAE-cellulose; (3) Sephadex G-200; (4) DEAE-cellulose with a very shallow gradient; (5) Sephadex G-150. All Z-disk-removing activity eluted in a single peak off each column. Z-disk-removing activity always coeluted with Ca2+-activated proteolytic activity, so Z-disk-removing activity in the P0-40 crude muscle extract is due to a single Ca2+-activated protease (CAF). The five column chromatographic procedures produced a 140-fold increase in specific activity of the Ca2+-activated proteolytic enzymic activity; because preparation of the P0-40 crude CAF fraction before chromatography produced a 127-fold increase in specific activity, the entire procedure described here produces a 17 800-fold increase in specific activity of CAF. This increase in specific activity suggests that muscle contains 3.4 mug of CAF per g of muscle fresh weight; this content is in reasonably good agreement with our yields of 0.25-0.76 mug of purified CAF per g of muscle. Purified CAF migrated as a single band during polyacrylamide gel electrophoresis in pH 7.5 Tris-HC1 buffer but migrated as two bands with molecular weights of 80 000 and 30 000 during polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Densitometric scans of sodium dodecyl sulfate-polyacrylamide gels show that the 80 000- and 30 000-dalton subunits make up 85 to 90% of the protein in purified CAF preparations and that these subunits are present in equimolar ratios.

Animals↗
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↗
PubMed1975

Purification of two Clostridium bacteriocins by procedures appropriate to hydrophobic proteins.

Two clostridocins distinguishable by their different modes of action on Clostridium pasteurianum have been isolated, namely, butyricin 7423 found in cultures of Clostridium butyricum NCIB 7423 and perfringocin 11105 produced by Clostridium perfringens type A, NCIB 11105. Both were trypsin-susceptible proteins which were soluble in concentrated aqueous ethanol and were able to bind large amounts of the nonionic detergent Triton X-100. In the presence of Triton X-100, butyricin 7423 behaved as a hydrophobic protein in being concentrated in the polyethylene glycol layer of a three-phase partition system of dextran-Ficoll-polyethylene glycol. Their capacity to bind Triton X-100 was exploited in a purification procedure applicable to both bacteriocins. After aqueous ethanol extraction of an ammonium sulfate-precipitated fraction (and, in the case of the perfringocin, a heat-treatment step), a bacteriocin-Triton X-100 adduct was purified by gel filtration through Sepharose 6B. The bacteriocin was then freed of Triton X-100 by chromatography on Sephadex LH-20. Samples of butyricin 7423 purified in this way from different sources contained variable amounts of carbohydrate. Yet sodium dodecyl sulfate-gel electrophoresis revealed the existence of a polypeptide component of 32,500 daltons (+/-10%), which displayed the biological activity of butyricin 7423 in the absence of any detectable associated carbohydrate (or lipid). Preparations of perfringocin 11105 contained no carbohydrate or lipid and migrated in sodium dodecyl sulfate-gel electrophoresis as a single protein component of 76,000 daltons (+/-10%). It was concluded that both bacteriocins behave as amphiphilic proteins, and some implications of this finding are considered.

Bacteriocins↗