Erik Slinde

Composition of muscle fibre types and connective tissue in bovine M. semitendinosus and its relation to tenderness.

The distribution of muscle fibre types and connective tissue in bovine M. semitendinosus is described. A parallel increase in the volume fraction of type I muscle fibres (from 10% to 30%) and a decrease in the IIB volume fraction (from 58% to 34%) was recorded from superficial to deep layers. A positive correlation was observed between the frequency and the cross-sectional area of both type I and IIB fibres. The elastic fibres formed irregularly shaped bundles that made up about 50% of the volume of the perimysium. Thin elastic fibres extended into the endomysium. The relative proportion of elastic fibres in the perimysial connective tissue increased towards the deeper layers of the muscle. A taste panel evaluation of the sensory properties was performed and the data were correlated to the histological observations. A gradual decrease in scores of four tenderness-related traits was recorded from the superficial to the deep layer of the muscle. The superficial layer was rated as most tender, whereas the consecutive layers were rated less tender. The possible relationship between the composition of muscle and the meat quality is discussed.

Effect of the hydrophile-lipophile balance of non-ionic detergents (Triton X-series) on the solubilization of biological membranes and their integral b-type cytochromes.

The solubilization of four integral membrane proteins (i.e. cytochrome b-561 of the chromaffin granule membrane, cytochrome b5 of the endoplasmic reticulum and the mitochondrial b-type cytochrome(s) as well as cytochrome c oxidase) has been studied at 0 degrees C using the non-ionic detergents of the Triton X-series having the common hydrophobic 4(1,1,3,3-tetramethylbutyl)phenoxy (t-octyl-phenoxy) group and a variable average number (n) of polar ethylene oxide units added. Following a pre-extraction of peripheral membrane and matrix proteins with low and high salt concentration and a weak non-ionic detergent (Tween 20, average hydrophile-lipophile balance (HLB) = 16.7), the amount of heme proteins solubilized by subsequent Triton X-solutions was measured. With the detergents tested the degree of solubilization decreased in the sequence cytochrome b-561 greater than cytochrome b5 greater than mitochondrial cytochrome(s) b and parallelled the effect of the detergents on light scattering and the phospholipid to protein ratio of the three membranes. For all the b-cytochromes, the solubilizing power of the detergent increased with decreasing average length of the polar ethylene oxide chain and the hydrophile-lipophile balance as long as clouding did not occur (e.g. Triton X-114,n = 7.5 and HLB = 12.4). Thus, the greatest difference in the degree os solubilization of the three cytochromes was observed with Triton X-405 (n = 40 and HLB = 17.9). All the cytochromes were most efficiently solubilized (i.e. approx. 90%) by Triton X-100 (n = 9.5 and HLB = 13.5).

A general and rational approach to the optimal recovery of mitochondria by differential centrifugation in homogenous media

Abstract The sedimentation coefficients ( S -values) of mitochondria have been determined in isotonic sucrose media for different mammalian tissues. Since the values show a relatively great variation, a general and rational approach to the optimal recovery of mitochondria from any tissue by differential centrifugation is presented. The method is primarily based on the determination of the average sedimentation coefficient at the actual isolation conditions. From the S -value thus determined and known centrifugation parameters, a specific procedure can be designed in each case.

The intracellular localization of long-chain acyl-CoA synthetase in brown adipose tissue

1. The acyl-CoA synthetase activity in brown adipose tissue of cold-exposed guinea pig has been studied by measuring the rate of palmitoylcarnitine formation in the presence of excess carnitine palmitoyltransferase. 2. The rate of palmitoylcarnitine formation in the mitochondria was found to be 161 plus or minus 64 nmol.mg-minus-1. min-minus-1 (n=9). 3. In the absence of added palmitate and bovine serum albumin a total of 35 plus or minus 1 nmol endogenous fatty acids.mg-minus-1 were activated with three different mitochondrial preparations. 4. Three different experimental approaches have been used to study the subcellular localization of the enzyme: (a) conventional differential centrifugation (De Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955) Biochem. J. 60, 604-617) (B) the determination of the sediterm of different marker enzymes (Slinde, E. and Flatmark. T. (1973) Anal. Biochem. 56, 324-340) and (c) the determination of the stoichiometry between the activities of these enzymes sedimented at higher centrifugal effects. 5. Throughout all fractionation procedures, the long-chain acyl-CoA synthetase follows strictly the amine oxidase generally considered to be exclusively located on the mitochondrial outer membrane.

VAriations in the activity of microsomal palmitoyl-CoA hydrolase in mixed micelle solutions of palmitoyl-coa and non-ionic detergents of the triton X series

The kinetics of palmitoyl-CoA hydrolase were influenced by both the availability of the substrate and formation of micelles. At palmitoyl-CoA concentrations below the critical micelle concentration, addition of non-ionic detergent increased the activity until the critical micelle concentration of the mixed micelles was reached. At palmitoyl-CoA concentrations above the critical micelle concentration, inhibitor of the activity was observed, but addition of detergents of the Triton X series reversed the inhibition. Maximum palmitoyl-CoA hydrolase activity was found when the ratios (w/v) of palmitoyl-CoA: Triton X-100 and palmitoyl-CoA: Triton X-405 were approximately 0.35 and 0.05, respectively. At these above the mixed critical micelle concentration. The results indicate that monomer palmitoyl-CoA is the substrate and that monomer forms of the non-ionic detergents of the Triton X series activate the enzyme. Isolated microsomal lipids activated the microsomal palmitoyl-CoA hydrolase, suggesting that a hydrophobic environment is advantageous for interaction between enzyme and substrate in vivo. The maximum activity in the presence of mixed micelles is discussed in relation to a model where mixed micelles are regarded as artificial membranes to which the enzyme may adhere in an equilibrium with the monomer substrate and detergent in the monomer form. It is suggested that intracellular membranes may resemble mixed micelles in equilibrium with detergent-active substrates such as palmitoyl-CoA.

A low-cost integrator for preparative ultracentrifuges

Abstract An integrator is described for the measurement of the time integral ∫ 0 t rpm 2 dt in preparative ultracentrifuge where linearity exists either (a) between tachometer generator ac voltage amplitude and rpm (e.g., Sorvall RC2-B) or dc voltage and rpm, or (b) between the square-wave frequency from the tachometer generator and rpm (e.g., Beckman L2-65B). The construction and the precision levels of an integrator for Sorvall RC2-B preparative ultracentrifuge in the range 0–10,000 rpm and for Beckman L2-65B preparative ultracentrifuge in the range 0–40,000 rpm are described.

On the polydispersity of bovine adrenal chromaffin granules Analytical differential centrifugation in isotonic homogeneous medium

Abstract The polydispersity of chromaffin granules of bovine adrenal medulla homogenates has been analyzed by analytical differential centrifugation in homogeneous media. Adrenaline and noradrenaline were determined by automated ion-exchange column chromatographic preseparation using ninhydrin as the subsequent detection reagent. 1. 1. Two populations of storage granules were detected in the large granule fraction, containing about 95% of the catecholamines in the homogenate, by sedimentation in a medium containing 0.25 M sucrose whether adrenaline or noradrenaline was used as the specific marker. The average sedimentation coefficents at 4°C ( s 4, B ) were estimated to s H = 12 440 ± 1850 S and s L = 3910 ± 280 S for the heavy and light population, respectively. The relative proportion of the two populations was estimated to be 65 ± 9% ( s L and 35 ± 8% ( s H ) . 2. 2. Several lines of the chromaffin granules in both types of adrenal medullary cells, i.e., their degree of maturation.

On the polydispersity of mitochondria in tissue homogenates and the determination of the average sedimentation coefficients of mixed populations of mitochondria.

Abstract The sedimentation profile (sediterm) of subcellular particles in homogenous media depends on the average sedimentation coefficient ( S value) and the size distribution. The present study has focused on the two common types of polydispersity, i.e., (i) a variable standard deviation in a normal (Gaussian) size distribution, and (ii) two populations of partieles with defined S values and size distributions. Theoretical considerations and experimental data indicate that rat liver mitochondria have a normal size distribution, ( r = 0.391 ± 0.063 μ m ) with much smaller standard deviation than previously assumed (σ = 0.118 μm) based on isokinetic gradient centrifugation and electron microscopy. Sedimentation of a mixture of rat liver and guinea pig ileal mitochondria having the S values 17,040 S and 5640 S, respectively, gave the expected profile (sediterm) of two populations of particles. Their S values were estimated to be identical to those obtained when the individual mitochondrial populations were sedimented. The ratio between the populations (based on the assay of marker enzyme) was found to be identical to the expected value.

Sedimentation coefficient, buoyant density, morphology, and optimal recovery of mitochondria from the ileal mucosa of guinea pigs.

Marker enzymes have been used to determine the average sedimentation coefficient (s-value) of mitochondria from the ileal mucosa of guinea pigs in isotonic sucrose medium. From the determined s-value, 5590 ± 320S, a procedure has been developed allowing optimal recovery of these mitochondria by differential centrifugation. Transmission electron micrographs of the isolated mitochondria show well-preserved mitochondria. Scanning electron microscopy reveals a homogenous population with low average diameters, 0.61 ± 0.09 μm (short axis) and 0.68 ± 0.09 μm (long axis). These values are consistent with the low s-value and the low measured buoyant density, D2020=1.167 g/cm3.

Inhibition of dopamine β-mono-oxygenase by non-ionic detergents of the triton X-series

Since the discovery that dopamine P-mono-oxygenase (3,4-dihydroxyphenylethylamine, ascorbate-oxygen oxidoreductase (P-hydroxylating), EC 1.14.17.1) is distributed equally between the soluble matrix phase and the inner membrane phase of the adrenal chromaffin granules [l], the non-ionic detergent Triton X-l 00 has been frequently used for the assay of the latent forms of this enzyme [l-S]. In some cases Triton X-100 has been reported not to affect the enzymic activity [ 1,5], whereas others have found a rather high degree of inhibition by this detergent [2,4]. During our studies on latent dopamine fl-mono-oxygenase activity in the bovine adrenal chromaffin granules, we have observed that Triton X-l 00 markedly inhibits the enzymic activity of matrix preparations [6]. In order to get a more clear idea of the mechanism of this inhibition and to find the most useful detergent for the assay of latent enzyme activity the effect of a series of related non-ionic detergents have been studied using highly purified dopamine /3-mono-oxygenase.