Douglas J. Horgan

Collagen crosslinks and their relationship to the thermal properties of calf tendons

The hydrothermal isometric tension and thermal transition temperature of collagen were determined in tendons from three different calf muscles. The levels of the nonreducible collagen crosslink, pyridinoline, and the collagen-associated Ehrlich chromogen were also measured in the three tendons. The reducible collagen crosslinks, hydroxylysinonorleucine, dihydroxylysinonorleucine, and histidinohydroxymerodesmosine were measured in two tendons. The thermal properties and levels of crosslinks were found to vary considerably between the different tendons, and also at different sites in two of the tendons. A strong correlation was observed between the thermal transition temperatures and the hydrothermal isometric tensions of the nine tendon sites examined. Both thermal properties correlated with the concentration of both pyridinoline and Ehrlich chromogen. The analogous behavior of the collagen-associated Ehrlich chromogen and the pyridinoline crosslink supports the role of the Ehrlich chromogen as a nonreducible crosslink.

The relationship between animal age and the thermal stability and cross-link content of collagen from five goat muscles.

The thermal stability of intramuscular collagen, as determined using differential scanning calorimetry, was measured in five muscles from 75 goats with known birth dates ranging in age from one day to 13 years. The collagen cross-link pyridinoline, and the collagen-associated, and putative cross-link, Ehrlich Chromogen were also measured. Five different muscles were examined and the effects of age compared to those found in the tendon of the longissimus dorsi muscle. The differences between intramuscular collagen and tendon collagen were found to be much greater than those between the intramuscular collagens of different muscles. Intramuscular collagen is more thermally stable than tendon collagen due to higher levels of heat-stable cross-links. However the increase in thermal stability of intramuscular collagen with age could not be explained simply in terms of the cross-links measured.

Isolation of transverse tubules by fractionation of sarcoplasmic reticulum preparations in ion-free sucrose density gradients.

A new method for the preparation of transverse tubules (T-tubules) from rabbit skeletal muscles is reported. When crude sarcoplasmic reticulum (SR) preparations were centrifuged on sucrose density gradients containing buffering ions (buffered gradients) 70-80% of the material sedimented as a single heavy band in the region of 43% sucrose. When this fraction (or crude SR) was recentrifuged on sucrose gradients prepared free of buffer or other ions (ion-free gradients) the heavy band dissociated into three fractions of different densities. The lightest fraction sedimented at 28% sucrose and was identified as T-tubules on the basis of its nitrendipine and ouabain binding properties. The enzymatic properties, cholesterol contents, and protein compositions of the fractions obtained when SR is centrifuged on buffered and ion-free sucrose density gradients were measured. The T-tubules were enriched in cholesterol and in marker enzymes for surface membranes while the other fractions were shown to be terminal cisternae and longitudinal cisternae on the basis of their (Ca2+,Mg2+)-ATPase activities and characteristic protein profiles.

Effect of pressure treatment on the sarcoplasmic reticulum of red and white muscles.

The effects of high pressure (150 MPa) on the sarcoplasmic reticulum of red and white muscles have been studied. When whole muscle is pressurised either pre-rigor or post-rigor the major effect on the SR is the loss of extra ATPase activity and the loss of several proteins including the 100,000 dalton ATPase and calsequestrin. When isolated SR is pressurised the extra ATPase activity is lost but there is no protein degradation. Measurements of muscle pH and the influence of pH on pressurisation effects indicate active proteolysis in white muscle when it is pressurised. In all these studies the basal ATPase activity was relatively unaffected. The effect of pressure treatment on the yield of SR protein varied with different muscles, being greatest in the muscles which had the highest concentration of extra ATPase and calcium uptake activities. These muscles also reached the lowest pH during pressurisation, thus favouring proteolysis.

Biochemical properties of purified transverse tubules isolated from skeletal muscle triads

Transverse tubules (t-tubules) were prepared from muscle by dissociation of intact triads during centrifugation in ion-free sucrose gradients. They were further purified by the removal of contaminating sarcoplasmic reticulum after loading with calcium phosphate. Purification was accompanied by enrichment in markers specific for t-tubules, e.g., nitrendipine binding sites. According to gel electrophoresis the purified t-tubules contained three major protein bands of 104, 70, and 30 kDa. When solubilized with detergents there was a two- to threefold increase in Mg2+-ATPase activity, and a corresponding increase in the 30-kDa protein band. The 104-kDa protein was shown to be a (Na+ + K+)-ATPase because of its phosphorylation by [gamma-32P]ATP in the presence of sodium ions. The orientation of the t-tubule membrane was predominantly inside-out.

A fluorometric assay for the potassium-dependent phosphatase activity of the (Na++K+)-adenosine triphosphatase

A fluorometric assay for the K+-dependent phosphatase activity of the (Na+ + K+)-ATPase in both purified and membrane-bound forms is described. The assay utilizes 3-O-methylfluorescein phosphate as substrate and measures the fluorescence of the 3-O-methylfluorescein produced by hydrolysis of the substrate. The assay described is an order of magnitude more sensitive than the assay employing p-nitrophenylphosphate, the substrate most commonly used to measure this activity. The assay is also suitable for the specific measurement of (Na+ + K+)-ATPase activities in membranes which contain high levels of other ATPase activities.

Simple spectrophotometric estimation of ATPase and calcium uptake activities of sarcoplasmic reticulum preparations

By adding 5–10 mM inorganic orthophosphate to an assay medium containing 10–100 μM added calcium it is possible to obtain from a single recorder tracing a measure of the “basal” and “extra” ATPase activities and an estimate of the rate of calcium uptake by sarcoplasmic reticulum preparations. The rates of calcium uptake as measured by this method are in good agreement with the rates obtained by the conventional 45Ca method. The method described has the advantages of being simple and rapid.

Modification of sarcoplasmic reticulum adenosine triphosphatase by adenosine triphosphate and magnesium

Abstract Lineweaver-Burk plots of Ca2+-activated adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum have been determined for a wide range of substrate concentrations. The plots measured at constant Mg2+ concentrations are normally nonlinear, but approach linearity either as the sarcoplasmic reticulum ages, or when small quantities of Triton-X100 are added. Titration with N-ethylmaleimide has the same effect on the activity of the ATPase measured either at high or low substrate concentrations. Lineweaver-Burk plots measured under conditions where the Mg2+ concentration is varied so as to be always equal to the ATP concentration are linear. These results have been interpreted as evidence that the adenosine triphosphatase has a single active site which uses MgATP as its substrate and which can be modified by free Mg2+.

The molecular location of Ehrlich chromogen and pyridinoline cross-links in bovine perimysial collagen.

Collagenous peptides containing the Ehrlich chromogen (EC), a trifunctional cross-link of proposed pyrrolic structure, were selectively isolated from a tryptic digest of bovine perimysial collagen by coupling to a diazotised support. Peptides containing pyridinoline (Pyr), another trifunctional cross-link but based on a 3-hydroxypyridinium ring, were isolated from the uncoupled material. The isolated cross-linked peptides were purified by chromatographic procedures and subsequently characterised by amino acid and sequence analyses. EC occurred in stoichiometric amounts in three-chained peptides derived from type I collagen cross-link regions. In contrast, Pyr was found in non-stoichiometric amounts in three-chained peptides where two of the chains were identified as the 76 amino-terminal residues of the α1 (III) collagen chain. The third chain in these Pyr cross-linked peptides was derived from the C-terminal helical cross-link region of either type III collagen or the corresponding region of type I collagen, with the former region predominating. These findings suggest that EC and Pyr cross-links of perimysial collagen are associated mainly with type I and type III collagen respectively.

Effect of high pressure on the regulation of phosphorylase activity in pre-rigor rabbit muscle

The effects of high pressure (150 MPa) on the regulation of phosphorylase activity in pre-rigor rabbit muscles have been studied at 35° and 0°C. At 35°C muscle contracts, phosphorylase is activated and the muscle pH falls to 5·8 in 2 min. Coinciding with these changes, phosphorylase phosphatase activity falls rapidly, while phosphorylase kinase, although active for longer, loses its activity as the pH falls. Both of these enzymes are completely inactivated after 5 min under pressure, while phosphorylase still retains 80% of its activity under these conditions. The effects of pressure on the activities of these enzymes in white and red muscles of rabbits were compared, with a greater effect being observed in white muscles. At 0°C, muscles subjected to high pressure did not contract, but at this temperature the three enzyme activities (phosphorylase, phosphorylase kinase and phosphorylase phosphatase) were all lost at a greater rate than at 35°C, although the pH of the muscle did not fall below 6·5. The effects of high pressure treatment on isolated phosphorylase a and b and phosphorylase kinase were also studied at both 0° and 35°C and the results obtained closely paralleled those observed in whole muscle.