147.
31. Haurowitz, Felix, The Chemistry and Function of Proteins, Academic
Press, New York, (1963).
32.
Setlow, Richard B. and Ernest C. Pollard, Molecular Biophysics,
Addison-Wesley Publishing Company, Reading, Mass. (1962).
,
DR. GOLL: Thank you, Harry, f o r t h e very c l e a r p i c t u r e
you havo given us concerning some of t h e rather d i f f i c u l t techniques which axe available t o study fibrous proteins. E a r l i e r
t h i s morning you heard a discussion of t h e structure of muscle.
I t h i n k you w i l l f i n d t h a t many of t h e s t r u c t u r a l c h a r a c t e r i s t i c s
o r s t r u c t u r a l features found in a muscle c e l l axe those which
we would o r d i n a r i l y expect i n a normal c e l l . A muscle c e l l has
a c e l l membrane, cytoplasm, sarcoplasmic reticulum o r endoplasmic
reticulum, mitochondria, nuclei -- many of t h e same s t r u c t u r a l
features which you would expect i n an ordinary c e l l . The
presence of t h e myofibril makes muscle and muscle c e l l s a highly
specialized c e l l , and Bob Cassens outlined f o r us some of t h e
unique features of muscle c e l l s . Dr. Szent-Gyorgyi then went
i n t o t h e c h a r a c t e r i s t i c s of t h e proteins, especially t h e prot e i n s which make up t h e myofibrils. W
e learned some of t h e
unique s t r u c t u r d c h a r a c t e r i s t i c s proceeding t o t h e molecular
l e v e l i n our discussion, p a r t i c u l a r l y i n t h e case of myosin,
about which t h e r e appears t o be more information than about
t h e other two major proteins found i n t h e rqyofibril. Then our
last speaker t h i s morning, Dr. Snyder, outlined f o r us i n
general terms t h e methods which are available t o separate and
s t u d y , t h e fibrous proteins which make up the bulk of muscle.
I think we're j u s t about r i g h t on time, so we should
have approximately f i f t e e n t o twenty minutes l e f t f o r discussion.
Are t h e r e any questions from t h e f l o o r ? I might add t h a t you
please state your name and your i n s t i t u t i o n before asking a
question f o r t h e benefit of our recorder.
DR. PAUL: I am Pauline Paul f r o m t h e University of
California. I am very much i n t e r e s t e d i n t h e problem of prot e i n separations and i n looking through t h e l i t e r a t u r e you f i n d
t h e sort of thing t h a t Dr. Szent-Gyorgyi mentioned concerning
a t e n p e r cent stroma content of rabbit muscle. Then you look
at t h e kind of thing that has been done with the beef round
muscles where you f i n d somewhere i n t h e neighborhood of maybe
two t o t h r e e p e r cent connective tissue. I am curious about
what else comes i n t o t h i s stroma t o bring it up. Coes anyone
have any information on t h i s ? We're t r y i n g t o match up some
of these basic d a t a and get some idea of how much we should
f i n d i n these d i f f e r e n t factions, you see, before w e s t a r t .
DR. GOLL: This i s an i n t e r e s t i n g topic. Perhaps
Dr. Szent-Gyorgyf would care t o comment on it. I understand
t h e nature of t h e proteins i n t h e Z-band i s not altogether
known and t h i s m a y contribute i n p a r t t o t h e disparity.
148.
DR. SZERC-GYORGYI: I am afraid it will depend on what type
of muscle you are using. You c e r t a i n l y w i l l have p r o t e i n i n t h e
Z-band. People suspect t h a t p a r t of it may be due t o tropomyosin,
but you may have various amounts of connective t i s s u e . I don't
t h i n k one can give a straightforward answer about what you will
f i n d o r what you should expect.
DR. PAUL: W e l l , t h e t h i n g I ' m puzzled about i s t h a t t h e
rabbit source has about t e n p e r cent stroma p r o t e i n on t h i s kind
o f a basis. The beef round muscles which would be expected t o have
a l o t more connective t i s s u e seem t o have a l o t less.
they are using d i f f e r e n t methods of separation.
Of course
DR. SZENT-GYORGYI: It would depend e n t i r e l y on what
method of extraction you use. You m a y be employing methods f o r
e x t r a c t i n g a c t i n o r actomyosin o r tropomyosin. But i f you use
20$ urea t h e p e r cent of soluble p r o t e i n will c e r t a i n l y be much
l a r g e r than i f you use simply high ionic strength KCI. If you
use potassium iodide of some s o r t you can make an e x t r a c t i o n of
95$ t o 96%. So it e n t i r e l y depends on how you define your ext r a c t i o n , how you i d e n t i f y your p r o t e i n s and then you have t o
f i n d out where t h e s e p r o t e i n s come from.
DR. PAUL: Has t h e r e been any work t o t r y and i d e n t i f y
what they c a l l these stroma p r o t e i n s ?
DR. SZENT-GYORGYI: Not at all. Part of t h e stroma
p r o t e i n f r a c t i o n m a y be tropoqyosin, which was c a l l e d a stroma
p r o t e i n about f i f t e e n years ago. Now it i s c a l l e d a f i b r i l l a r
p r o t e i n o r a p r o t e i n of t h e myofibril. It i s not associated
with t h e sarcolemma at least, and now we s t a r t t o suspect it
m a y be a c t u a l l y part of the Z-band but it i s a long process,
you see. I t h i n k you c a l l it a stroma p r o t e i n i f you don't
know anything about it.
(Laughter)
DR. T € W " :
I am from Oscar Mayer & Company. I
would l i k e t o d i r e c t a question t o D r . Szent-Gyorgyi. A t r i g o r
mortis how constant do you consider t h e stoichiometric combinat i o n of t h e m l e c u l a r r a t i o s of a c t i n and myosin, and can t h i s
be influenced by t h e physiological condition of t h e muscle
t i s s u e at t h a t t i m ?
DR. SZENT-GYORGYI: Well, t h e amount of a c t i n and myosin
i n t h e muscle i s not a question. I t h i n k t h e question i s what
are t h e number of cross linkages present during r i g o r mortis, and
I have t o speculate here. It has been concluded by others, t h a t
t h e development of r i g i d i t y , or rigor, coincides with t h e d i s appearance of ATP. It develops only when t h e A!TP l e v e l decreases.
I cannot answer your question d i r e c t l y , however, I would l i k e
t o know t h e answer.
DR. TAKI: T a k i , from t h e University of Florida. I am
a l i t t l e b i t confused about t h e method of e x t r a c t i o n for t h e
149.
d i f f e r e n t muscle proteins. Different people seem t o use d i f f e r ent procedures f o r extraction of t h e d i f f e r e n t protein coqonents,
such as myofibrillar proteins. What i s t h e difference i n t h e
extraction, whether we use, s a y pH 6 , o r an ionic strength of
0 . 1 o r 0.51 Are we extracting t h e same protein or not? Also,
concerning sarcoplasmic protein extraction, should we use water
o r buffer f o r t h e extraction of these proteins? What i s our
reference? What i s the standard procedure, o r i s n ' t there any?
DR. SNYIER: I think you h i t on it. The trouble i s we
don't have any reference, and t h e r e i s no standard procedure.
You have t o consider t h e f a c t t h a t t h e r e are many, many proteins
involved. I don't t h i n k it i s surprising t h a t each of these
proteins would require j u s t a l i t t l e b i t of change i n ionic s t r u c t u r e o r pH o r temperature o r some of t h e other conditions t o get
them out exclusively i n r e l a t i o n t o all of t h e other proteins t h a t
are i n muscle. I don't think t h i s i s anything t o worry about.
It i s t o be expected. But of course t h e problem i s t o f i n d t h e
r i g h t condition f o r t h e protein you want, and t h i s i s s t r i c t l y
quesswork as f a r as I know.
DR. TAKI: Ik we g e t a l l t h e protein out of t h e muscle,
a l l of t h e myofibrillar protein out, f o r instance, using a c e r t a i n
volunve at a c e r t a i n strength o r not? Using t h e micro-kjildahl
determination we s a y t h a t 16% of muscle i s protein. Is t h i s
determination true? CO we get all t h i s protein out of t h e muscle?
DR. SNYDER:
DR. GOLL:
Maybe D r . Go11 could answer t h a t .
I didn't hear t h e question.
DR. SNYDER: I j u s t don't know. I c a n ' t say whether
you can get a l l of t h e rnyofibrillar proteins out of muscles. It
depends on how you grind t h e thing up t o start with, and d i f f e r ent workers are using d i f f e r e n t mthods, yet they all a r r i v e at
almost t h e same answer.
DR. GOLL: They have used numerous d i f f e r e n t solutions.
I believe that complete extraction of myofibrillar protein has
been demnstrated by using strong K I extracting solutions. This
i s your best bet, i f you want quantitative extraction of t h e
myofibrillar proteins.
DR. TAKT: How i s one sure t h a t he g e t s a l l of the
protein out of t h e muscle?
DR. SNYDER: I don't t h i n k you would be sure, not at
t h e state of t h e knowledge we have r i g h t now.
are sure.
DR. SZENT-GYORGYI:
DR. GOLL:
You measure it. That i s how you
You measure what i s l e f t ?
150.
DR. SZENT-GYORGYI: No, you measure the ATPase a c t i v i t y
of t h e e n t i r e muscle and then t h e A!TPase a c t i v i t y of t h e extracted
protein. If these are not t h e same, then you have not extracted
all of the myofibrillar protein f o r almost a l l of t h e ATPase
a c t i v i t y i s i n t h e myofibrillar protein f r a c t i o n . If you have not
extracted a l l of t h e protein, then you check your e x t r a c t procedure. You know t h a t extract procedure does not destroy ATPase
a c t i v i t y . By t h i s procedure you can measure, somewhat, how comp l e t e your extraction has been. With o t h e r proteins, i t ' s a b i t
tougher, but it can be done. If you do t h i s measurement, how
much t h e r e was and how much is remaining, then you know. But f o r
extracting t h e other myofibrillar proteins you need d i f f e r e n t
procedures.
DR. TAKI: W e l l , i n your c l a s s i f i c a t i o n of muscle prot e i n s , there i s a general statement t h a t you can extract a l l of
these proteins by using a buffer of high ionic strength, about
0.5 o r so. If one used t h i s strength, should one expect t o get
a l l these proteins?
DR. SZENT-GYORGYI: If you cut t h e muscle up before
extraction. If you don't cut it up, you don't expect anything,
o r very little-sayt ten per cent of what i s there. By varying
t h e degree t o which t h e muscle i s minced before extraction, you
may change t h e amount of p r o t e i n extracted considerably. But
whether completely extracted o r not, t h a t depends upon t h e muscles
used, how you do your experiments, how long you extract and so on.
But you can determine what i s present i n t h i s type of muscle and
what i s l e f t after extraction. If you don't determine t h i s ,
nobody guarantees you t h a t you get 100 per cent extraction either.
DR. GOLL: J u s t a couple mre questions.
DR. WANDERSTOCK: Dr. Szent-Gyorgyi, I wonder i f you
would elaborate on t h e most current evidence describing t h e
mechanics of r i g o r .
DR. SZENT-GYORGYI: As f a r as r i g o r mortis i s concerned,
I t h i n k t h e best study i s s t i l l S c h i l l e r ' s , where they followed
t h e appearance of r i g o r and measured muscle metabolism during
t h e development of r i g o r o r r i g i d i t y of t h e muscle. They found
t h a t it was correlated only with t h e disappearance of ATP, and
I think I am correct i n saying t h a t t h e simplest p i c t u r e i s t h a t
r i g o r i s due t o formation of actomyosin, because of t h e f a c t
t h a t AC
!P disappears. ATP present i n the r e s t i n g s t a t e of muscle
i s necessary t o keep t h e l i n k s between a c t i n and myosin separated,
and you have got t o have enough ATP, of course, t o make it
contract. Muscle cannot be stretched i n r i g o r . The muscle w i l l
r a t h e r break than s t r e t c h . What happens later i s d i f f i c u l t t o
s a y . It i s probably due t o some proteolytic changes.
DR. MULLINS: Mullins, from Louisiana. I would l i k e
t o d i r e c t a question t o Dr. Snyder. I s it possible t o i s o l a t e
r e t i c u l i n from collagen?
151.
DR. SNYDER: I am going t o pass on that one. The expert
i s up on t h e podium as f a r as collagen i s concerned. I n my own
estimation, r e t i c u l i n hasn't been well characterized. I don t
have information on it myself.
DR. GOLL: I would s a y it would be extremely d i f f i c u l t .
Perhaps you could make d i f f e r e n t use of t h e thermostability of
protein.
I t h i n k at t h i s point t h a t I w i l l t u r n t h e program
back t o D r . Henrickson.
DR. HENRICKSON: It appears t h a t we have only spectators i n t h e
o t h e r room. The reciprocators are a l l i n t h i s room. I don't know where
Chairman Briskey i s . Are there any announcements, Quinn?
DR. KOLB: We are very pleased t o have t h e Usinger Sausage people
serve as hosts t o t h e c h a r t e r members of t h e American Meat Science Associat i o n t h i s noon at 12:OO o'clock over at L o w e l l H a l l .
DR. HENRICKSON:
We are adjourned u n t i l 10:15.
(Recess)
DR. HENRICKSON: Gentlemen, Prof. Quinn Kolb has an announcement
before we proceed w i t h t h e program. Quinn?
PROF, KOLB: Thank you, Bob. We have one thing t h a t I hope we
can g e t across t o you, i f we can t a l k t o t h e coffee drinkers and o t h e r s t o o .
When we assigned you parking places w e hoped you would continue t o use those
parking places t h a t were assigned t o you. If your c a r i s i n another spot
you m a y w e l l be i n a place where t h e owner of t h a t parking lot may have
you removed at your expense. So t h i s c r e a t e s a l i t t l e problem t h a t we
t h i n k we can a l l e v i a t e by working with you, and so we won't pick out i n dividuals but i f your c a r i s not i n t h e place o r i g i n a l l y assigned t o you,
would you t r y by noon o r s h o r t l y after t o get it back i n t h a t o r i g i n a l
spot. W e have a couple of f r a t e r n i t i e s who feel t h a t using t h e i r spot perhaps j u s t i s n ' t exactly Hoyle. So i f you folks who perhaps have changed
your parking place could get back i n your o r i g i n a l spot, it would help t h e
problems of our parking committee a g r e a t deal and then we perhaps wouldn't
have t h e problem of t r y i n g t o l o c a t e your c a r i n one of t h e p o l i c e l o t s
here i n t h e c i t y . So we want you t o enjoy your s t a y here. The o t h e r
announcement we want t o make again, i n case you missed it, was t h a t all of
you people and your guests t h a t you rnay have here are i n v i t e d t o t h e noon
luncheon and I pointed t h i s out t o you before t h a t t h e Usinger people are
pleased t o serve as h o s t s t o t h e chaster members and t h e i r guests of t h e
American Meat Science Association. So we w i l l look f o r you at 12:OO
o'clock over at L o w e l l H a l l and we wish you would get t h i s message t o a l l
of your guests and f r i e n d s here. The wives are not i n v i t e d t o t h e luncheon
t h i s noon.
152.
One o t h e r announcement, tonight we are serving w e l l over 300 at
t h e banquet at t h e Memorial Union and we would urge you t o go over as soon
as you can after t h e session i s f i n i s h e d t h i s afternoon. We are going t o
t r y and serve at 6:OO o'clock, on t i m e , and our f o l k s at t h e Union are
quite punctual. We w i l l need your cooperation t o g e t i n t h e building up t h e
stairs and i n t o t h e main h a l l at 6:OO o'clock. Very f i n e . Enjoy yourself
t h i s afternoon and t h i s evening.
DR. €ENRICKSON: Thank you, Quinn. Proceeding with our program,
you note t h a t t h e o t h e r selected t o p i c w a s physiological phases of t h e basic
research on muscle t i s s u e . To head t h i s section i s Robert Sayre of t h e
Western U t i l i z a t i o n Research and Development Division of ARS, USDA at
Albany, California.
DR. SAYRJ3: Thank you. I would l i k e t o t a k e t h i s opportunity t o
express my appreciation t o t h e o t h e r members of my comrmtttee f o r t h e i r
excellent help and advice during t h e development of t h i s program. D r . Fred
Deatherage, Dr. Harold Hedrick, Dr. Paul Lewis and Dr. J i m Stouffer.
We are q u i t e f o r t u n a t e t o have with u s Dr. John Whitaker from t h e
University of California. He i s p r e s e n t l y on a one year sabbatical leave
and i s working i n t h e Department of Chemistry at Northwestern University.
D r . Whitaker received h i s Ph.D. degree under Dr. Eeatherage at Ohio S t a t e
and after a t o u r i n the army has been at t h e University of C a l i f o r n i a at
Davis. H i s Ph.D. work w a s i n t h e l i n e of p r o t e i n h y d r o l i s i s on ion exchange
resins, and work since then has been with p r o t e o l y t i c enzymes. A t t h i s
time it gives me g r e a t pleasure to present Dr. John Whitaker.
DR. WHITmR:
Thank you, D r . S a p e .
############