A method for the preparation of skeletal muscle sarcolemma

Westort

1969

SUMMARY– Sarcolemmae are usually identified solely by morphological characteristics. We have determined several chemical and enzymic properties of sarcolemmae from chicken breast muscle prepared by homogenization of aged muscle in dilute CaCl2 solution, washing 4 times in NaCl‐histidine at pH 7.4, extraction with water buffered to pH 7 and isolation by differential centrifugation on a discontinuous sucrose gradient. The phospholipid content of the sarcolemmae was low, representing only 2 to 3% by weight compared with the 20 to 35% usually found in membraneous systems. This discrepancy may be due to the relatively small proportion of plasma membrane in the sarcolemma.Analyses indicate little contamination by nuclei or mitochondria. The sarcolemmae have, like the microsomal fraction, high contents of RNA and glucose‐6‐phosphatase activity. The sarcolemma is either rich in these elements or is contaminated by other subcellular elements, such as the transverse tubules (T system), which are. The sarcolemmae display a Mg+2‐ activated ATPase activity which is typical for membraneous systems. Lactate dehydrogenase was shown to be associated with the sarcolemmae. Whether this represents the situation in vivo or is an artifact of preparation is not clear. The sarcolemmae are capable of binding soluble LDH.

Section: The Sarcolemmamentioning

confidence: 98%

Sarcolemmae from Chicken Skeletal Muscle. 2. Properties

Westort

1969

“…The sarcolemma of the striated muscle cell, however, can be isolated with a remarkable retention of form and this, along with its importance in the contractile process, has led to its preparation in several laboratories (Kono et al, 1961;McCollester, 1962;Rosenthal et al, 1965 ;Abood et al, 1966;Westort et al, 1966).…”

Section: Sarcolemmae From Chicken Skeletal Muscle I Preparationmentioning

confidence: 99%

“…In some procedures muscle cell segments are washed with strong salt solutions (Kono et al, 1961 ;Abood et al, 1966) ; and in others, moderate salt concentrations and an incubation at 37°C with a limited pH range are used (McCollester, 1962 ;Rosenthal et al, 1965).…”

Section: Sarcolemmae From Chicken Skeletal Muscle I Preparationmentioning

confidence: 99%

Sarcolemmae from Chicken Skeletal Muscle. 1. Preparation

Westort

1969

SUMMARY– The outer sheath, or sarcolemma, of the muscle cell plays an important role in the contractile process and may, therefore, be significant in the changes which occur in muscle post‐mortem. It has been suggested that a breakdown in a cytoskeleton of the cell is necessary before the contractile proteins become accessible to water and extractable.One procedure for preparing sarcolemmae involves homogenization of the muscle in CaCl2 solution, washing with NaCl‐histidine, and an incubation for 30 min at 37°C. Several factors which govern the ability to prepare sarcolemmae from chicken breast muscle largely free of other cellular components have been studied in this report. These include CaCl2 concentrations during homogenization, NaCl and histidine concentrations during the washes, the pH of the wash solutions, the number of washes required, the necessity of the incubation, and the pH of the extracting water.The purpose of these experiments was to find a procedure which would allow variation of the preparatory conditions so that the association of enzymes with the sarcolemma could be studied. It is known that the association of enzymes with particulate fractions of the cell are sensitive to environmental conditions. A necessary step to allow restrictive conditions of preparation, such as low pH and ionic strengths, to be used was aging of the whole excised muscle for 4 hr at 0–4°C. The function of this aging process is not known. It does not appear to involve solubilization of protein nor major changes in several classes of phosphorus compounds.

“…It has been suggested that contraction of muscle is initiated by depolarization of the T-system with a concomitant release of calcium from the terminal sacs of the sarcoplasmic reticulum (Constantin et al, 1965). Several workers have reported on methods useful in the preparation of sarcolemmae from muscle cells (Abood et al, 1966;Kono et al, 1961;McCollester, 1962 ;Rosenthal et al, 1965;Westort et al, 1966). Methods of preparation have been of two types.…”

Section: Introductionmentioning

confidence: 99%

“…In an attempt to use a procedure more nearly physiological, McCollester (1962) employed dilute salt solutions and near neutral pH values with an incubation of the cell segments at 37°C or a longer one at 4°C. Modifications of this procedure have been made (Rosenthal et al, 1965;Westort et al, 1966).…”

Section: Introductionmentioning

confidence: 99%

Role of Flavin Adenine Dinucleotide in Stabilization of Cytoskeleton of Chicken Muscle Cell

Stanley

Sawyer

1968

SUMMARY Aging of muscle had previously been shown in our laboratory to increase the propensity of properly treated muscle cell segments to empty on extraction with water. It has been suggested that this emptying is caused by breakdown of a cytoskeleton, and, further, that this cytoskeleton is stabilized by flavin adenine dinucleotide (FAD). Due to the possible relationship of cytoskeletal breakdown to quality changes in meat post‐mortem, the role of FAD in the preservation of cytoskeletal structure in chicken breast muscle was studied. No significant differences in FAD decomposition or extraction were found between samples handled in a manner such as to produce very large differences in the extent of emptying, the measure of cytoskeletal breakdown. Similarly, adding FAD to suspensions of muscle cell segments could not inhibit emptying under conditions where the supernatant fraction of a muscle homogenate could. It was concluded that FAD plays no role in the stabilization of the cytoskeleton of chicken breast muscle.