ISOLATION
A N D PROPERTIES OF MUSC LS LYSOSOMES*
.
JOHN W . C B I R D
DEPARTMENT OF PHYSIOLOGY
RWERS UNIVERSITY
I n a classical series of papers beginning with B e r t h e t and
(1951) and summarized by d e Duve (1959) it was demonstrated
t h a t i n o s m o t i c a l l y p r o t e c t e d homogenates of r a t l i v e r c e r t a i n
h y d r o l y t i c enzymes w i t h a c i d pH optimum d i s p l a y e d t h e phenomenon of
l a t e n c y , The l a t e n c y p r o p e r t y was demonstrated by t h e fact t h a t t h e
enzymes were p r a c t i c a l l y u n r e a c t i v e t o t h e i r substrates when c o n d i t i o n s
t o keep t h e p a r t i c l e s i n t a c t were maintained. The enzymes became
maximally a c t i v e toward s u b s t r a t e s only when t h e homogenates were
s u b j e c t e d t o physico-chemical t r e a t m e n t s which would d i s r u p t membranes,
On t h e basis of d i f f e r e n t i a l c e n t r i f u g a t i o n experiments, it was
proposed t h a t t h e hydrolases belonged t o a d i s t i n c t p a r t i c l e , and
t h e name "lysosome" w a s proposed (de me, e t a l . , 1935). de Duve
(1959) suggested t h a t t h e lysosomal enzymes were p r i m a r i l y c a t a b o l i c
i n f u n c t i o n , and t h a t being s e g r e g a t e d from surrounding cytoplasm by
a bounding membrane t h e y were prevented from d i g e s t i n g t h e i r h o s t c e l l ,
It was a l s o stated, u n f o r t u n a t e l y , that under c e r t a i n c i r c m s t a n c e s
t h e lysosomes acted as " s u i c i d e bags" by releasing t h e i r c o n t e n t s
i n t o t h e c e l l and t h u s r e s u l t i n g i n c e l l d e a t h (de D w e , 1963). I
say "unfortunate,
because t h e " s u i c i d e bag concept" masked f o r
s e v e r a l y e a r s t h e profound s i g n i f i c a n c e of t h e lysosome i n n o m a 1
c e l l physiology.
de h v e
'I
Many d i s c i p l i n e s have c o n t r i b u t e d t o t h e development of t h e
lysosome concept, e s p e c i a l l y i n d e f i n i n g lysosomal f u n c t i o n s with
r e s p e c t t o t h e normal economy o f t h e c e l l . It i s now thought t h a t
t h e s e v e r a l t y p e s o f lysosomes form a complex i n t r a c e l l u l a r d i g e s t i v e
system, whereby macromolecules brought i n t o t h e c e l l , o r "worn o u t "
c e l l u l a r components are organized i n t o vacuoles, which i n t u r n f u s e
with primary lysosomes formins: secondary lysosomes. Digestion o c c u r s
i n t h e secondary lysosomes and t h e end products are t r a n s p o r t e d i n t o
t h e c e l l ' s cytoplasm by p a s s i v e d i f f u s i o n o r a c t i v e t r a n s p o r t (de Duve
and Wattiaux, 1966). In some c e l l s t h e end products o f d i g e s t i o n may
Undigestible material
be s e c r e t e d from t h e c e l l as u s e f u l by-products.
1s e i t h e r e x c r e t e d by e x o c y t o s i s , o r may remain i n t h e c e l l i n t h e
form o f a residual body such as l i p o f u s c i n g r a n u l e s i n muscle and
nerve t i s s u e (Novikoff, 1962).
*
.
Presented st t h e 24th Annual Reciprocal Meat Conference o f t h e
Anerican M e a t Science Association, 1971
68
S e v e r a l workers have enphasieed, however, that t h e lysosomes and
o t h e r component8 o f t h e v a c u o l a r a p p a r a t u s a r e not developed t o t h e
same d e g r e e of complexity i n a l l c e l l t y p e s (de Dwe, 19678 Novikoff,
1962; and S t r a u s , 1967). The v a c u o l a r a p p a r a t u s is a morphological
s p e c i a l i e a t i o n which is normally p r e s e n t i n t h o s e cells nhose
p h y a i o l o g i c a l f u n c t i o n requires a n e f f i c i e n t and economical catabolic
machinery. A s u r v e y o f t h e number of lysosomes found i n d i f f e r e n t
t y p e s of cells shows that lysosomes are more numerous i n e p i t h e l i a l
c e l l s o f organs having a phagocytic, a b s o r p t i v e , o r s e c r e t o r y
f u n c t i o n (Straw, 1967) where a h i g h l y developed c a t a b o l i c machinery
is e s s e n t i a l t o t h e c e l l o r organ. I n s k e l e t a l muacle fibers, which
do n o t have phagocytia or s e c r e t o r y f u n c t i o n s , microscopy had f a i l e d
t o demonstrate t h e presence of lysosomes or lysosome-like g r a n u l e s .
The failure t o f i n d lysosome-like morphological e n t i t i e s i n normal
n u e c l e c e l l s sparked a c o n t r o v e r s y a few y e a r s ago as t o t h e o r i g i n
o f a a i d h y d r o l a s e a a t i v i t y i n muscle t i s s u e . Tappel (1966) stated
t h a t t h e particles d e s c r i b e d by bioohemical s t u d i e s m e t o t a l l y
c o n t r i b u t e d by t h e non-muscle c e l l a of nurscle t i s s u e . Furthermore,
i n c e r t a i n myopathies a dramatic i n c r e a s e i n macrophages, l e u c o c y t e s
and lymphocytes occurs which is c o i n c i d e n t w i t h i n c r e a s e s i n t o t a l
lyaosomal ensyme a c t i v i t i e s . S i n c e t h e c e l l s that comprise t h e
c o n n e c t i v e t i s s u e components of muscle a r e known t o have among t h e
h i g h e s t c o n c e n t r a t i o n s of lysosomal enzymes, Kohn (1969) has c a l c u l a t e d
that under c e r t a i n s i t u a t i o n s t h e y could account f o r a l l t h e lysosomal
enzyme a c t i v i t y found i n muscle t i s s u e .
Van Flaet, e t a l . (1968) r e p o r t e d that lysosome-like s t r u c t u r e s
were not found i n n o r n a l s k e l e t a l muscle f i b e r s of r a b b i t s . P e l l e g r i n o
and F’ranzini (1963) e m p h a t i c a l l y stated t h a t no lysosome has e v e r been
detected i n normal muscle fibers by e l e c t r o n microscopy. Maier and
Z a i n a n (1965) concluded that a c i d phosphatase, t h e lysosomal marker
enzyme most f r e q u e n t l y used i n h i s t o c h e m i c a l s t u d i e s , could not be
demonstrated i n normal rat s k e l e t a l n u s a l e f i b e r s , Smith (1965)
r e p o r t e d t h a t t h e h i s t o c h e m i c a l r e a c t i o n s f o r acid phosphatase i n
normal r a b b i t muscle is s c a n t y and c o n f i n e d t o bloed v e s s e l s ( i . e . ,
e n d o t h e l i a l connective c e l l s ) .
The experimental c e n c l u s i o n s of t h e morphologists and biochemists
have been d i f f i c u l t t o r e c o n c i l e . Thua, t h e experiments I w i l l
d i s c u s s are an a t t e m p t t o r e s o l v e t h e e x i s t i n g c o n t r o v e r s y over t h e
n a t u r e of t h e lysosomes i n normal s k e l e t a l musale. I must a l s o
confess my p r e j u d i c e s . My b a s i c working h y p o t h e s i s I s that a l l
normal musole p r o t e i n catabolism is i n i t i a t e d and completed by
lysosomal a c i d p r o t e a a e s , which axe indigenous t o t h e muscle f i b e r s .
I an n o t aware o f any c a r e f u l l y c h a r a c t e r i z e d o r proven n e u t r a l
p r o t e a s e s i n muscle t i s s u e . The a l k a l i n e p r o t e a s e of Kescalka and
Miller (1960) and t h e n e u t r a l p r o t e a s e of Kohn (1965) have r e c e n t l y
been shown, i n an e l e g a n t s t u d y by Willemot, I a l a n n e and B e r l i n g u e t
(1969), t o be x a n t h i n e oxidase. I am c e r t a i n l y n o t opposed t o
n e u t r a l p r o t e a s e s , as the- occurence would s i m p l i f y matters g r e a t l y ,
I l o o k forward w i t h great a n t i c i p a t i o n t o a paper c u r r e n t l y &press
by Noguchi and Kandatsu, o f t h e University of Tokyo, on t h e p u r i f i c a t i o n and p r o p e r t i e s o f a new a l k a l i n e p r o t e a s e i n rat s k e l e t a l
muscle
Chem., personal communication from Noguchi),
I should a l s o r e p o r t that P r o f e s s o r B, Sylven demonstrated a
p u r i f i e d lysosomal c a t h e p s i n B from tumors t h a t had a broad pH
spectrum from 3.5 t o 8.0, w i t h good a c t i v i t y at pH 7.0, u s i n g urea
denatured E d e s t r i n as s u b s t r a t e ( I n t e r n a t i o n a l Research Conference
o f Lysosomes, Louvain, 1970)
(e,
e.
There have been o c c a s i o n a l s u g g e s t i o n s t h a t a c i d pH is
"unphysiological" and t h a t a c i d hydrolases can, t h e r e f o r e , have
l i t t l e s i g n i f i c a n c e i n l i v i n g organisms (Barrett ' 1969). Although
t h e pH optima o f t h e s e enzymes a r e well removed from n e u t r a l i t y ,
s e v e r a l of them have been shown t o have s u f f i c i e n t a c t i v i t y a t t h e
h i g h e r pH t o be h i g h l y s i g n i f i c a n t i n t h e l o n g time-scale of
p h y s i o l o g i c a l processes. Moreover, t h e a v a i l a b l e evidence from t h e
use of i n d i c a t o r s (Rous, 1925) p o i n t s t o a n extremely a c i d pH i n t h e
d i g e s t i v e wicuoles w i t h i n phagocytic c e l l s .
For today's d i s c u s s i o n , first l e t us review t h e e s s e n t i a l
hiochemical c r i t e r i a f o r lysosomes i n muscle t i s s u e ;
Sedimentation p r o p e r t i e s (figure 1).
Using d i f f e r e n t i a l c e n t r i f u g a t i o n , and e x p r e s s i n g t h e enzyme
a c t i v i t y as mean r e l a t i v e specific a c t i v i t y o f t h e f r a c t i o n s v e r s u s
t h e i r mean r e l a t i v e p r o t e i n c o n t e n t , t h e area o f each block r e p r e s e n t i n g
a f r a c t i o n is t h u s p r o p o r t i o n a l t o t h e percentage of a c t i v i t y recovered
i n t h e corresponding f r a c t i o n , and i t s h e i g h t t o t h e degree o f
p u r i f i c a t i o n achieved over t h e homogenate (de Dwe e t al., 1955). The
f r a c t i o n s are r e p r e s e n t e d on t h e abscissa i n t h e o r d e r i n which t h e y
were i s o l a t e d , i.e., from l e f t t o r i g h t : N, n u c l e a r , M, heavy
mitochondrial$ L, l i g h t mitochondrial; P, microsomal; and S, f i n a l
s u p e r n a t a n t f r a c t i o n . It is s e e n that t h e h i g h e s t RSA is found i n
t h e l i g h t m i t o c h o n d r i a l f r a c t i o n , which has been demonstrated t o be
t h e lysosome-rich f r a c t i o n i n o t h e r t i s s u e s ,
S t r u c t u r e - l i n k e d l a t e n c y (table 1 ),
If t h e acid hydrolase is c o n t a i n e d w i t h i n a non-permeable
membrane, chemical o r p h y s i c a l f o r c e s which d i s r u p t t h i s membrane
should a l l o w a c c e s s i b i l i t y of t h e enzyme t o its s u b s t r a t e , In t h i s
s l i d e we see t h a t i n c r e a s i n g t h e d u r a t i o n o f homogenization i n c r e a s e s
t h e amount o f free a c t i v i t y , i . e . , i n c r e a s e s t h e amount o f enzyme
available t o react with substrate.
The thermal l a b i l i z a t i o n of acid h y d r o l a s e s from l i v e r , kidney
and muscle lysosomes i s shown i n figure 2. Muscle lysosomes seem
more r e s i s t a n t t o thermal i n c u b a t i o n t h a n a r e l i v e r and kidney
lysosomesr When t h e homogenates a r e incubated i n n e u t r a l o r a c i d
media, t h e rate which lysosomal enzymes are r e l e a s e d i n t o t h e nonsedimentable f r a c t i o n is s e v e r a l times greater for l i v e r and kidney
t h a n f o r muscle,
70
0
2
I
Q
0
l2
I
I
JJO
s
I
r:
0
c
c
3
d
a
N
8
z
m
i
c
Q)
A
0
C
I
-
rc)
0
Y
71
TABLE 1.
INFLUENCE OF PRELIMINARY BLENDING OF
MUSCLE ON ENZYME LATENCY
X VirTis
Aryl S u l f a t a s e
Ribonuclease
1
2
4
1
2
4
Enzyme a c t i v i t y "
l?ree
Total
101.o
59.8
75.6
106.8
33.1
117.0
106.7
58.6
60.0
62.3
39.1
65.3
Free
(%a>
59.2
64.6
100.1
56.4
65.2
104.8
Muscles were cut i n t o small p i e c e s w i t h s c i s s o r s , t h e n blended
i n 0.25 M s u c r o s e with a V i r T i s "45" homogenizer a t t o p speed
for 2 seconds, f o r t h e number of times i n d i c a t e d i n t h e table.
This was followed by t h r e e up-and-down passes i n a Dual homog e n i z e r . Homogenates were assayed f o r free and t o t a l
a c t i v i t i e s as d e s c r i b e d i n t e x t , except t h a t i n c u b a t i o n times
were 1 5 minutes,
*RNase = A O.D./mg p r o t e i n p e r h r x 10-5; a r y l s u l f a t a s e =
m p o l e s nitrocathechol/ma p r o t e i n p e r hr
.
72
pK 5.0
8
p-NPPa 8 e
f4
0
20
10
,o
20
'
pGluc
6
10
0
40
I
20
0
tu
1
2
INCUBATION TZME (Hours)
Figure 2 .
Thermal l a b i l i z a t i o n of a c i d hydrolases from l i v e r ,
kidney and muscle lysosomal p a r t i c l e s .
e, kidney; a , l i v e r ; 0, muscle (from Canonico, 1969).
The effect of freezing and thawing on lysosomes is presented i n
f i g u r e 3. A t o t a l mitochondrial p r e p a r a t i o n was frozen by immersing
t h e t e s t t u b e i n a mixture o f d r y ice and a c e t o n e , and thawed by
p l a c i n g t h e t u b e s i n a 3 8 O C i n c u b a t o r . It i s noted t h a t one f r e e z e t h a n t r e a t m e n t released 5% of t h e c a t h e p s i n a c t i v i t y , whereas
subsequent freeze-thaw c y c l e s released a n a d d i t i o n a l 10% o f enzyme.
The following f i g u r e (figure 4 ) demonstrates t h e effect o f
f'reezing rate on t h e s o l u b i l i z a t i o n of c a t h e p s i n D. Whole g a s t r o cnemius muscles were frozen by e i t h e r plunging t h e muscle and its
c o n t a i n e r i n t o l i q u i d n i t r o g e n (130°C/min, measured by a n a t t a c h e d
thermocouple) o r by a c o n t r o l l e d Linde BF-4 b i o l o g i c a l f r e e z i n g
a p p a r a t u s . The "S" under t h e second set o f bar graphs r e p r e s e n t s
experiments from shredded muscle. The r e s u l t s i n d i c a t e that t h e
lysosomes are kept i n t a c t i f t h e f r e e z i n g rates are i n t h e range o f
10C/min, e s p e c i a l l y i f t h e muscle is p r o t e c t e d by immersion i n 0.25
M s u c r o s e media, i n d i c a t e d by "M" under t h e last pair of bar graphs.
Additional experiments i n d i c a t e d that muscles fYozen a t loC/min and
s t o r e d f o r up t o 16 days under l i q u i d n i t r o g e n had no i n c r e a s e i n
solubilized cathepsin,
S t r u c t u r e - l i n k e d l a t e n c y can be demonstrated by s e v e r a l a d d i t i o n
methods, i n c l u d i n g d e t e r g e n t t r e a t m e n t s , hypotonic s o l u t i o n s , s o n i c a t i o n , and t h e use of p h y s i o l o g i c a l l a b e l i z e r s such as vitamin A .
Our first c l u e for t h e e x i s t e n c e o f lysosomes i n muscle c e l l s
came from some morphological s t u d i e s using a c r i d i n e orange (Canonico
and Bird, 1969). Koenig (1963) and A l l i s o n and Young (1964) had
presented convincing evidence that t h i s v i t a l dye w a s c o n c e n t r a t e d
w i t h i n lysosomes, c a u s i n g them t o appear as b r i g h t orange g r a n u l e s i n
f l u o r e s c e n c e microscopy, Our o b s e r v a t i o n s o f teased muscle fibers
and f r o z e n s e c t i o n s o f normal gastrocnemius muscle showed o c c a s i o n a l
f l u o r e s c e n t g r a n u l e s i n t h e p e r i n u c l e a r r e g i o n and w i t h i n t h e muscle
f i b e r s . However, a f t e r s u b j e c t i n g t h e rats t o 6 days o f s t a r v a t i o n
b e f o r e dye i n j e c t i o n , r e l a t i v e l y l a r g e numbers o f orange g r a n u l e s were
seen i n t h e p e r i n u c l e a r r e g i o n , and throughout t h e muscle fiber,
Dingle and B a r r e t t (1968) r e c e n t l y r e p o r t e d t h a t actual counts
of orange f l u o r e s c e n t g r a n u l e s i n g r a d i e n t c e n t r i f u g a t i o n f r a c t i o n s
a r e i n good agreement w i t h s p e c i f i c a c t i v i t i e s o f lysosomal enzymes,
W
e have extended t h e s e o b s e r v a t i o n s by t h e s p e c t r a p h o t o f l u o r m e t r i c
determination of a c r i d i n e orange i n c e l l f r a c t i o n s (figure 5 ) and I
t h i n k have c l e a r l y shown that t h e dye d i s t r i b u t i o n i s s i m i l a r t o t h e
d i s t r i b u t i o n p a t t e r n o f particle bound a c i d h y d r o l a s e s , M h e r m o r e ,
t h e sedimentation c h a r a c t e r i s t i c s o f lysosomal p r t i c l e s d i d not
a p p e a r t o be a l t e r e d by a c r i d i n e orange, s i n c e similar a c i d hydrolase
d i s t r i b u t i o n p a t t e r n s were o b t a i n e d from muscles o f normal and a c r i d i n e
orange-injected animals. I w i l l s a y more about t h i s l a t e r ,
Because of our biochemical and morphological evidence f o r t h e
p o s s i b l e e x i s t e n c e o f lysosomes i n muscle f i b e r s w e designed some
60
40
20
.
I
2
3
4
.TIMES FROZEN AND THAWED.
F'igure 3.
The effect o f freezing and thawing on lysosomes
(from Bond and Bird, 1966).
5
1
GAST ROCNEMIUS
CONTROL
1
FROZEN
I4
I
I
I
E
I
.
)
130
s
15
FREEZING RATES
P
I
@/MI$
-
I
I
I
M
L
4,
ACRIDINE
ORANGE
c
0
1
20
I
40
I
60
2 TOTAL PROTEIN
Figure 5.
I
I
'80
I
100
I E s t r i b u t i o n p a t t e r n o f a c r i d i n e orange
after d i f f e r e n t i a l c e n t r i f u g a t i o n of muscle
homogenate (f'rom Canonico and Bird, 1969).
77
experiments t o d i f f e r e n t i a t e t h e c o n t r i b u t i o n o f lysosomes from
v a r i o u s p o s s i b l e c e l l u l a r s o u r c e s i n s k e l e t a l muscle t i s s u e . Our
first experiments u t i l i z e d t h e t e c h n i q u e s o f r a t e - z o n a l c e n t r i f u g a t i o n
(Canonico and Bird, 1970). The experimental design is shown i n
figure 6. The d i s t r i b u t i o n o f particle bound a c t i v i t y i s shown i n
figure 7. I would l i k e t o c a l l your a t t e n t i o n t o t h e fact t h a t we
have a bimodal d i s t r i b u t i o n o f our enzyme a c t i v i t y ! a slower sedimenting peak a t f r a c t i o n 9 and a fast sedimenting peak a g a i n s t t h e
g r a d i e n t cushion a t f r a c t i o n 26. Also n o t e that our d i s t r i b u t i o n i s
heterogeneous w i t h r e s p e c t t o t h e i n d i v i d u a l enzymes, s i n c e 30% o f
t h e t o t a l a c t i v i t y of @glucuronidase, RNase and aryl s u l f a t a s e i s
found a g a i n s t t h e cushion, as compared t o 18%of t h e t o t a l c a t h e p s i n
and a c i d phosphatase.
W
e t h e n r e p e a t e d t h e s e experiments with animals i n j e c t e d w i t h
Dextran-500, which h a s been shown t o i n c r e a s e t h e d e n s i t y o f lysosomes.
The data showed a decreased a c t i v i t y i n t h e slower sedimenting peak
with a concomitant i n c r e a s e d a c t i v i t y a g a i n s t t h e cushion f o r t h e
three enzymes 3-glucuronidase, RNase and aryl s u l f a t a s e , w i t h no
change i n t h e p a t t e r n f o r c a t h e p s i n D and a c i d phosphatase, W
e
t h e n i n j e c t e d our animals with Triton-1339, which normally r e s u l t s
i n l a r g e r but less dense lysosomes, I n t h i s experiment t h e r e was a
decrease i n t h e enzyme a c t i v i t y a g a i n s t t h e cushion f o r t h e three
enzymes 3-glucuronidase, RNase and a r y l s u l f a t a s e , w i t h a g a i n no
effects on t h e o t h e r two enzymes.
The d i f f e r e n t sedimentation rates and t h e a p p a r e n t h e t e r o g e n e i t y
of t h e enzymes i n t h e s e two p a r t i c l e p o p u l a t i o n s suggested t o u s t h a t
w e were d e a l i n g w i t h more t h a n one group o f lysosomes. Furthermore,
one o f t h e s e p o p u l a t i o n s was capable o f phagocytyzing t h e i n j e c t e d
e t h e n designed some isopycnics u b s t a n c e s , while t h e o t h e r was n o t . W
zonal c e n t r i f u g a t i o n experiments, where t h e p a r t i c l e s c o u l d be
separated s o l e l y on t h e basis o f d e n s i t y . The experimental p r o t o c o l
is d e p i c t e d i n figure 8.
The equilibrium d e n s i t y histograms of t h e acid hydrolases from
normal muscle demonstrate a broad d i s t r i b u t i o n with a modal e q u i l i brium d e n s i t y o f 1. I 8 i n s u c r o s e ( f i g u r e 9). The h e t e r o g e n e i t y i n
d i s t r i b u t i o n o f t h e acid h y d r o l a s e s is f u r t h e r demonstrated by
c a t h e p s i n and acid phosphatase histograms b e i n g skewed toward l i g h t e r
d e n s i t i e s , while RNase, 3-glucuranldase and aryl s u l f a t a s e a r e skewed
toward t h e d e n s e r p o r t i o n s o f t h e g r a d i e n t . Cytochrome oxidase is
c o n c e n t r a t e d w i t h i n a narrow-density span w i t h a m o d a l d e n s i t y
s l i g h t l y greater t h a n 1.18. W
e a l s o s t u d i e d t h e enzyme muramldase
(lysozyme), a lysosomal hydrolase found i n l e u c m y t e s and macrophages,
The muramidase d a t a i s not p r e s e n t on t h i s figure, but had a bimodal
d i s t r i b u t i o n with peaks at 1.15 and 1.20.
L i t t l e rnuramidase a a t i v i t y was found a t 1.18, t h e modal e q u i l i b r i u m
d e n s i t y peak f o r t h e o t h e r lysosomal enzymes. The bimodal d i s t r i b u t i o n
o f muramidase i n t h i s s t u d y i s i n agreement w i t h t h e f i n d i n g s f o r
l e u c o c y t e s by B a g g i o l i n i , e t a l . (1969).
78
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35
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Figure 7.
10-5
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D i s t r i b u t i o n p a t t e r n o f enzymes, a c r i d i n e orange, and p r o t e i n a f t e r
f r a c t i o n a l of post-nuclear muscle homogenates by r a t e - z o n a l
c e n t r i f u g a t i o n (a2t = 410 x IO7 radZ/sec).
..
- ..
..
..-
ISOPYCNIC
CEN TR IFUGAT ION
Figure 8,
Experimental d e s i g n of isopycnic-zonal technique
(from Canonico and Bird, 1970).
..
- .
81
82
W
e t h e n r a n i s o p y c n i c s t u d i e s on our muscle t i s s u e s after
i n j e c t i n g t h e a n i m a l s w i t h Dextran-500 o r T r i t o n 1339 (figure I O ) .
After i n j e c t i n g w i t h Dextran-500, t h e m o d a l d e n s i t y o f t h e f i v e a c i d
h y d r o l a s e s was n o t s h i f t e d from t h e c o n t r o l v a l u e o f 1.18.
However,
a p o r t i o n of t h e a c t i v i t y was shifted t o t h e more dense part of t h e
gradkent. This s h i f t w a s greater f o r t h e three enlsylaes @-glucuronidase,
RNase and a r y l s u l f a t a s e , t h a n f o r c a t h e p s i n or s c i d phosphatase, and
i n d i c a t e d that a small group of l y s o s o n e s r i c h i n t h e s e enzymes were
capable o f a l t e r i n g t h e i r equilibrium d e n s i t y by t h e accumulation and
e a l s o s e e a similar s h i f t i n
s t o r a g e o f exogenous materials. W
d i s t r i b u t i o n , a l t h o u g h not as dramatic, from animals i n j e c t e d w i t h
T r i t o n 1339 (Mgure 11) with t h e d i f f e r e n c e t h a t t h e a c t i v i t y i s
s h i f t i n g t o t h e more b o n p n t p o r t i o n of t h e g r a d i e n t .
S i n c e t h e a d n i n i s t r a t i o n of exogenous m a t e r i a l s d i d n o t affect
t h e equilibrium d e n s i t y o f t h e large lysosome group, which w e
s u s p e c t e d as b e i n g of muscle fiber o r i g i n , s t a r v a t i o n was used 8s
an endogenous s t i m u l u s , t o h o p e f u l l y promate t h e development of large
secondary lysosomes (figure 12). Swift and Hruban (196b) suggested
several y e a r s ago that t h e formation of a u t o p h a g i c v a c u o l e s is a
defense mechanism of normal o e l l s t o s t a r * a t i o n . Since muscle i s
t h e major store of amino acids, after, o f c o u r s e , r a p i d d e p l e t i o n of
l i v e r and t h e free animo acid pool, it is n e t s m p r i s i n g that autophagic vaouoles would be formed f o r t h e d i g e s t i o n of muscle d u r i n g
prolonged s t a r v a t i o n . Returning t o t h e figure, we s e e that s t a m a t i o n
caused a d e c r e a s e , and o m only t r e a t m e n t c a u s i n g a d e c r e a s e , i n t h e
modal e q u i l i b r i u m d e n s i t y o f a l l f i v e a c i d h y d r o l a s e s . Cathepsin and
acid phosphatase were decreased t o 1.16 and t h e o t h e r t h r e e enzymes
t o 1.165.
Our i n t e r p r e t a t i o n of t h e i s o p y c n i c data is sumraarieed on t h e
next figure (figure 13). Considering first t h e d i s t r i b u t i o n h i s t o g r a m kbla Dextran-500 t r e a t e d a n i m a l s , a best f i t c u r v e was drawn
through t h e l e f t face o f t h e equilibrium d e n s i t y d i s t r i b u t i o n , w i t h
t h e apex u o l n c i d i n g w i t h t h e m o d a l d e n s i t y peak a t 1.18. A mirror
image of t h i s curve was r e p r o d w e d on t h e r i g h t side of t h e apex so
as t o c o n s t r w t a bell-shaped ourve. The d i f f e r e n c e between t h i s
bell-shaped c u r v e and t h e e x p e r i m e n t a l d a t a waa used t o c o n s t r u c t
a smaller bell-shaped curve. These saae larger curves fYoa t h e
Dextran-jOO data were t h e n super-imposed on t h e r i g h t f a o e of t h e
data fron t h e a n i m a l s i n j e c t e d w i t h T r i t o n 1339. Notice t h e good
f i t of t h e curve w i t h t h e e x p e r h e n t a l data. Then, small bell-shaped
curves were o b t a i n e d as b e f o r e . By t h i s t e c h n i q u e t n o groups of
lysosomes w i t h bell-shaped d i s t r i b u t i o n were r e s o l v e d . The smaller
group c o n t r i b u t e d approximately 25% of t h e a r y l s u l f a t a s e , 8-glucuronidase and RNase, 5% of t h e c a t h e p s i n D and acid phosphatase, and
almost a l l o f t h e muramidase. The larger group, on t h e o t h e r hand,
c o n t a i n s approximately 75% o f t h e 0-glucuronidase, RNase and a x y l
sulfatase, and 95% of t h e c a t h e p s i n and acid phosphatase a c t i v i t y .
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Effect o f s t a m a t i o n on t h e d e n s i t y d i s t r i b u t i o n p a t t e r n s
o f r a t muscle a c i d h y d r o l a s e s , Animals were fasted 6 days
but provided with t a p w a t e r and s a l i n e s o l u t i o n , Dotted
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Figure 13,
bell-shaped curves o f b e s t - f i t drawn through t h e e q u i l i b r i u m
d e n s i t y d i s t r i b u t i o n d a t a , o f a c i d hydrolases i n muscles o f
dextran-500 and T r i t o n N3-1339-treated a n i m a l s , T h e o r e t i c a l
curves a r e e x t r a p o l a t e d beyond l i m i t s o f g r a d i e n t (from
h n o n i c o and Bird, 1970).
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e a r e persuaded that t h e larger group o f lysosomes with a modal
e q u i l i b r i u m d e n s i t y o f 1.18 a r e t h o s e lysosomes indigenous t o t h e
muscle fiber, and t h a t t h e smaller group of lysosomes are d e r i v e d
from phagocytic c e l l s .
I would l i k e t o show a n o t h e r slide which may be o f more
i n t e r e s t t o some o f you, i n t h a t it concerns a commonly e d i b l e
muscle (figure 14). These data r e p r e s e n t a comparison o f isopycnic
zonal c e n t r i f u g a t i o n d a t a o f f i s h muscle from rainbow t r o u t acclimated
a t three d i f f e r e n t t e m p e r a t u r e s t 4 O C , 12OC, and 18OC (Milanesi and
Bird, 1971). 12OC i s t h e optimum temperature for growth and reprod u c t i o n i n t r o u t , whereas 5OC and 18OC r e p r e s e n t t h e u s u a l environmental l i m i t s f o r t r o u t , W
e see that t h e d i s t r i b u t i o n p a t t e r n s a r e
e s s e n t i a l l y t h e same f o r t h e 1 2 O C acclimated f i s h as for rat s k e l e t a l
muscle, This p a r t i c u l a r s t u d y i n d i c a t e s t h a t temperature is a l s o a n
indigenous s t i m u l u s f o r changes i n lysosome p o p u l a t i o n s , 1 2 O C
acclimated f i s h had a m o d a l d e n s i t y of 1.18 f o r t h e i r a c i d h y d r o l a s e s ;
whereas a d a p t i n g t h e f i s h t o t h e lower and h i g h e r temperatures changed
t h e modal d e n s i t i e s t o 1.16 and 1.19, r e s p e c t i v e l y ,
It i s c e r t a i n l y a l l t o t h e well and good t o have n i c e enzyme
r e c o v e r i e s , and experimental data t h a t n i c e l y f i t s t h e t h e o r e t i c a l
c u r v e s . However, t h e fact remains that t h e r e has been a c e r t a i n
amount of d i s c u s s i o n i n t h e l i t e r a t u r e concerning t h e n o t a b l e absence
of t h e usual t y p e of lysosomes i n normal s k e l e t a l muscle fibers. If
one r e a d s t h e l i t e r a t u r e c a r e f u l l y , most a u t h o r s make a s t a t e m e n t t o
t h e effect t h a t "no lysosomes were observed," which i s q u i t e
d i f f e r e n t from stating t h a t t h e r e a r e "no lysosomes i s s k e l e t a l
muscle fibers." I t h i n k t h e r e are two main hinderances i n o u r
t h i n k i n g . Most o f us come away from our biochemical t r a i n i n g
thoroughly convinced that t h e body i s one b i g p i e c e o f l i v e r , o r
On t h e o t h e r hand, t h e r e are many
more r e c e n t l y , a large E.
d i s t i n g u i s h e d l a b o r a t o r i e s i n t e r e s t e d i n t h e physiology and biochemistry
o f muscle, b u t t h e primary focus f o r t h e past couple decades has been
concerned with t h e c o n t r a c t i l e a p p a r a t u s . My p o i n t is, I don't t h i n k
a s e r i o u s e f f o r t has been made t o look f o r lysosomes per s e i n muscle
fibers,
e.
A y e a r ago, w h i l e working i n P r o f e s s o r d e Duve's l a b o r a t o r y i n
Lowain, we made some i n i t i a l a t t e m p t s i n t h i s area. I n t h a t we were
anxious t o see a c t i v e lysosomes w i t h i n normal p h y s i o l o g i c a l l i m i t s ,
we chose t h e s t a r v a t i o n model as being t h e most l i k e l y p r o s p e c t .
Before looking a t t h e e l e c t r o n micrographs l e t us first t a k e a look
at t h e s t a t e o f t h e animal d u r i n g t h i s s t a r v a t i o n process ( f i g u r e 15).
W
e see a 40% decrease i n wet weight of t h e gastrocnemius muscle f o r
t h e e i g h t day period; v e r y l i t t l e , i f any change in t h e r a t i o of
gastrocnemius weight t o body weight, i n d i c a t i n g that t h e decrease i n
gastrocnemius weight is n o t d i s p r o p o r t i o n a t e t o t h e o v e r a l l d e c r e a s e
i n body weighti v e r y l i t t l e change i n gastrocnemius p r o t e i n concent r a t i o n d u r i n g t h e first 6 days o f s t a r v a t i o n , t h e n a decrease i n
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ir, s x c i f i c a c - i v i t y o f n u s c l e a c i d hydrolases ixin:
ci slays o f foocl r e s r r i c t i o n .
p r o t e i n c o n c e n t r a t i o n ; and f i n a l l y , t h e changes i n dry weight of t h e
gastrocnemius, As noted, t h e r e was a s i g n i f i c a n t i n c r e a s e i n d r y
weight of t h e muscle between t h e t h i r d and f o u r t h day. I am n o t
s u r e w h a t caused t h i s muscle dehydration, b u t because of t h e r a p i d
o n s e t and r a p i d t e r m i n a t i o n , I am tempted t o s a y that it may be a
hormonal response. As you w i l l s e e i n l a t e r s l i d e s , we t h i n k that
muscle autophagy is i n i t i a t e d around t h e f i f t h day o f s t a x v a t i o n .
It would be n i c e t o f i n d a s p e c i f i c hormone t o r e g u l a t e or t r i g g e r
autogaphy w i t h i n normal p h y s i o l o g i c a l limits!
The n e x t figure (figure 16) shows t h e changes i n s p e c i f i c
a c t i v i t y of our enzymes d u r i n g t h i s p e r i o d of s t a r v a t i o n . W
e see
l i t t l e change i n t h e t h r e e enzymesB-glucuronidase, a r y l s u l f a t a s e
and R N a a e for t h e first 5 t o 6 days, t h e n a d e c r e a s e i n a c t i v i t y .
Acid phosphatase and c a t h e p s i n on t h e o t h e r hand, have a d i s t i n c t
i n c r e a s e between days 1 and 5 , t h e n a sharp d r o p i n a c t i v i t y . Our
p r e l i m i n a r y i n t e r p r e t a t i o n is t h a t t h e r e i s an a c t i v a t i o n of pree x i s t i n g enzyme, o r s y n t h e s i s of new enzyme d u r i n g t h e s e first 5
days; t h e n , f u s i o n of t h e lysosome w i t h a newly formed autophagic
vacuole, and i n i t i a t i o n of i n t r a c e l l u l a r d i g e s t i o n . With prolonged
s t a n r a t i o n , t h e r e may be i n v a s i o n of t h e t i s s u e by macrophages,
b r i n g i n g i n a d d i t i o n a l lysosomes,
I n o u r next experiment (figure I?), we followed t h e f r e e
a c t i v i t y changes i n our muscle t i s s u e from starved a n i m a l s . With
our homogenizing t e c h n i q u e s , muscles from normal a d u l t a n i m a l s
usually have around 50% free a c t i v i t y , I n t h i s experiment t h e r e w a s
l i t t l e change i n fYee a c t i v i t y u n t i l t h e f i f t h day of s t a m a t i o n ,
where it i n c r e a s e d t o approximately 65%, and on t h e s i x t h day was as
e a t t r i b u t e t h e i n c r e a s e i n free a c t i v i t y t o a n
high as 95%. W
i n c r e a s e i n s i z e , and hence a n i n c r e a s e i n f r a g i l i t y o f t h e lysosomes
t o t h e homogenizing procedure, of c o u r s e , t h e o t h e r p o s s i b i l i t y
remains t h a t some of t h e enzyme is s o l u b i l i z e d i n v i v o ,
I n o u r e l e c t r o n microscope s t u d i e s w i t h whole t i s s u e s , t h e medial
head of the gastrocnemius muscle w a s used from animals s t a m e d 6 days.
The t i s s u e was placed i n a s p r i n g d e v i c e which kept t h e t i s s u e a t
approximately r e s t i n g l e n g t h . The t i s s u e s were f i x e d i n 2% g l u t a r a l d e hyde b u f f e r e d t o 7.4, and c o n t a i n i n g 0.25 M s u c r o s e ; t h i s was followed
by p o s t - f i x i n g i n 1% osmium t e t r o x i d e , t h e n d e h y d r a t i o n i n a s e r i e s o f
a l c e h o l a , and imbedding i n epon. The s e c t i o n s were s t a i n e d w i t h
u r a n y l a c e t a t e and Reynolds s o l u t i o n ,
Before commenting on t h e rniCrograph8, I would l i k e t o r e f r e s h
your memory on t h e morphology o f t h e s s r c o p l a s m i c r e t i c u l u m . As you
w i l l r e c a l l , t h e r e m e l o n g i t u d i n a l t u b u l e s which completely enmesh
t h e m y o f i b r i l , l i k e a sausage s k i n , and each s e c t i o n t e r m i n a t e s at
lateral cisternae, which a b u t a g a i n s t "T" o r t r a n s v e r s e t u b u l e s ,
forming t h e s o - c a l l e d "triad." I n mammals t h e triads are l o c a t e d
over t h e A-I b o u n d r i e s , so t h a t w e have 2 triads p e r saxcomere, and
an a d d i t i o n a l t u b u l e system e x t e n d i n g from t h e A-I boundry of one
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sarcomere a c r o s s t h e 2 l i n e t o t h e A-I boundry o f t h e a d j o i n i n g
sarcomere
I nould now l i k e t o show some micrographs demonstrating 1 p o sones; autophagic vacuoles c o n t a i n i n g o r g a n e l l e s being d i g e s t e d ;
suggestive evidence as t o n h e r e t h e lysosomes are o r i g i n a t i n g f'romt
p i c t u r e s o f lysosome from f r a c t i o n s prepared by c e n t r i f u g a t i o n ! and
some h i s t o c h e m i s t r y a t t h e EM l e v e l (about 10 micrographs).
The question that I now t h i n k has t o be answered d e f i n i t i v e l y
is where do muscle lysosomes o r i g i n a t e from?
The S-R i s one o f t h e most i n t e r e s t i n g s p e c i a l i z a t i o n s of t h e
v a c u o l a r a p p a r a t u s , It was discovered at t h e change o f t h e c e n t u r y
by Verattl and completely n e g l e c t e d u n t i l 1953 when P r o f e s s o r P o r t e r
and h i s c o l l e a g u e s published t h e first e l e c t r o n micrographs of t h i s
structure. It was brought t o prominence w i t h its demonstrated r o l e
i n e x c i t a t i o n - c o n t r a c t i o n coupling.
A few y e a r s ago Pearce (1965) suggested a n o t h e r p o s s i b l e f u n c t i o n ,
He noted e l e c t r o n dense material i n t h e lateral sacs o f t h e S.R. and
a f t e r s t a i n i n g f o r a c i d phosphatase noted t h e r e a c t i o n product t o be
p a r t i c u l a r l y l o c a l i z e d i n t h e r e g i o n o f t h e triad, and a smaller
m o u n t o f t h e enzyme r e l a t e d t o t h e l o n g i t u d i n a l t u b u l e s . It was h i s
opinion t h a t "if lysosomes e x i s t , t h e y d i d so not as separate
e n t i t i e s , b u t as part o f t h e l o n g i t u d i n a l s a r c o t u b u l a r system," and
he coined t h e term ~lsarcotubulo-lysosomal system. '*
In a n o t h e r paper by FBwcett and McNutt (1969) t h i s past y e a r ,
t h e y r e p o r t e d on p r e v i o u s l y undescribed dense bodies i n h e a r t
v e n t r i c l e , These v e s i c l e s were i n t h e v i c i n i t y of t h e 2 l i n e , and
similar i n appearance t o o t h e r vesicles found i n t h e g o l g i r e g i o n
and beneath t h e plasma membrane, They a l s o r e p o r t e d a n o c c a s i o n a l
c o n t i n u i t y of t h e s e v e s i c l e s w i t h t h e S-R i n t h e Z-band r e g i o n ,
s u g g e s t i n g t h a t t h e vesicles were e i t h e r a r i s i n g from, o r c o a l e s i n g
with, t h e r e t i c u l u m . Because o f our own o b s e r v a t i o n s , and t h o s e
j u s t mentioned, ne a r e p r e s e n t l y persuaded t h a t some muscle lysosomes
can a r i s e from t h e S-R when needed by t h e animal,
The mechanism o f muscle d e g e n e r a t i o n i n d i s e a s e states remains
t o be e l u c i d a t e d . Honever, we a r e reminded t h a t a common o b s e r v a t i o n
i n muscle disease is t h e complete d e g e n e r a t i o n o f a s i n g l e muscle
f i b e r , o r segment o f t h e fiber, l a y i n g a l o n g s i d e a p p a r e n t l y normal
muscle f i b e r s . And a l s o , a c c o r d i n g t o Van Breeman (1960) and o t h e r s ,
t h e first morphological change a t t h e u l t r a s t r u c t u r a l l e v e l i n
p r o g r e s s i v e muscle diseases, i s d i a l a t i o n o f t h e sarcoplasmic
e are i n t r i g u e d w i t h t h e p o s s i b i l i t y o f t h i s t u b u l a r
reticulum. W
sleve around t h e muscle f i b r i l t u r n i n g i n t o a large lysosome.
93
Acknowleaements
The author wishes t o acknowledge the research contributions of
D r s . Judith Bond, Marilyn Pollack, 'Pond Berg, Peter Canonico,
Albert Milanesi, Mr. U i l l l a m Stauber, and Professor Christian de Duve.
This nork was supported i n part by USPHS grants NS-07180 and HD4334,
a N I H Research Career Developnent A w a r d and a F'ulbright Researah
Scholarship,
References
Allison, A . C . and M. R . Young,
Eaggiolini, M . ,
40I 529.
1964.
Life Sci. 3(12);1407,
J . G . Hirsch and C, de Duve.
1969. J . C e l l .
w.
.
Baxrett, A , J
1969. In: Lysosomes i n Biology and Medicine, J , T ,
Dingle and H . B . F e l l , eds., Amsterdam; North Holland Publishing
CO., pp. 245-312.
1951.
Berthet, J . and C. de Duve.
BlLochem. J. 50r174,
Bird, J.W.C.,
T . Berg and J . H . Leathern.
Med. 127r182.
1968.
-
Pr0c. SOC, Expl. B i o l .
Bird, J.W.C., T . Berg, A. Milanesi and W . T . Stauber.
Bioohem. Physiol. 30r4-57.
Bond, J
. and J .W .C , Bird.
1966.
1969. Comp,
Federation ProceedinRs 25 r242.
Canonico, P . G . and J . W . C . Bird.
1969, Cytobios l A t 2 3 .
Canonico, P . G. and J . W . C . Bird.
1970.
J. C e l l . B i o l . 45(2):321.
de b e , C . 1959. I n ; Lysosomes, a new group o f cytoplasmic
p a r t i c l e s , T. Hayashi, ed. New Yorkr Ronald Press, pp. 128-159.
de Duve, C . 1963. I n : Lysosomes, A . V . S , de Reuck and M . P . Cameron,
eds. Boston: L i t t l e Brown and Co., pp. 1-31,
de Duve, C .
1967.
Protoplasna 63(1-3);35.
B . C , PTesmnan, R . Gianetto, R . Wattiaux and F. Appelmans,
Biochern. J. 60r604.
de Duve, C . ,
1955.
de Duve, C . and R . Wattiaux.
1966. E .
&.
Physiol. 281435.
Dingle, J . T . and A . J . E h m e t t .
1968.
h w c e t t , D . W , and N , S, McNutt.
1969. J. C e l l , Biol. 41 1 1 ,
Koenig, H .
K O ~ ,R.
1963.
R.
1965.
Kahn, R . R ,
1969.
Biochem. J. 109(3):19P.
J . Histochern. Cytochem. 111120.
&.
J. Pathol. 471315,
E.I n v e s t ,
20,202.
95
Koszalka, T . R . and I . L . Miller.
Novikoff, A . B .
Mirsky, eds.
,
P.
1925,
Smith, Barbara,
Bird.
1971.
Chem. 2351665.
Cytochem. 14151396,
Comp, Biochem. Physiol.
1962. In: The Cell, Vol. 11. J , Brachet and A . E .
New Yorkr Academic Press. pp. 423-488.
Pollack, M . S . and J.W.C.
ROUS,
-J . -B i-o l .
1965. 2. Histochen.
Maier, D . M . and H . Zaiman.
Milanesi, A , A , and J .W .C
I n Press.
1960.
Bird.
1968.
Am.
J . Physiol. 2151716.
J. Exp. Med. 41 1399.
1965. Res. Muscular J&strophx Proc, 3 r d Symp. 133.
Straus, W. 1967. I n r Enzyme Cytology, D . E . Roodyn, ed.
Academic PTess, I n c . pp. 239-319.
Swift, H. and Z. Hruban.
1964.
Fed.
New Yorkr
Proc. 23r1026.
Tappel, A . L . 1966. In; The Physiology and Biochemistry o f Muscle
as a Food, E . J . Brinkey, R . G. Cassens and J . E . Trautman, eds,
Madison! University of Wisconsin Press. pp. 237-249.
Van Breeman, V . L .
1960,
-.
J. Path. 371333.
Van F l e e t , J . F , , B . V . Hall and J . Simon,
52(5)11067.
1968. h e r , J, Pathol,
Willermot, J . , M. Loulanne and L, Berlinguet.
B i o p h s 1331350.
1969. Arch. Biochem.
D. E . GOLL: Thank you v e r y much, D r . Bird, f o r t h a t most
i n t e r e s t i n g and up-to-date summary of muscle lysosomes. It has
r e a l i m p l i c a t i o n s t o t h e meat i n d u s t r y and shows some p o s s i b i l i t i e s
w e want t o e x p l o r e i n t h e d i s c u s s i o n period. The next s p e a k e r on
t h i s afternoon's program i s Rr. Fred F a m i s h from t h e Department
of Animal Science a t Iowa S t a t e University.
I promised Fred I
would f o r e g o any long i n t r o d u c t i o n s and s t o r i e s about some o f h i s
a n t i c s a t Iowa S t a t e , Fred r e c e i v e d h i s B.S. and M.S. and Ph.D.
degrees from t h e U n i v e r s i t y o f Missouri and came t o Iowa S t a t e i n
January o f 1965 and even though we g r e e t e d him with 25 below z e r o
temperatures, he has s t a y e d and maintained a c o n t i n u i n g i n t e r e s t
i n h i s Ph.D. problem which was t h e r o l e of lysosomes i n meat q u a l i t y .
The t i t l e o f Red's t a l k t h i s a f t e r n o o n is Extent and Role of
P r o t e o l y s i s i n Post-mortem Muscle,
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