Idea Transcript
114.
M O L E C U L A R STRUCTURE OF T H E M Y O F I B R I L R. 6. C A S S E M S
The purpose of t h i s paper i s t o acquaint t h e audience with t h e myofibrillar s t r u c t u r e of muscle. I n order t o appreciate t h e f i n e structure of t h e myofibril however, one must begin with t h e gross structure of muscle, and then proceed t o the molecular l e v e l keeping i n mind how t h e large u n i t s are dependent f o r t h e i r structure upon t h e smaller u n i t s . Succeeding papers t h i s morning will be concerned with discussing the actual muscle proteins. The remarks i n t h i s paper w i l l be confined t o s t r i a t e d muscle. Several reviews concerned primarily w i t h striated vertebrate muscle have served as useful guides i n t h e preparation of t h i s manuscript. (see l i s t of general references).
Gross s t r u c t u r e of muscle A muscle, as seen with t h e naked eye o r w i t h low power magnification, i s separated i n t o d i f f e r e n t levels of organization by t h e connective t i s s u e components. A t h i c k layer of connective t i s s u e , termed t h e epimysium, covers o r invests any given muscle. The epimysium p r o j e c t s i n t o t h e muscle and divides it i n t o d i f f e r e n t orders c a l l e d t h e bundles o r f a s c i c u l i w h i c h are merely bundles of muscle fibers or c e l l s . The connective t i s s u e projections defining t h e muscle bundles, are termed t h e perimysium. The endomysium i s a d e l i c a t e connective t i s s u e extension from t h e perimysium which Projects into t h e bundles and envelops t h e separate muscle fibers. The endowsium i s a network which c a r r i e s c a p i l l a r i e s , and it should not be confused with t h e sarcolemma of t h e muscle fibers (Ham and Leeson 1961, Bloom and Fawcett 1962). The muscle f i b e r i s a long, c y l i n d r i c a l multinucleated c e l l which may vary i n thickness from 10 t o 100 u o r w r e and can be 1 t o 40 mm. i n length. The muscle f i b e r i s invested by a t h i n membrane c a l l e d t h e sarcolemma. Muscle nuclei are elongated o r ovoid and generally are found at t h e periphery of t h e f i b e r i n t h e space between t h e sarcolemma and t h e myofibrils--this c h a r a c t e r i s t i c serving as a c r i t e r i o n of i d e n t i f i c a t i o n i n striated muscle. The sarcoplasm i s the cytoplasm of t h e muscle c e l l i n which t h e myofibrils or c o n t r a c t i l e elements are embedded. Mitochondria, l i p i d inclusions, Golgi apparatus and other granules are present i n the sarcoplasm but are not o r d i n a r i l y v i s i b l e without t h e aid of special techniques.
The c h a r a c t e r i s t i c c r o s s - s t r i a t e d appearance of skeletal muscle arises from t h e orderly alignment of t h e anisotropic and isotropic segments of t h e q y o f i b r i l s and resembles t h e stacking of l i g h t and dark segments. Myofibrils are t h e elongated i n t r a c e l l u l a r c o n t r a c t i l e elements which measure about 1 u i n thickness, and as j u s t mentioned give rise t o t h e chara c t e r i s t i c banded o r s t r i a t e d p a t t e r n of s k e l e t a l muscle. With polarized
115. l i g h t t h e discs t h a t seem darker w i t h t h e ordinary l i g h t microscope are anisotropic (bifreringent) while those t h a t appear l i g h t e r with t h e l i g h t microscope are i s o t r o p i c . As a r e s u l t of these properties, t h e darker d i s c s are c a l l e d A bands and t h e l i g h t e r d i s c s are c a l l e d I bands. I n addition, a dark l i n e termed t h e Z l i n e b i s e c t s t h e I band. A l i g h t area i n t h e center of t h e A band i s known as t h e H band and bisecting t h i s band i s a dark M l i n e . Less frequently a dark s t r i a t i o n termed t h e N band may be seen midway between t h e Z l i n e and t h e A band. The sarcomere, o r u n i t of muscle structure, is t h e area between two adjacent Z l i n e s (Ham and Leeson 1961, Bloom and Fawcett 1962, Structure and Function of m s c l e . Vol. I . 1960).
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The sarcoplasmic reticulum was described by Veratti (1902) using t h e l i g h t microscope, as an extensive network surrounding t h e myofibrils. More recently Voyle and Lawrie (1963) have published some excellent p i c t u r e s of t h i s system i n beef muscle. This brief discussion has indicated t h e basic features of muscle structure which can o r d i n a r i l y be observed within t h e limits of ordinary l i g h t miscroscopy. Coincident with t h e increased usage of electron microscopy i n t h e biological sciences, tremendous progress has been m a d e i n t h e knowledge of muscle s t r u c t u r e . The advent of t h i n sectioning, shadow casting and negat.ive staining, and t h e constant improvement i n h i s t o l o g i c a l techniques f o r electron microscopy have a l l added f u r t h e r impetus t o t h e progress i n t h i s f i e l d . A detailed description of t h e u l t r a s t r u c t u r e of t h e sarcolemma, sarcoplasmic reticulum, mitochondria and t h e myofibrils w i l l follow, but no f u r t h e r information w i l l be given concerning s t r u c t u r a l d e t a i l s of t h e nuclei, t h e Golgi apparatus o r other sarcoplasmic inclusions. The sarcolemma
A description of t h e f i n e s t r u c t u r e of t h e sarcolemma would be incomplete without f i r s t c i t i n g t h e e a r l y work of Schwann (1839) and Bowman (1840). Bowman described a delicate, structureless, tubular membrane which invested each striated muscle fiber o r multinucleated c e l l ; Schwann had previously deemed it t o be t h e muscle c e l l membrane. The sarcolemma (Robertson 1956, Porter and Palade 1957, Fawcett and Selby 1958) has been shown t o be about 100 A t h i c k with t w o peaks of density about 50-60 A apart (each peak of density i s about 25 A wide) between which l i e s a trbngh of lesser density. This s t r u c t u r e i s known as t h e u n i t membrane and i s c h a r a c t e r i s t i c of animal c e l l membranes i n general. Mauro and Adams (1961) have described four regions at t h e periphery of t h e muscle c e l l . They are as follows, beginning at t h e o u t e m s t region: (1) a mesh of extremely d e l i c a t e filaments which appear t o lack s t r i a t i o n s , (2) another filamentous layer composed of collagen filaments--the density o r concentration of both these layers f l u c t u a t e s widely, (3) a uniform, s t r u c t u r e l e s s layer measuring 300-500 A width. This layer i s commonly known as t h e basement membrane o r ground substance and i s usually r i c h i n polysaccharide, and, (4) t h e plasma membrane. The problems associated with passage of ions and molecules through t h e sarcolemma, and t h e possible r o l e of t h e sarcolemma i n coupling e x c i t a t i o n t o contraction o f f e r i n t e r e s t i n g fields of research.
116. The Sarcoplasmic Reticulum The sarcoplasmic reticulum i s a complicated system of tubules and vesicles located i n t h e sarcoplasm and enmeshing t h e myofibrils. The papers of Bennett and Porter (1953) and Porter and Palade (1957) have been i n s t r u mental i n i n i t i a l l y describing t h e sarcoplasmic reticulum. Other e a r l y s u b s t a n t i a l contributions include t h e r e s u l t s of B e p e t t (1955, 1956, 1958), Edwards anfl Ruska (1955), Edwards e t al. (1956), Sjostrand (1956), Andersson (1957), Sjostrand and Andersson-Cedergren (1957). The membranes of the sarcoplasmic reticulum conform t o t h e u n i t membrane design and resemble t h e endoplasmic reticulum of other c e l l s . Muscatello and Andersson-Cedergren (1962) have discussed t h e function of t h e endoplasmic reticulum as a s i t e of protein biosynthesis--this i s quite generally accepted i n c e l l s such as those of l i v e r and pancreas. However, they have noted t h a t t h e sarcoplasmic reticulum of muscle i s evidently d i f f e r e n t i n morphological organization and physiological properties f r o m t h e endoplasmic reticulum of other c e l l s . Relatively f e w granules o r p a r t i c l e s are found i n muscle and they a r e not regularly attached t o t h e sarcotubular membranes.
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A description of t h e arrangement of sarcoplasmic reticulum i n rat s a r t o r i u s muscle (Porter and Palade 1957) w i l l be given i n order t o b e t t e r p i c t u r e one type of arrangement t h e membranes might assume (these authors suggested t h r e e different, but s t i l l similar, arrangements of t h e sarcoplasmic reticulum i n rat sartorius, larval salamander and rat cardiac muscle). Porter and Palade (1957) suggested t h a t t h e reticulum was represented as being disposed i n two alternating lace-like sets of anastomosing tubule-like channels, resembling bracelets, surrounding t h e myofibrils. One s e t w a s located with a plane of symmetry at each 2 band and extended as f a r on each side as t h e N band o r A - I junction. Alternating between each of these members were somewhat wider sets of i n t e r l a c i n g tubules, symmetric a l at t h e planes of t h e M bands, lying opposite t h e A bands. They d i d not f i n d any longitudinal members connecting adjacent sets across the A - I junctions. Between each alternating set, at l e v e l s close t o t h e A - I junction, Porter and Palade discerned rows of minute sausage-shaped vesicles. It i s t h i s combination of opposed c i s t e r n a l d i l a t i o n s and intermediary vesicles t h a t c o n s t i t u t e s t h e "triad". One must r e a l i z e of course that t h i s description i s given only as an example, and t h a t i n r e a l i t y t h e arrrangement, complexity and extent of t h e sarcoplasmic reticulum v a r i e s g r e a t l y among d i f f e r e n t muscles and species.
Further investigation of t h e small transverse vesicles (Fawcett and Revel 1361, Revel 1962) showed t h e middle element of t h e t r i a d t o be a tubule running at r i g h t angles t o t h e long axis of the fiber. It was r e cently demonstrated (Franzini-Armstrong 1963, Fahrenbach 1963) i n skeletal muscle t h a t t h e transverse tubular system i s connected with t h e sarcolemma and t h a t t h e space within it i s i n communication with t h e e x t r a c e l l u l a r space. It has also been shown i n sheep heart muscle (Simpson and Oertelis 1962) and i n human and rabbit myocordial c e l l s (Nelson and Benson 1963) t h a t t h e transverse tubular system of t h e reticulum i s i n contact with t h e e x t e r i o r of t h e c e l l . The experiments of A. Huxley and Taylor (1955a, b) and A. Huxley (1957a,b) demonstrated t h e existence i n s k e l e t a l muscle of a s t r u c t u r e which could transmit a signal leading t o contraction f r o m t h e sarcolemma
117. transversely t o t h e i n t e r i o r of t h e f i b e r . I n frog semitendinosus they applied t h e t i p of an electrode micropipette t o d i f f e r e n t locations on t h e surface of t h e sarcolemma, and applied s t i n d a t i n g currents very much weaker than t h e one required f o r i n i t i a t i n g propagated a c t i v i t y of t h e membrane of t h e f i b e r . No c o n t r a c t i l e response could be observed at many places on t h e surface of t h e fiber, but c e r t a i n d i s c r e t e l o c i did give a response. Those spots which did give a response were found along t h e middle of t h e I band, centering on a projection of the 2. A t these spots t h e two adjacent half-sarcomeres contracted f o r a number of microns i n t o t h e fiber. These r e s u l t s are extremely i n t e r e s t i n g i n l i g h t of t h e suggestions t h a t t h e transverse tubules communicate through t h e sarcolemma at t h e l e v e l of the triad (i.e. from 2 l i n e t o A-I junction). F u r t h e m r e an association e x i s t s between the amount o r characteri s t i c s of t h e sarcoplasmic reticulum and t h e speed o r use of t h e muscle. Peachy and Huxley (1963) found t h a t t h e myofibrils of twitch fibers from f r o g were always w e l l delineated by sarcoplasmic elements; no triads were found i n any of t h e slow fibers but were present i n each of t h e twitch fibers. Revel (1962) has reported t h a t t h e extremely f a s t acting B a t crycothyroid muscle possesses a very well-developed sarcoplasmic reticulum. Fahrenbach (1963) has indicated t h a t an elaborate sarcoplasmic reticulum i s coxmionly associated with fast-acting muscles, and t h a t t h e complexity of t h e tubular system i s a function of myofibrillar geometry, whereas the degree of development of t h e c i s t e r n a l system i s related t o contraction speed of t h e muscle. Fahrenbachs (1963) evidence indicated t h a t with increasing contraction speeds, the c i s t e r n a l p o r t i o n of t h e system becomes more extensive while t h e p o t e n t i a l impulse conducting system of tubules remains e s s e n t i a l l y constant i n i t s degree of development. Slautterback (1963) has shown t h a t t h e sarcoplasmic reticulum i s very much less prominent i n cardiac myofibers of t u r t l e s kept at'0 C . as compared t o those exercised at 370 C. (the heart rate of t h e cold t u r t l e s 1/10 t h a t of t h e warm t u r t l e s ) . The p a r t i c u l a t e relaxing f a c t o r of muscle consists of fragments of t h e sarcoplasmic reticulum (Nagai e t al. 1960, Ebashi and Lipmann 1962). Whether t h e relaxing granules arise from t h e tubular middle elements of t h e triad, t h e outer elements of t h e triad o r t h e rest of t h e reticulum remains a question.
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H i l l (1960, 1962) has reported t h a t adenine nucleotides and creatine phosphate were both strongly concentrated at d e f i n i t e positions within t h e I band. H i l l ( i n press, J . Cell. Biol.) has more recently shown t h a t t h e adenine nucleotides, concentrated i n t h e I band s i t e , l i e not within t h e f i b r i l s but i n t h e spaces between them, and almost c e r t a i n l y i s a component of t h e reticulum.
The importance and complexity of t h e sarcoplasmic reticulum i s evident. Two functions are generally assigned t o t h e sarcoplasmic reticulum--the t r a n s f e r of mzhabolites o r t o a c t as an impulse conducting system.
118.
The Mitochondria The mitochondria of muscle have been described by Palade (1952, 1953) and many other researchers. The d i s t r i b u t i o n of mitochondria i n muscle i s variable but they are usually found longitudinally oriented i n t h e spaces between myofibrils, immediately under t h e sarcolemma or clustered at t h e poles of nuclei. Quite often they are found i n a horizontal plane at t h e l e v e l of t h e 2 l i n e o r A-I junction. The double membrane and c r i s t a e of mitochondria have been extensively described, but recently a higher degree of organization has been elucidated i n t h e mitochondria of Blowfly f l i g h t muscle (Smith 1963). Negative stained preparations of disrupted s a r c o s o ~ srevealed t h a t both t h e outer limiting membrane and c r i s t a e membrane bore large numbers of small p a r t i c l e s . The p a r t i c l e s consisted of a subspherical "headt' and c y l i n d r i c a l " s t a l k " and were arranged on t h e membranes e i t h e r randomly o r were collected i n t o c i r c u l a r o r elongated groups. Mitochondria contain both Krebs cycle and electron t r a n s f e r systems. Krebs cycle enzymes are s i t u a t e d i n the matrix o r at l e a s t are not t i g h t l y bound. Molecules of t h e respiratory chain and associated phosphorylating sytems are intimately organized i n t o replicated u n i t s o r assemblies which appear t o represent subportions of organized s t r u c t u r a l elements o r membranes of mitochondria.
The Myofibril We have previously indicated t h e s i z e and appearance of t h e myofibril as revealed by l i g h t microscopy, and will now proceed t o discuss i n more detail t h e structure of t h e myofibril as revealed by electron microscopy. The c l a s s i c a l work of H. Huxley ( H u l e y 1953, 1957, Huxley and Hanson 1957) as well as excellent refforts by other researchers. (Bennett and Porter 1953, Hodge 1955, Sjostrand and Andersson-Cedergren 1957) have revealed many of t h e u l t r a s t r u c t u r a l details of s t r i a t e d muscle myofibrils. The basic structure of t h e rqyofibrils, which i s responsible f o r i t s c r o s s - s t r i a t e d appearance, has been shown t o be due t o i t s composit i o n of overlapping arrays of two kinds of filaments. The array of t h i c k filaments (about 110 A diameter) i s dense and anisotropic and composes the A band, while t h e array of t h i n filaments (about 50 A diameter) i s present alone i n t h e l e s s dense I band. The density of t h e A band i s greatest where t h e two sets of filaments overlap. The H zone, o r l i g h t e r zone, i n t h e center of t h e A band i s t h e area where only thick filaments are present. I n cross section, t h e t h i c k filaments of vertebrate skeletal muscle are arranged hexagonally with t h e centers of the t h i c k filaments being 450 A apart. Where t h e arrays overlap, each t h i c k filament i s encircled by s i x t h i n ones and each t h i n filament i s shared by t h r e e t h i c k ones. Each of t h e t h i c k filaments bears a large number of regularly spaced short lateral projections termed "bridges". There are six longitudinal rows of pro ject i o n s t h a t are staggered so t h a t a projection occurs every 60-70 A along t h e t h i c k filaments. The rows are arranged so t h a t they occur opposite t h e s i x t h i n filaments. These cross-bridges have been implicated i n cont r a c t i o n as t h e t h i c k and t h i n filaments move p a s t one another. Although some evidence has been offered t h a t t h e A band shortens during contraction i n limulus muscle (EeVillafranca and Marschhaus 1963), recent extensive work by Page and Huxley (1963) has shown t h a t l i t t l e i f any change occurs i n filament lengths of striated muscle during contract i o n . They concluded t h a t filament lengths were t h e same i n both r e s t i n g and excited muscles at a l l sarcomere lengths greater than 2 . 1 u .
119. The arrangement of filaments during t h e formation o f contraction bands (A. Huxley and Niedergerke 1954, 1958, A. Hwcley and Gordon 1962, Hoyle and McAlear 1963 and Cassens et al. 1963) requires f u r t h e r research. The association of t h e filaments w i t h t h e muscle proteins, myosin and actin, has been examined by d i f f e r e n t methods. D i f f e r e n t i a l extraction of t h e proteins has been reported by Hanson and Hwrley (1957), and r e a c t i v i t y of t h e myos$n and a c t i n t o fluorescent l a b e l l e d antibodies has been studied
by Szent-Gyorgi and Holtzer (1963a,b). Present evidence indicates t h a t myosin i s located i n t h e A band and a c t i n i n t h e I band--from t h e p o s i t i o n of t h e filaments t h i s would lead t o t h e conclusion t h a t myosin i s located i n t h e t h i c k filaments and a c t i n i s located i n t h e t h i n filaments. The Z l i n e
The c o n t i n u i t y of t h e t h i n filaments through t h e Z l i n e has been a subject of controversy, but evidence on s t r i a t e d muscle by Danish workers (Knappeis and Carlsen 1962) has shown t h a t t h e myofilaments terminate at t h e Z l i n e . The I band filaments terminate as rod-like projections on e i t h e r side of t h e Z l i n e w i t h one I band filament on one side lying between two I band filaments on t h e opposite side. I n cross section through t h e Z l i n e region, t h e I band filaments s i t i n t h e corners of squares while oblique Z l i n e filaments form t h e s i d e s of squares. The t e t r a g o n a l p a t t e r n formed by t h e Z l i n e filaments i s r o t a t e d 45O w i t h respect t o t h e tetragonal p a t t e r n formed by I band filaments on both s i d e s of t h e Z l i n e . This
s t r u c t u r a l arrangement i s i n t e r p r e t e d t o indicate t h a t each I band filament on one s i d e o f t h e Z l i n e faces t h e c e n t e r of t h e space between four I band filaments on t h e opposite s i d e of t h e Z region and t h a t t h e interconnection from each I band filament i s formed by f o u r Z l i n e filaments. Suggestions have been made (H. Huxley 1963) t h a t these Z region filaments are formed from tropoqyosin and t h a t t h e tropomyosin furthermore extends along t h e t h i n filaments of t h e I band. Hanson and Lowy (1963) suggested that each I band filament i n vivo contains two tropomyosin threads as well as two a c t i n threads and t h a t t h e f o u r threads continue i n t o t h e Z l i n e as t h e f i n e filaments previously described.
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The myofilaments
Hwcley (1963) has succeeded i n fragmenting muscle t o t h e filament level and has then examined these preparations w i t h t h e e l e c t r o n microscope. H e has characterized t h e t h i c k filaments as being 100-120 A i n diameter and predominantly 1.5--1.6 u long with tapered ends. Because of imperfect separation these t h i c k filaments o f t e n have t h i n filaments attached t o them. These filaments are bound together v i a t h e "bridges" o r projections mentioned earlier. There were a l a r g e number of these projections on t h e surface of t h e t h i c k filaments but they appeared absent f r o m t h e c e n t r a l zone ( .15- .2 u) A s l i g h t thickening w a s observed i n t h e middle of t h i s bare zone. The t h i n filaments were shown t o be 60-70 A i n diameter w i t h a v a r i a b l e length of 0.5-1.0 u o r longer. Some t h i n filaments were found s t i l l attached t o t h e Z region and extended about 1 u on e i t h e r side. Both the separated and attached t h i n filaments had a c h a r a c t e r i s t i c beaded appearance. T h i s beaded appearance (Hanson and Lowy 1963) of t h e t h i n f i l a ments arises f r o m t h e inference t h a t they c o n s i s t of two h e l i c a l l y wound strands composed of sub-units which appear t o be a l i k e and approximately spherical. There i s evidence t h a t each of t h e globular subunits seen i n t h e e l e c t r o n microscope represents one a c t i n monomer.
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120. If p u r i f i e d myosin i s p r e c i p i t a t e d from .6 M KC1 by lowering t h e concentration t o .2--.l u, rod-shaped p a r t i c l e s become v i s i b l e with dimensions of about 150 A diameter by 2 u long. The p a r t i c l e s are spindle-shaped with a rough surface due t o a large number of projections. Many of t h e p a r t i c l e s show t h e r e l a t i v e l y bare c e n t r a l shaft previously mentioned.
The s i z e and shape of t h e protein molecule and how it may give rise t o t h e c h a r a c t e r i s t i c s t r u c t u r e of t h e filaments w i l l be covered i n t h e following paper by Dr. A. G . Szent-Gygrgyi. A brief discussion of t h e s t r u c t u r e of muscle as revealed by l i g h t microscopy was given. The u l t r a s t r u c t u r e of t h e sarcolemma, sarcoplasmic reticulum, mitrochondria and myofibrils was discussed. Research concerning t h e s t r u c t u r e o r structural changes occuring i n muscle t i s s u e o f f e r s atremendous f u t u r e i n t h e f i e l d of meat research.
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Epimysium
Figure 1 Cross section of muscle showing connective tissue components. (Ham and Leeson 1961).
125.
I
I BAND
t
A BAND
-
H ZONE
-1
n
Z LINE
+0 . 8 4 ~ -
I BAND
z LINE
0.w
I.5p _IC1 MYOFIBRIL SHOWING BAND PATTERN AT RESTING LENGTH Figure 2
Magram of nruscle band pattern.
Figure 3 Three-dimensional d r a g of a block of muscle.
(Bennett 1956).
126.
Figure 4 The Sarcolemma (Cassens Univ. of Wisconsin Thesis 1963).
A
H A
7-
L
Figure 5 Diagrammatic reconstruction of sarcoplasmic reticulum surmmdbg one myofibril (Franzini-Armstrong 1963)
127
Figure 6 Muscle section showing sarcoplasmic reticulum and "triads" (Fwcett and Revel 1961).
128.
Sarcoplasm Sarco/emma
2atr’cuIar f i b e r s
Endomys;a/ co//aS.cn and fine e l a s t i n f i b c 9 t - s Figure 7
Diagrammatic s k e t c h of muscle f i b e r .
-
Meat Products 1960).
I
m
z
Figure 8
A
m H n
------
(The Science of Meat and
I
-
z
Diagram showing arrangement of muscle qyofilaments.
129.
,
Figure 9 Section of pig muscle showing bending pattern (Cassens, R. G , E. J. Briskey and W. G. Hoekstra., J. Food S c i . 28, 680 (1963).
r
c
F i w e 10 Diagram of Z line region (Knappeis and Carlsen 1962).
130.
DR. WILL: Thank you, Bob, f o r that interesting paper on t h e structure of muscle. If you hare any questions about these presentations w e would appreciate it i f you would hold them u n t i l t h e discussion period following the first three papers t h i s morning. Having now learned something about t h e s t r u c t u r a l features of muscle -fibrils, it appears logical t o t u r n our attention t o t h e fibrous proteins which make up these myofibrils. We are especially pleased and honored t h i s morning t o have someone who has contributed as much as any single person to our present stake of k n o w l eQe of muscle myofibrillar proteins. Among t h e numerous contributions which Dr. Szent-Gyorgyi has made i n t h i s area i s t h e discovery, i n t h e e a r l y 1950ts, of t h e rapid p m t o l y t i c cleavage of nlyosln t o l i g h t and heavy merowosin. Dr. Szent-Gyorgyi's mst recent publioations, 86 indicated by Dr. Cassens i n h i s paper, have d e a l t with the fluorescent antibody labeling of muscle proteins. It is a privilege t o welcome at t h i s time Dr. Andrew G. Szent-Gyorgyi of the Dartrmuth Medical School, who w l l l discuss "Proteins of t h e Myofibril". Dr. Szent-Gyorgyi. (Applause)