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Techniques for Study of Protein Synthesis 283. TECHNIQUES FOR STUDY OF PROTEIN SYNTHE'SIS F. C. PARRISH, JR. IOWA STATE UNIVERSITY ............................................................................... A study of skeletal muscle protein biosynthesis requires a number of both simple and sophisticated techniques because it involves a study of cellu- lar subunits and molecules and their reactions. Many of these techniques h?-ve already been successfully used to acquire a considerable body of knowledge about muscle biochemistry and ultrastructure. Consequently, adaptation of these techniques provides a strong potential for obtaining some very inter- esting and illuminating information about muscle protein biosynthesis and development. Other aids in the study of muscle protein biosynthesis hwe been the knowledge supplied by the molecular biologist on the biosynthetic mecha- nism of cells and on the behavior of actin and myosin in solution. Recent research, utilizing many of the same techniques as those used for the study of protein chemistry and structure, has provided us with some very profound information about myof ibrillogenesis of mammalian skeletal muscle. Naturally, the subject of myofibrillogenesis is of much interest and concern to the muscle biologist and meat scientist because the proteins of the myofibril are the ones that most directly affect muscle growth and development, contraction, rigor mortis, meat tenderness, water binding, emulsification and human nutri- tion. If we are able to make substantial improvement in the quantitative, qualitative, and nutritive characteristics of meat we must turn to the use of techniques that will yield information on how muscle proteins are synthesized and formed into meat at the cellular and molecular level. With knowledge gained at these levels we can then begin to regulate those mechanisms and compounds affecting muscle growth and composition. The purpose of this paper is to present information about certain preparative and analytical techniques used in the study of myofibrillar protein biosynthesis. The kind of information obtained, rather than the mechanics of the technique, will be emphasized. Also, the techniques listed are not intended to be dl inclusive, usually only one investigator will be cited, although these same or similar techniques have been used by other researchers, and in some instances some of these techniques could be classified as both preparative and analytical. Furthermore, many specific techniques are embodied within a general technique. First of all, I would like to describe very briefly several general techniques. For a detailed description, one should refer to the materials and methods section of the original paper. Then I would like to specifically refer to the morphological techniques of Allen and Pepe (1965) and Fischman (1967) and the biochemical techniques of Heywood and coworkers (1967, 1968a, 1968b, 1968c, 1969). When consideration is given to the large number and variety of constituents and reactions involved in protein synthesis, it becomes very obvious why the techniques for protein biosynthesis are numerous and sophisticated (Figure 1). Techniques become even more complex when a study is made of the protein biosynthetic machinery of eucaryotes as opposed to procaryotes. The three major features observed in Figure 1 are replication, transcription and translation. Replication or exact duplication of DNA is essential because DNA contains the genetic information for the synthesis of specific protein molecules. DNA polymerase is the enzyme responsible for 284. the catalysis of the exact replication of DNA. Transcription describez the events in which mRNA takes the genetic information from DNA in the nucleus and carries it to the ribosomes in the cytoplasm. The combinatim of rrXNA and ribosomes in the cytoplasm constitute the elements upon which the pAroce;s of protein synthesis takes place. Transcription is catalyzed by the enzyne, RNA polymerase. The translational process involves the arrival of mino acids in the form of activated amino acyl-t-RNA, the sequential assembly of these mino acids, and the formation of peptide bonds. Eventually the formation of a protein molecule will take place. Obviously, then, the study of a system of this kind requires a number of preparative and analytical techniques. Table 1 contains a list of preparative techniques essential for a study of muscle protein biosynthesis. Quantitative analyses refers to those techniques required to principally determine nitrogen, protein, and nucleic acids. Munro and Fleck (1966c,b; 1969) have presented excellent reviews of these techniques. Isolation media and homogenization are neces- sary for proper sample collection. These techniques vary according to investigators but the techniques of Heywood et d. (1967) have been success- fully employed to prepare highly active polysomes.-- Differential and density gradient centrifugation are indispensable tools for isolating and fraction- ating ribosomes (Heywood --et al., 1967). Tissue culture represents an excellent technique for the study of --in vitro muscle cell differentiation, development, and protein synthesis. Holtzer and his coworkers (Bischoff and Holtzer, 1970; Nameroff and Holtzer, 1969; Okajaki and Holtzer, 1965; 1966) have successfully employed these techniques. Recently, Reporter (1969) used tissue culture to study conver- sion of histidine to 3 methylhistidine and its turnover rate in actin and myosin. Analytical techniques are enumerated in Table 2. Spectrophotometry is very useful in determining the kind and quantity of nucleic acids (Heywood --et ELL., 1967). Electrophoresis has been very useful in the identification of the synthesized products, particularly myosin (Heywood et a.l., 1967). It has also been useful in the study of ribosomal proteins7LG --et al., 1969; Spiegel --et al., 1970). Chromatography has been useful in identification (Heywood and Rich, 1968) and purification (Baril and Hermann, 1967) of myosin. Kabat and Rich (1969) have successfully used autoradiography to show that muscle fibers are the major site of muscle ribosome synthesis. Fluorescence microscopy has provided information about myogenesis (Okajaki and Holtzer, 1965; Coleman and Coleman, 1968). Electron microscopy of muscle differentia- tion and development have been elegantly done by Allen and Pepe (1965) and Fischman (1967) . Message (1968) has also investigated muscle development using electron microscopy. Enzyme assays are vaLuable techniques because they provide evidence for the appearance or existance of certain components during differentiation and development. AMP-deaminase (Kendrick-Jones and Perry, 1967), ATPase (Trayer and Perry, 1966; Obinato, 1969), DNA polymerase (O'Neill and Strohman, 1969), creatine kinase (Coleman and Coleman, 1968) , RNA polymerase (Marchok and Wolff, 1968; Breuer and Florini, 1966; Florini and Brewer, 1966) and thymidylate kinase (Scholl et al., 1968) are examples of enzymes associated with muscle development. The technique of isotopic amino acid incorporation is essential in determining the ability of ribo- somes to synthesize proteins (Heywood --et al., 1967). Now I would like to discuss in somewhat more detail the structural and biochemical evidence for myofibrillogenesis. Allen and Pepe (1965), with the electron microscope, were able to show that thin filaments (actin) appeared before thick f ilanent s (myosin) in developing chick embryo muscle cells (Figure 2). The thin filaments appeared in stage 16 (Figure Za) and by stage 18 large numbers of wavy thin filaments appeared (Figure 2b) and also thin filaments were observed interspersed with thick filaments (Figure Zc). Thick filaments were only observed in aggregates of thin and thick filaments. Also at this stage the first appearance of large poly- ribosomes were observed. In stage 20 (Figure 3) an increase of thin and thick filaments into myofibrils occurred. After four days of development, stage 24 myotond cells represent every stage of development (Figure 4) . The typical banding pattern of mature myofibrils are observed at stage 28 (Figure 5). Figure 6 shows the polyribosome structure containing 70-75 ribosomes thought to be associated with thick filaments (myosin) synthesis. A number of questions still remain unanswered about the appearance and relationship of certain constituents and particularly about the time of morphological appearance of thin and thick filaments. Fischman (1967), although disagreeing with Allen and Pepe (1965) about the time of appear- ance of thin filaments, also has done some very interesting work on chick embryo muscle cell differentiation and development with the electron microscope. Figure 7 shows a cross section through regions of three adjacent developing muscle fibers and their constituents. To be noted is the hexagonal array of thick and thin filaments in the myofibril and the free thick and thin filaments in the cytoplasm. A longitudinal section of this same material shows the various stages of myofibril formation (Figure 8). Free actin and myosin filmnts as well as two different stages of myofibril formation can be clearly observed. The first stage (Mfl) contains thick and thin filamnts, but without visible Z lines, and a later stage (Mfz) contains 2, 1, and A bands. Thin filaments were always observed to be in a 7 to 10 fold excess of thick filaments. Fischman (1967) concluded that thin filaments did not appear before thick filaments, although all of his work was done on 12-day chick embryo muscle. Figure 9 shows a fully formed myofibril with
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