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R M Robson

Publications and source records attributed to R M Robson.

At least 55 records · Page 3Linked to original sources
PubMed1985

Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments.

After dialysis against 10 mM-Tris-acetate (pH 8.5), vimentin that has been purified in the presence of urea is present in the form of tetrameric 2 to 3 nm X 48 nm rods known as protofilaments. These building blocks in turn polymerize into intermediate filaments (10 to 12 nm diameter) when they are dialyzed against a solution of physiological ionic strength and pH. By varying the ionic conditions under which polymerization takes place, we have identified two classes of assembly intermediates whose structures provide clues as to how an intermediate filament may be constructed. The structure of the first class, seen when assembly takes place at 10 to 20 mM-salt at pH 8.5, strongly suggests that one of the initial steps of filament assembly is the association of protofilaments into pairs with a half-unit axial stagger. Increasing the ionic strength of the assembly buffer leads to the emergence of short, full-width intermediate filaments at approximately 50 mM-salt at pH 8.5. In the presence of additional protofilaments, these short filaments elongate to many micrometers when the ionic strength and pH are further adjusted to physiological levels. The electron microscope images of the assembly intermediates suggest that vimentin-containing intermediate filaments are made up of eight protofilaments, assembled such that there is an approximately 22 nm axial stagger between neighboring protofilaments. We propose that this half-unit staggering of protofilaments is a fundamental feature of intermediate filament structure and assembly, and that it could account for the 20 to 22 nm axial repeat seen in all intermediate filaments examined so far.

Animals↗
PubMed1984

Properties of smooth muscle vinculin.

Vinculin, isolated from turkey gizzard smooth muscle, was purified by chromatography on CM-cellulose after isolation from a DEAE-cellulose column. Two-dimensional gel electrophoretic analysis of crude muscle fractions demonstrated that: 1) much of the approximately 130,000-dalton protein present in smooth muscle did not co-isoelectrically focus with the purified 130,000-dalton vinculin and 2) the purified vinculin consisted of three major, closely spaced isoelectric variants that were present only in small amounts in the original smooth muscle sample. Purified vinculin sedimented as a single peak with a sedimentation coefficient S0 20,w of 5.9. Circular dichroism spectra of purified vinculin indicated a considerable degree of secondary structure, with an alpha-helical content of approximately 50% as measured at 208 nm. The ultraviolet absorption spectrum of vinculin gave a measured E1%(278) of 4.64. Digestion of vinculin, much of which is located at the cytoplasmic surface of the cell membrane, with Ca2+-activated neutral protease purified from skeletal muscle yielded major fragments with molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 98,000, 85,000, and 26,000. The factor(s) in DEAE-cellulose-purified vinculin responsible for decreasing the low shear viscosity of actin was removed and found in a crude fraction isolated by CM-cellulose chromatography. The purified vinculin had a small, but positive effect on the MgCl2-induced polymerization of actin as measured by low shear viscometry.

Amino Acids↗
PubMed1984

Growth and differentiation of chicken embryo muscle cell cultures derived from fast- and slow-growing lines. Intrinsic differences in growth characteristics and insulin response.

Primary myogenic cell cultures derived from 12-day embryos of genetically fast-growing chickens (fast cultures) and slow-growing chickens (slow cultures) were grown under identical conditions to examine differences in growth and differentiation at the cellular level. The two types of cultures exhibited significant (P less than 0.01) differences in proliferation, protein accumulation, response to the addition of insulin to the culture medium and the amount of insulin bound per nucleus. The fast cultures exhibited a larger number of both total nuclei and fused nuclei at 48, 72 and 96 h in culture, accumulated more protein per nucleus at 24, 48 and 72 h in culture and demonstrated a greater response to the addition of insulin to the culture medium, as reflected by increased fusion rate and protein accumulation at 24 h in culture. Maximal response to insulin in both types of cultures was obtained at 24 h to added insulin concentrations of 10(-10)-10(-9) M. Slow cultures bound more [125I]-insulin than fast cultures at 24 h in culture. These experiments suggest that different muscle growth potentials in animals of the same species are at least partly due to intrinsic cellular differences in the myogenic cells that give rise to adult muscle tissue.

Animals↗
PubMed1984

Development of methods to measure activity of polysomes and cytoplasmic enzymes from bovine skeletal muscle in in vitro protein-synthesis assays.

A cell-free, protein-synthesis system containing components from bovine skeletal muscle was developed as prerequisite to attempts to learn whether polysomes or cytoplasmic enzymes limit rate of muscle protein synthesis in the current population of domestic animals. Amino acid incorporation into trichloroacetic acid-precipitable protein was optimal at pH 7.5 and, if Tris buffer was used, at K+ and Mg2+ concentrations of 40 mM and 4 mM, respectively. Optimal concentrations of compounds that provide energy for amino acid incorporation were 1 mM ATP, .2 mM GTP, 20 micrograms creatine phosphokinase/ml and 20 mM creatine phosphate; .03 mM of each of the 20 amino acids was required for assays lasting up to 60 min. Neither rate of tRNA acylation nor availability of aminoacyl-tRNA was rate-limiting in the cell-free system established; hence, the cytoplasmic enzyme fraction from bovine skeletal muscle contains ample tRNA and has aminoacyl-tRNA synthases that are sufficiently active to form aminoacyl-tRNA faster than these compounds are used to form polypeptides in the cell-free system developed. Reinitiation of ribosomes onto new mRNA occurred very slowly, if at all, in the protein-synthesis system developed. Cytoplasmic enzymes were rate-limiting whenever cytoplasmic enzyme protein to polysomal protein ratios were 3.2:1 or lower in the cell-free system. Polysomes were rate-limiting whenever cytoplasmic enzyme protein to polysomal protein ratios were 400:1 or higher in the cell-free system. These ratios define the conditions needed to assay activity of cytoplasmic enzymes or polysomes from different animals quantitatively.

Adenosine Triphosphate↗
PubMed1984

Relative roles of polysomes and cytoplasmic enzymes in regulating bovine skeletal muscle protein synthesis.

Activities of cytoplasmic enzyme or polysomal fractions, both prepared from longissimus muscle of bovine animals differing markedly in muscle growth were measured in cell-free, protein synthesis assays. Two groups of animals were used: 1) eight animals comprising two "double-muscled" animals fed a high-energy diet, two "double-muscled" animals fed a low-energy diet, two "normal" animals fed a high-energy diet and two "normal" animals fed a low-energy diet and 2) six animals differing in age and sex and including two bull calves, 87 and 145 kg; two 18- to 24-mo-old animals, 411 and 596 kg and two cows, 594 and 681 kg. Ability of cell-free, protein-synthesis systems prepared from muscle of different animals to incorporate amino acids into TCA-precipitable protein differed significantly and was in direct correlation with phenotypic muscle growth. Mixing experiments using cytoplasmic enzymes from one set of animals with polysomes from another set showed that source of cytoplasmic enzymes had a greater effect on rate of in vitro protein synthesis than source of polysomes. Cytoplasmic enzymes and polysomes from double-muscled animals and animals fed a high-energy diet supported more rapid in vitro protein synthesis than the same components from normal animals or animals fed low-energy diets. The "double-muscling" characteristic had a greater effect on rate of in vitro protein synthesis than did high or low-energy diets. Cytoplasmic enzymes and polysomes from calves supported a greater rate of in vitro protein synthesis than the same components from 18- to 24-mo-old animals or cows. Based on these limited results in one species and with two groups of animals in that species, cytoplasmic enzymes may have a greater role in limiting rate of muscle protein synthesis in the current population of domestic animals than polysomes. Composition of the crude cytoplasmic enzyme fraction used in this study is unknown, but it likely contains elongation and termination factors in addition to aminoacyl-tRNA synthases and regulatory RNA.

Age Factors↗
PubMed1983

Properties of soleus muscle Z-lines and induced Z-line analogs revealed by dissection with Ca2+-activated neutral protease.

Rat soleus muscle Z-lines and Z-line anomalies induced by neostigmine methyl sulfate (NMS) and cat soleus muscle Z-lines and Z-line anomalies induced by tenotomy were examined by electron microscopy before and after dissection of muscle fibers with Ca2+-activated neutral protease (CAF) to elucidate structural properties of Z-lines and related Z-line-type structures. In both normal and treated muscles, interdigitation of thin (6-7 nm) filaments, which were continuous with I-filaments (actin) from adjacent sarcomeres, was observed at the Z-line in longitudinal section. Both neostigmine methyl sulfate and tenotomy treatments induced muscle atrophy associated with Z-line degradation, streaming, and irregular distribution and accumulation of Z-line material and Z-rod formation. Tenotomized muscle also was characterized by the presence of N-line-like bands and I-Z-I brushes. CAF digestion removed the electron-dense covering material from Z-rods and revealed a backbone of actin filaments. The origin of Z-rods, their structural similarity to Z-lines in longitudinal and cross section, and their susceptibility to CAF indicate that Z-rods are directly related to native Z-lines and are probably lateral polymers of a basic Z-line unit. The regular square net alignment (22 nm) of I-filaments (actin) in cross sections of I-Z-I brushes which contain no N-lines suggests that the I-square net arrangement near the Z-line is determined by Z-filament-actin filament interaction rather than by the N-line or other factors. The results suggest that I-filaments (actin) penetrate the mammalian Z-line and are Z-line constituents and that the width of Z-lines and the length of Z-rods are determined by the amount of overlap of actin filaments. The perpendicular periodicity of Z-rods and the zigzag-oblique arrowheadlike appearance seen in longitudinal sections of Z-lines are attributed to alpha-actinin.

Animals↗
PubMed1983

Evidence for actin involvement in cardiac Z-lines and Z-line analogues.

Canine and feline cardiac Z-lines and Z-rods were examined by electron microscopy before and after digestion of muscle fibers with Ca2+-activated protease (CAF). Removal by CAF of electron-dense material which covers Z-lines and Z-rods exposed interdigitating longitudinal filaments (6-7 nm in diameter) apparently continuous with thin filaments of the respective I-bands. The newly exposed longitudinal filaments of CAF-treated Z-lines and of CAF-treated Z-rods bound heavy meromyosin and therefore are actin. The width of Z-lines and length of Z-rods are determined by the amount of overlap of actin filaments of opposite polarity. The oblique filaments in Z-lines and Z-rods are responsible for the perpendicular periodicity of Z-lines and Z-rods, and are attributed to alpha-actinin.

Actinin↗
PubMed1982

Nemaline myopathy rod bodies. Structure and composition.

Ca2+-activated protease (CAF) digestion of glycerinated nemaline myopathy muscle removed the electron-dense material covering rods and Z-lines and exposed longitudinal backbone filaments, 6-7 nm wide, which span the lengths of the original rods. Decoration of the exposed filaments (which are responsible for the periodicity parallel to the long axis of intact nemaline rods) with heavy meromyosin (HMM) proved they are actin filaments. After CAF treatment, cross-striated periodical patterns in longitudinal sections and Z-filament-like proteins connecting actin filaments seen in cross-section disappeared. This suggests that alpha-actinin may be involved in formation of this pattern because of the specificity of CAF toward alpha-actinin. Gel electrophoresis of CAF-treated nemaline muscle showed that most alpha-actinin is released into the supernatant, whereas the residue is mainly actin and myosin. Electron microscope examination of longitudinal sections of intact rods shows an oblique filament pattern, thin (7 nm) lines, thick (11 nm) lines, and an amorphous-appearance previously observed in normal Z-lines, patterns observed depend on sectioning angle and section thickness. In cross-section, rods show small square net (SS) and basket-weave (BW) forms. The SS form predominates and coexistence of the 2 forms, which also occur in normal Z-lines, is observed. Results support the idea that rods are lateral polymers of Z-line units. We think that the length of rods, as well as the width of Z-lines, is determined by the amount of overlap of actin filaments of opposite polarity. Initiation of rod formation may be due to deregulation of actin filament length.

Animals↗
PubMed1981

Effect of alpha-actinin on actin structure. Actin ATPase activity.

Alpha-Actinin increases the ATPase activity of actin by up to 84%, depending un pH, divalent cations present and the added Mg2+: ATP ratio. Dithiothreitol decreases actin ATPase activity approx. 20% but does not reduce the ability of alpha-actinin to increase actin ATP activity. Increasing amounts of added alpha-actinin up to 1 mos alpha-actinin to 49 mol actin cause in increasing increment in actin ATPase activity, but adding alpha-actinin beyond 1 mol alpha-actinin to 49 mol actin elicits only small additional increments in activity. Actin ATPase activity ranges from approx 100 nmol Pi/mg actin per h (4.3 mol Pi/mol actin per h) at high levels (10 mM) of ATP in the presence of lower amounts (1 mM) of added mg2+ to approx. 12.5 nmol Pi/mg actin per h (0.52 mol Pi/mol actin per h) at high pH (8.5) or at low levels (0.5-1.0 mM) of ATP in the presence of higher amounts (10 mM) of added Mg2+ ATp uncomplexed with Mg2+ inhibits the ability of alpha-actinin to increase F-actin ATPase activity. Activities with different divalent cations showed that the actin ATPase in these studies, which was 1/100 as great as Mg2+-modified actomyosin ATPase activity, was not due to trace amounts of myosin contaminating the actin preparations. The results are consistent with the concept that alpha-actinin can alter the structure of actin monomers.

Actinin↗
PubMed1981

Effect of alpha-actinin on actin structure. Release of bound nucleotide.

We have examined the alpha-actinin-F-actin interaction by measuring the effect of highly purified alpha-actinin on bound nucleotide exchange in F-actin. Exchange was followed by measuring the release of actin-bound [14C]ADP in the presence of ATP using an ultrafiltration technique. Alpha-Actinin increases by about 60 to 70% the rate of release of F-actin bound nucleotide when incubated for 1 h in the presence of 1 mM ATP/1 mM MgCl2/0.05 mM CaCl2/0.5 mM dithioerythritol/100 mM KCI/20 mM Tris-acetate, pH 7.5, at 37 degrees C. The ability of alpha-actinin to enhance nucleotide exchange was maximal when alpha-actinin was added at a level near 10% of actin present by weight (molar ratio of 1 alpha-actinin to 49 actin monomers). The potentiating effect of alpha-actinin on the nucleotide exchange rate of F-actin was not highly related to the Mg2+: ATP ratio present in the incubation mixture. Alpha-actinin also increased the rate of bound nucleotide exchange of f-actin was present in a reconstituted actomyosin suspension. The results are consistent with th possibility that one alpha-actinin can affect the structure of multiple actin monomers present in an actin filament.

Actinin↗
PubMed1981

Effect of alpha-actinin on actin structure: viscosity studies.

The effect of ATP on ability of alpha-actinin to increase viscosity of F-actin was measured in three different solutions: 100 mM KCl; 100 mM KCl/l mM Mg2+; and Mg2+ alone at concentrations of 1-6 mM. When ATP and Mg2+ are added at equimolar ratios or at added [ATP] to added [Mg2+] greater than equimolar, alpha-actinin has no effect on F-actin viscosity in the absence of KCl. ATP decreases viscosity of alpha-actinin/F-actin mixtures by 20% even in the presence of KCl, evidently because ATP affects the alpha-actinin-F-actin interaction. Molar ratios of 1 alpha-actinin to 49 actins increase specific viscosity of F-actin approx. 2-fold at 37 degrees C in the presence of 1 mM ATP, so ATP does not prevent the alpha-actinin-F-actin interaction.

Actinin↗
PubMed1981

Purification of desmin from adult mammalian skeletal muscle.

A method has been developed for preparation of purified desmin from mature mammalian (porcine) skeletal muscle. A crude desmin-containing fraction was prepared by modification of procedures used for isolation of smooth-muscle intermediate-filament protein [Small & Sobieszek (1977) J. Cell Sci. 23, 243-268]. The desmin was extracted in 1 M-acetic acid/20 mM-NaCl at 4 degrees C for 15h from the residue remaining after actomyosin extraction from washed myofibrils. Successive chromatography on hydroxyapatite and DEAE-Sepharose CL-6B in 6M-urea yielded desmin that was routinely more than 97% 55 000-dalton protein and that had no detectable actin contamination. Removal of urea by dialysis against 10mM-Tris/acetate (pH 8.5)/1 mM dithioerythritol and subsequent clarification at 134 000 g (rav. 5.9 cm) for 1 h results in a clear desmin solution. Dialysis of purified desmin against 100 mM-NaCl/1 mM-MgCl2/10 mM-imidazole/HCl, pH 7.0, at 2 degrees C resulted in the formation of synthetic desmin filaments have an average diameter of 9-11.5 nm. The present studies demonstrate that the relatively small amount of desmin in mature skeletal muscle can be isolated in sufficient quantity and purity to permit detailed studies of its properties and function. Although 10nm filaments have not been unequivocally demonstrated in mature muscle in vivo, that the purified skeletal-muscle desmin will form 10 nm filaments in vitro lends support to their possible existence and cytoskeletal function in mature skeletal-muscle cells.

Actins↗
PubMed1981

Immunoelectron and immunofluorescence localization of desmin in mature avian muscles.

Antisera or affinity-purified antibodies shown to be specific for avian gizzard desmin antigen by double immunodiffusion, antigen-blocking, and immunoautoradiography experiments have been used in indirect immunofluorescence and indirect immunoelectron microscopy to demonstrate localization of desmin in myofibrils from mature avian muscles. The light microscope results agree with the work of others in that they suggest that desmin is primarily at or near the periphery of Z-lines of striated muscle myofibrils. Immunoperoxidase labelling more clearly shows that the reactive desmin antigen is located almost entirely between Z-lines of adjacent parallel myofibrils and that there is no obvious correlation between locations of T-tubules and desmin structures. The electron-dense reaction product often followed an approximately linear course between Z-lines of adjacent myofibrils and indicated the desmin antibodies had decorated a small number of filaments spanning this region. These results suggest that the desmin found in close association with myofibrils of mature striated muscle is in the aggregated form of 10-nm filaments.

Animals↗
PubMed1981

Disassembly of synthetic 10-nm desmin filaments from smooth muscle into protofilaments.

Synthetic 10-nm filaments formed from highly purified turkey gizzard desmin have a helically oriented substructure and disassemble into 2 to 2.5 nm protofilaments and 3.5 to 5 nm subfilaments after treatment with 1 or 2 M urea or with low-ionic-strength buffer (8 mM Tris, pH 8.2). SDS-gels of 10-nm filaments treated with these solutions show that a single 55 000-dalton band is present before and after all treatments and indicate that the newly revealed substructure is not caused by proteolysis. Antibodies to electrophoretically purified desmin react, in immunodiffusion, only with the antigen and decorate both the subfilamentous particles and the synthetic 10-nm filaments. These studies indicate that a synthetic 10-nm desmin filament is a rope-like structure constructed from the 2 to 2.5 nm diameter protofilaments.

Animals↗
PubMed1980

Disproportionate accumulation of myosin and tropomyosin in cultured muscle cells.

Accumulation of two major myofibrillar proteins, myosin and tropomyosin, was monitored in differentiating skeletal muscle cultures. The tropomyosin subunit to myosin heavy chain accumulation rate ratio was more than twice the stoichiometric ratio of tropomyosin subunit to myosin heavy chain in mature skeletal muscle myofibrils and crude myofibrils from cultured muscle cells. Electron microscopy revealed normal patterns of myofibril assembly in these muscle cultures. Therefore, the observed disproportionate accumulation of tropomyosin and myosin heavy chain may be reflecting normal cellular conditions for de novo myofbril assembly.

Animals↗