Isolation and the Properties of Muscle Lysosomes

ISOLATION AND PROPERTIES OF MUSC LS LYSOSOMES* JOHN W. C . BIRD DEPARTMENT OF PHYSIOLOGY RWERS UNIVERSITY

In a classical series of papers beginning with Berthet and de hve (1951) and summarized by de Duve (1959) it was demonstrated that in osmotically protected homogenates of rat liver certain hydrolytic enzymes with acid pH optimum displayed the phenomenon of latency, The latency property was demonstrated by the fact that the enzymes were practically unreactive to their substrates when conditions to keep the particles intact were maintained. The enzymes became maximally active toward substrates only when the homogenates were subjected to physico-chemical treatments which would disrupt membranes, On the basis of differential centrifugation experiments, it was proposed that the hydrolases belonged to a distinct particle, and the name "lysosome" was proposed (de me, et al., 1935). de Duve (1959) suggested that the lysosomal enzymes were primarily catabolic in function, and that being segregated from surrounding cytoplasm by a bounding membrane they were prevented from digesting their host cell, It was also stated, unfortunately, that under certain circmstances the lysosomes acted as "suicide bags" by releasing their contents into the cell and thus resulting in cell death (de Dwe, 1963). I say "unfortunate, 'I because the "suicide bag concept" masked for several years the profound significance of the lysosome in noma1 cell physiology.

Many disciplines have contributed to the development of the lysosome concept, especially in defining lysosomal functions with respect to the normal economy of the cell. It is now thought that the several types of lysosomes form a complex intracellular digestive system, whereby macromolecules brought into the cell, or "worn out" cellular components are organized into vacuoles, which in turn fuse with primary lysosomes formins: secondary lysosomes. Digestion occurs in the secondary lysosomes and the end products are transported into the cell's cytoplasm by passive diffusion or active transport (de Duve and Wattiaux, 1966). In some cells the end products of digestion may be secreted from the cell as useful by-products. Undigestible material 1s either excreted by exocytosis, or may remain in the cell in the form of a residual body such as lipofuscin granules in muscle and nerve tissue (Novikoff, 1962).

* Presented st the 24th Annual Reciprocal Meat Conference of the Anerican Meat Science Association, 1971 . 68

Several workers have enphasieed, however, that the lysosomes and other component8 of the vacuolar apparatus are not developed to the same degree of complexity in all cell types (de Dwe, 19678 Novikoff, 1962; and Straus, 1967). The vacuolar apparatus is a morphological specialieation which is normally present in those cells nhose phyaiological function requires an efficient and economical catabolic machinery. A survey of the number of lysosomes found in different types of cells shows that lysosomes are more numerous in epithelial cells of organs having a phagocytic, absorptive, or secretory function (Straw, 1967) where a highly developed catabolic machinery is essential to the cell or organ. In skeletal muacle fibers, which do not have phagocytia or secretory functions, microscopy had failed to demonstrate the presence of lysosomes or lysosome-like granules. The failure to find lysosome-like morphological entities in normal nuecle cells sparked a controversy a few years ago as to the origin of aaid hydrolase aativity in muscle tissue. Tappel (1966) stated that the particles described by bioohemical studies me totally contributed by the non-muscle cella of nurscle tissue. Furthermore, in certain myopathies a dramatic increase in macrophages, leucocytes and lymphocytes occurs which is coincident with increases in total lyaosomal ensyme activities. Since the cells that comprise the connective tissue components of muscle are known to have among the highest concentrations of lysosomal enzymes, Kohn (1969) has calculated that under certain situations they could account for all the lysosomal enzyme activity found in muscle tissue.

Van Flaet, et al. (1968) reported that lysosome-like structures were not found in nornal skeletal muscle fibers of rabbits. Pellegrino and F’ranzini (1963) emphatically stated that no lysosome has ever been detected in normal muscle fibers by electron microscopy. Maier and Zainan (1965) concluded that acid phosphatase, the lysosomal marker enzyme most frequently used in histochemical studies, could not be demonstrated in normal rat skeletal nusale fibers, Smith (1965) reported that the histochemical reactions for acid phosphatase in normal rabbit muscle is scanty and confined to bloed vessels (i.e., endothelial connective cells).

The experimental cenclusions of the morphologists and biochemists have been difficult to reconcile. Thua, the experiments I will discuss are an attempt to resolve the existing controversy over the nature of the lysosomes in normal skeletal musale. I must also confess my prejudices. My basic working hypothesis Is that all normal musole protein catabolism is initiated and completed by lysosomal acid proteaaes, which axe indigenous to the muscle fibers. I an not aware of any carefully characterized or proven neutral proteases in muscle tissue. The alkaline protease of Kescalka and Miller (1960) and the neutral protease of Kohn (1965) have recently been shown, in an elegant study by Willemot, Ialanne and Berlinguet (1969), to be xanthine oxidase. I am certainly not opposed to neutral proteases, as the- occurence would simplify matters greatly, I look forward with great anticipation to a paper currently &press by Noguchi and Kandatsu, of the University of Tokyo, on the purification and properties of a new alkaline protease in rat skeletal muscle (e,e. Chem., personal communication from Noguchi), I should also report that Professor B, Sylven demonstrated a purified lysosomal cathepsin B from tumors that had a broad pH spectrum from to 8.0, with good activity at pH 7.0, using urea denatured Edestrin3.5 as substrate (International Research Conference of Lysosomes, Louvain, 1970)

There have been occasional suggestions that acid pH is "unphysiological" and that acid hydrolases can, therefore, have little significance in living organisms (Barrett ' 1969). Although the pH optima of these enzymes are well removed from neutrality, several of them have been shown to have sufficient activity at the higher pH to be highly significant in the long time-scale of physiological processes. Moreover, the available evidence from the use of indicators (Rous, 1925) points to an extremely acid pH in the digestive wicuoles within phagocytic cells.

For today's discussion, first let us review the essential hiochemical criteria for lysosomes in muscle tissue;

Sedimentation properties (figure 1 ). Using differential centrifugation, and expressing the enzyme activity as mean relative specific activity of the fractions versus their mean relative protein content, the area of each block representing a fraction is thus proportional to the percentage of activity recovered in the corresponding fraction, and its height to the degree of purification achieved over the homogenate (de Dwe et al., The fractions are represented on the abscissa in the order in which1955). they were isolated, i.e., from left to right: N, nuclear, M, heavy mitochondrial$ L, light mitochondrial; P, microsomal; and S, final supernatant fraction. It is seen that the highest RSA is found in the light mitochondrial fraction, which has been demonstrated to be the lysosome-rich fraction in other tissues,

Structure-linked latency (table 1 ),

If the acid hydrolase is contained within a non-permeable membrane, chemical or physical forces which disrupt this membrane should allow accessibility of the enzyme to its substrate, In this slide we see that increasing the duration of homogenization increases the amount of free activity, i.e., increases the amount of enzyme available to react with substrate.

The thermal labilization of acid hydrolases from liver, kidney and muscle lysosomes is shown in figure 2. Muscle lysosomes seem more resistant to thermal incubation than are liver and kidney lysosomesr When the homogenates are incubated in neutral or acid media, the rate which lysosomal enzymes are released into the non- sedimentable fraction is several times greater for liver and kidney than for muscle, 70

0 2

I Q

0 l2

II I 0r: s c JJO

c 3 d 8 m z N a i Q)c

I 0 rc)- 0 C Y 71

TABLE 1. INFLUENCE OF PRELIMINARY BLENDING OF MUSCLE ON ENZYME LATENCY

Enzyme activity" X VirTis l?ree Total Free (%a>

Aryl Sulfatase 1 59.8 101 .o 59.2 2 75.6 117.0 64.6 4 106.8 106.7 100.1 Ribonuclease 1 33.1 58.6 56.4 2 39.1 60.0 65.2 4 65.3 62.3 104.8

Muscles were cut into small pieces with scissors, then blended in 0.25 M sucrose with a VirTis "45" homogenizer at top speed for 2 seconds, for the number of times indicated in the table. This was followed by three up-and-down passes in a Dual homogenizer. Homogenates were assayed for free and total activities as described in text, except that incubation times were 15 minutes,

*RNase = A O.D./mg protein per hr x 10-5; aryl sulfatase = mpoles nitrocathechol/ma protein per hr . 72

pK 5.0 8 p-NPPa 8 e

20 pGluc 6

' 10

I 20 t 0 1 2 INCUBATION TZME (Hours)u

Figure 2. Thermal labilization of acid hydrolases from liver, kidney and muscle lysosomal particles. e, kidney; a, liver; 0, muscle (from Canonico, 1969). The effect of freezing and thawing on lysosomes is presented in figure 3. A total mitochondrial preparation was frozen by immersing the test tube in a mixture of dry ice and acetone, and thawed by placing the tubes in a 38OC incubator. It is noted that one freeze- than treatment released 5% of the cathepsin activity, whereas subsequent freeze-thaw cycles released an additional 10% of enzyme. The following figure (figure 4) demonstrates the effect of f'reezing rate on the solubilization of cathepsin D. Whole gastrocnemius muscles were frozen by either plunging the muscle and its container into liquid nitrogen (130°C/min, measured by an attached thermocouple) or by a controlled Linde BF-4 biological freezing apparatus. The "S" under the second set of bar graphs represents experiments from shredded muscle. The results indicate that the lysosomes are kept intact if the freezing rates are in the range of 10C/min, especially if the muscle is protected by immersion in 0.25 M sucrose media, indicated by "M" under the last pair of bar graphs. Additional experiments indicated that muscles fYozen at loC/min and stored for up to 16 days under liquid nitrogen had no increase in solubilized cathepsin,

Structure-linked latency can be demonstrated by several addition methods, including detergent treatments, hypotonic solutions, soni- cation, and the use of physiological labelizers such as vitamin A.

Our first clue for the existence of lysosomes in muscle cells came from some morphological studies using acridine orange (Canonico and Bird, 1969). Koenig (1963) and Allison and Young (1964) had presented convincing evidence that this vital dye was concentrated within lysosomes, causing them to appear as bright orange granules in fluorescence microscopy, Our observations of teased muscle fibers and frozen sections of normal gastrocnemius muscle showed occasional fluorescent granules in the perinuclear region and within the muscle fibers. However, after subjecting the rats to 6 days of starvation before dye injection, relatively large numbers of orange granules were seen in the perinuclear region, and throughout the muscle fiber,

Dingle and Barrett (1968) recently reported that actual counts of orange fluorescent granules in gradient centrifugation fractions are in good agreement with specific activities of lysosomal enzymes, We have extended these observations by the spectraphotofluormetric determination of acridine orange in cell fractions (figure 5) and I think have clearly shown that the dye distribution is similar to the distribution pattern of particle bound acid hydrolases, Mhermore, the sedimentation characteristics of lysosomal prticles did not appear to be altered by acridine orange, since similar acid hydrolase distribution patterns were obtained from muscles of normal and acridine orange-injected animals. I will say more about this later,

Because of our biochemical and morphological evidence for the possible existence of lysosomes in muscle fibers we designed some -Jc

. I 2 3 4 5 .TIMES FROZEN AND THAWED.

F'igure 3. The effect of freezing and thawing on lysosomes (from Bond and Bird, 1966). 1 GAST ROCNEMIUS

CONTROL 1 FROZEN

I - I I

E I.) P

130 15 I I s M FREEZINGI RATES @/MI$ 4 4, L

ACRIDINE ORANGE

c 1 I I I I 0 20 40 60 '80 100 2 TOTAL PROTEIN

Figure 5. IEstribution pattern of acridine orange after differential centrifugation of muscle I homogenate (f'rom Canonico and Bird, 1969). 77 experiments to differentiate the contribution of lysosomes from various possible cellular sources in skeletal muscle tissue. Our first experiments utilized the techniques of rate-zonal centrifugation (Canonico and Bird, 1970). The experimental design is shown in figure 6. The distribution of particle bound activity is shown in figure 7. I would like to call your attention to the fact that we have a bimodal distribution of our enzyme activity! a slower sedimenting peak at fraction 9 and a fast sedimenting peak against the gradient cushion at fraction 26. Also note that our distribution is heterogeneous with respect to the individual enzymes, since 30% of the total activity of @glucuronidase, RNase and aryl sulfatase is found against the cushion, as compared to 18% of the total cathepsin and acid phosphatase.

We then repeated these experiments with animals injected with Dextran-500, which has been shown to increase the density of lysosomes. The data showed a decreased activity in the slower sedimenting peak with a concomitant increased activity against the cushion for the three enzymes 3-glucuronidase, RNase and aryl sulfatase, with no change in the pattern for cathepsin D and acid phosphatase, We then injected our animals with Triton-1339, which normally results in larger but less dense lysosomes, In this experiment there was a decrease in the enzyme activity against the cushion for the three enzymes 3-glucuronidase, RNase and aryl sulfatase, with again no effects on the other two enzymes.

The different sedimentation rates and the apparent heterogeneity of the enzymes in these two particle populations suggested to us that we were dealing with more than one group of lysosomes. Furthermore, one of these populations was capable of phagocytyzing the injected substances, while the other was not. We then designed some isopycnic- zonal centrifugation experiments, where the particles could be separated solely on the basis of density. The experimental protocol is depicted in figure 8.

The equilibrium density histograms of the acid hydrolases from normal muscle demonstrate a broad distribution with a modal equilibrium density of 1 .I8 in sucrose (figure 9). The heterogeneity in distribution of the acid hydrolases is further demonstrated by cathepsin and acid phosphatase histograms being skewed toward lighter densities, while RNase, 3-glucuranldase and aryl sulfatase are skewed toward the denser portions of the gradient. Cytochrome oxidase is concentrated within a narrow-density span with a modal density slightly greater than 1.18. We also studied the enzyme muramldase (lysozyme), a lysosomal hydrolase found in leucmytes and macrophages, The muramidase data is not present on this figure, but had a bimodal distribution with peaks at 1.15 and 1.20.

Little rnuramidase aativity was found at 1.18, the modal equilibrium density peak for the other lysosomal enzymes. The bimodal distribution of muramidase in this study is in agreement with the findings for leucocytes by Baggiolini, et al. (1969). 78

, 45

35 n yl 25 ; P 0 IS g

OXIDAS C -4- 5 0 i 4 10-5 20 2;- - FRACTION NULIBER Figure 7. Distribution pattern of enzymes, acridine orange, and protein after fractional of post-nuclear muscle homogenates by rate-zonal centrifugation (a2t = 410 x IO7 radZ/sec). aJ0

.. - ...... -.

..-

ISOPYCNIC CENTR IFU GAT ION

Figure 8, Experimental design of isopycnic-zonal technique (from Canonico and Bird, 1970). 81 82

We then ran isopycnic studies on our muscle tissues after injecting the animals with Dextran-500 or Triton 1339 (figure IO). After injecting with Dextran-500, the modal density of the five acid hydrolases was not shifted from the control value of 1.18. However, a portion of the activity was shifted to the more dense part of the gradkent. This shift was greater for the three enlsylaes @-glucuronidase, RNase and aryl sulfatase, than for cathepsin or scid phosphatase, and indicated that a small group of lysosones rich in these enzymes were capable of altering their equilibrium density by the accumulation and storage of exogenous materials. We also see a similar shift in distribution, although not as dramatic, from animals injected with Triton 1339 (Mgure 11) with the difference that the activity is shifting to the more bonpnt portion of the gradient.

Since the adninistration of exogenous materials did not affect the equilibrium density ofthe large lysosome group, which we suspected as being of muscle fiber origin, starvation was used 8s an endogenous stimulus, to hopefully promate the development of large secondary lysosomes (figure 12). Swift and Hruban (196b) suggested several years ago that the formation of autophagic vacuoles is a defense mechanism of normal oells to star*ation. Since muscle is the major store of amino acids, after, of course, rapid depletion of liver and the free animo acid pool, it is net smprising that autophagic vaouoles would be formed for the digestion of muscle during prolonged starvation. Returning to the figure, we see that stamation caused a decrease, and om only treatment causing a decrease, in the modal equilibrium density of all five acid hydrolases. Cathepsin and acid phosphatase were decreased to 1.16 and the other three enzymes to 1.165. Our interpretation of the isopycnic data is sumraarieed on the next figure (figure 13). Considering first the distribution histo- gram kbla Dextran-500 treated animals, a best fit curve was drawn through the left face of the equilibrium density distribution, with the apex uolnciding with the modal density peak at 1.18. A mirror image of this curve was reprodwed on the right side of the apex so as to constrwt a bell-shaped ourve. The difference between this bell-shaped curve and the experimental data waa used to construct a smaller bell-shaped curve. These saae larger curves fYoa the Dextran-jOO data were then super-imposed on the right faoe of the data fron the animals injected with Triton 1339. Notice the good fit of the curve with the experhental data. Then, small bell-shaped curves were obtained as before. By this technique tno groups of lysosomes with bell-shaped distribution were resolved. The smaller group contributed approximately 25% of the aryl sulfatase, 8-glucuronidase and RNase, of the cathepsin D and acid phosphatase, and almost all of the5% muramidase. The larger group, on the other hand, contains approximately 75% of the 0-glucuronidase, RNase and axyl sulfatase, and 95% of the cathepsin and acid phosphatase activity. El'I 4 01 '1 81 'I 91 'K

s i(

IJ H 3 0 i;.e m0 oz 'I 81 'I 91 'I 'IT

oz 'I 8'1 '1 91 '1 fiT '1* STARVED

RNase 12 c

l2 t -GLUC

...... A r-i l-44 rl v EQUILIBRIIR.1 DENSITY A Figure 12, Effect of stamation on the density distribution patterns of rat muscle acid hydrolases, Animals were fasted 6 days but provided with tap water and saline solution, Dotted lines show normal distribution. KID RIBONUCLEASE B-GLUCURONIDASE

t L, : * , L

z 0 at c z W V z 0 V ,ATHhPSIN D W> F 4 -1 w a

DENSITY Figure 13, bell-shaped curves of best-fit drawn through the equilibrium density distribution data,of acid hydrolases in muscles of dextran-500 and Triton N3-1339-treated animals, Theoretical curves are extrapolated beyond limits of gradient (from hnonico and Bird, 1970). 86

n.-

aoaww w ccc

i...,;,, U?

We are persuaded that the larger group of lysosomes with a modal equilibrium density of 1.18 are those lysosomes indigenous to the muscle fiber, and that the smaller group of lysosomes are derived from phagocytic cells.

I would like to show another slide which may be of more interest to some of you, in that it concerns a commonly edible muscle (figure 14). These data represent a comparison of isopycnic zonal centrifugation data of fish muscle from rainbow trout acclimated at three different temperaturest 4OC, 12OC, and 18OC (Milanesi and Bird, 1971). 12OC is the optimum temperature for growth and repro- duction in trout, whereas 5OC and 18OC represent the usual environ- mental limits for trout, We see that the distribution patterns are essentially the same for the 12OC acclimated fish as for rat skeletal muscle, This particular study indicates that temperature is also an indigenous stimulus for changes in lysosome populations, 12OC acclimated fish had a modal density of 1.18 for their acid hydrolases; whereas adapting the fish to the lower and higher temperatures changed the modal densities to 1.16 and 1.19, respectively,

It is certainly all to the well and good to have nice enzyme recoveries, and experimental data that nicely fits the theoretical curves. However, the fact remains that there has been a certain amount of discussion in the literature concerning the notable absence of the usual type of lysosomes in normal skeletal muscle fibers. If one reads the literature carefully, most authors make a statement to the effect that "no lysosomes were observed," which is quite different from stating that there are "no lysosomes is skeletal muscle fibers." I think there are two main hinderances in our thinking. Most of us come away from our biochemical training thoroughly convinced that the body is one big piece of liver, or more recently, a large E. e.On the other hand, there are many distinguished laboratories interested in the physiology and biochemistry of muscle, but the primary focus for the past couple decades has been concerned with the contractile apparatus. My point is, I don't think a serious effort has been made to look for lysosomes per se in muscle fibers,

A year ago, while working in Professor de Duve's laboratory in Lowain, we made some initial attempts in this area. In that we were anxious to see active lysosomes within normal physiological limits, we chose the starvation model as being the most likely prospect. Before looking at the electron micrographs let us first take a look at the state of the animal during this starvation process (figure 15). We see a 40% decrease in wet weight of the gastrocnemius muscle for the eight day period; very little, if any change in the ratio of gastrocnemius weight to body weight, indicating that the decrease in gastrocnemius weight is not disproportionate to the overall decrease in body weighti very little change in gastrocnemius protein concentration during the first 6 days of starvation, then a decrease in + 3

V 0 E I- v) 0a z 0- c wa Q? ca tn L 0

tn 2n

0 Ln 0 r: c c u? c?c swvt19 lN33t13d w9w lN33U3d NORMALIZED SPECIFIC ACTIVITY / 100 gm BODY WEIGHT

ARYL SULFATASE -GLUCURONIDASE RNASE 2 1 I I I I I I I I

CATALASE 1; P-N-P PHOSPHATASE

3 CATHEPSIN D

DAYS OF STARVATION -. i-iir~1‘. :.Llanh;cs ir, sxcific ac-ivity of nuscle acid hydrolases ixin: ci slays of foocl resrriction. protein concentration; and finally, the changes in dry weight of the gastrocnemius, As noted, there was a significant increase in dry weight of the muscle between the third and fourth day. I am not sure what caused this muscle dehydration, but because of the rapid onset and rapid termination, I am tempted to say that it may be a hormonal response. As you will see in later slides, we think that muscle autophagy is initiated around the fifth day of staxvation. It would be nice to find a specific hormone to regulate or trigger autogaphy within normal physiological limits!

The next figure (figure 16) shows the changes in specific activity of our enzymes during this period of starvation. We see little change in the three enzymesB-glucuronidase, aryl sulfatase and RNaae for the first 5 to 6 days, then a decrease in activity. Acid phosphatase and cathepsin on the other hand, have a distinct increase between days 1 and 5, then a sharp drop in activity. Our preliminary interpretation is that there is an activation of pre- existing enzyme, or synthesis of new enzyme during these first 5 days; then, fusion of the lysosome with a newly formed autophagic vacuole, and initiation of intracellular digestion. With prolonged stanration, there may be invasion of the tissue by macrophages, bringing in additional lysosomes, In our next experiment (figure I?), we followed the free activity changes in our muscle tissue from starved animals. With our homogenizing techniques, muscles from normal adult animals usually have around 50% free activity, In this experiment there was little change in fYee activity until the fifth day of stamation, where it increased to approximately 65%, and on the sixth day was as high as 95%. We attribute the increase in free activity to an increase in size, and hence an increase in fragility of the lysosomes to the homogenizing procedure, of course, the other possibility remains that some of the enzyme is solubilized in vivo,

In our electron microscope studies with whole tissues, the medial head of the gastrocnemius muscle was used from animals stamed 6 days. The tissue was placed in a spring device which kept the tissue at approximately resting length. The tissues were fixed in 2% glutaralde- hyde buffered to 7.4, and containing 0.25 M sucrose; this was followed by post-fixing in 1% osmium tetroxide, then dehydration in a series of alcehola, and imbedding in epon. The sections were stained with uranyl acetate and Reynolds solution,

Before commenting on the rniCrograph8, I would like to refresh your memory on the morphology of the ssrcoplasmic reticulum. As you will recall, there me longitudinal tubules which completely enmesh the myofibril, like a sausage skin, and each section terminates at lateral cisternae, which abut against "T" or transverse tubules, forming the so-called "triad." In mammals the triads are located over the A-I boundries, so that we have 2 triads per saxcomere, and an additional tubule system extending from the A-I boundry of one r.: 0 n -. I .bJ 0 .rl k -tJ r/: 8 vr > 3 e- 0 ckl + CCI I lDWJOU 0 d t. t-- > d - 0 1 t-o u Q: 0 I IDWJOU I W W t 4-10 (1L 0a LL L 0)

C.1 $1 GI r: r: I ID W JOU .ri

I I I I I I I I I I 1 ooo(3ooooooo 0'00~cDmucr)cv-- lN33t13d 92 sarcomere across the 2 line to the A-I boundry of the adjoining sarcomere . I nould now like to show some micrographs demonstrating 1po- sones; autophagic vacuoles containing organelles being digested; suggestive evidence as to nhere the lysosomes are originating f'romt pictures of lysosome from fractions prepared by centrifugation! and some histochemistry at the EM level (about 10 micrographs).

The question that I now think has to be answered definitively is where do muscle lysosomes originate from? The S-R is one of the most interesting specializations of the vacuolar apparatus, It was discovered at the change of the century by Verattl and completely neglected until 1953 when Professor Porter and his colleagues published the first electron micrographs of this structure. It was brought to prominence with its demonstrated role in excitation-contraction coupling.

A few years ago Pearce (1965) suggested another possible function, He noted electron dense material in the lateral sacs of the S.R. and after staining for acid phosphatase noted the reaction product to be particularly localized in the region of the triad, and a smaller mount of the enzyme related to the longitudinal tubules. It was his opinion that "if lysosomes exist, they did so not as separate entities, but as part of the longitudinal sarcotubular system," and he coined the term ~lsarcotubulo-lysosomal system. '*

In another paper by FBwcett and McNutt (1969) this past year, they reported on previously undescribed dense bodies in heart ventricle, These vesicles were in the vicinity of the 2 line, and similar in appearance to other vesicles found in the golgi region and beneath the plasma membrane, They also reported an occasional continuity of these vesicles with the S-R in the Z-band region, suggesting that the vesicles were either arising from, or coalesing with, the reticulum. Because of our own observations, and those just mentioned, ne are presently persuaded that some muscle lysosomes can arise from the S-R when needed by the animal, The mechanism of muscle degeneration in disease states remains to be elucidated. Honever, we are reminded that a common observation in muscle disease is the complete degeneration of a single muscle fiber, or segment of the fiber, laying alongside apparently normal muscle fibers. And also, according to Van Breeman (1960) and others, the first morphological change at the ultrastructural level in progressive muscle diseases, is dialation of the sarcoplasmic reticulum. We are intrigued with the possibility of this tubular sleve around the muscle fibril turning into a large lysosome. 93 Acknowleaements

The author wishes to acknowledge the research contributions of Drs. Judith Bond, Marilyn Pollack, 'Pond Berg, Peter Canonico, Albert Milanesi, Mr. Uilllam Stauber, and Professor Christian de Duve. This nork was supported in part by USPHS grants NS-07180 and HD4334, a NIH Research Career Developnent Award and a F'ulbright Researah Scholarship, References

1964. Life Sci. 3(12);1407, Allison, A. C. and M. R. Young, Eaggiolini, M., J. G. Hirsch and C, de Duve. 1969. J. Cell. w. 40 I 529. Baxrett, A, J . 1969. In: Lysosomes in Biology and Medicine, J, T , Dingle and H. B. Fell, eds., Amsterdam; North Holland Publishing CO., pp. 245-312.

Berthet, J. and C. de Duve. 1951. BlLochem. J. 50r174,

Bird, J.W.C., T. Berg and J. H. Leathern. 1968. Pr0c. SOC, Expl. Biol. -Med. 127r182. Bird, J.W.C., T. Berg, A. Milanesi and W. T. Stauber. 1969. Comp, Bioohem. Physiol. 30r4-57. Bond, J . and J .W .C , Bird. 1966. Federation ProceedinRs 25 r242. Canonico, P. G. and J.W.C. Bird. 1969, Cytobios lAt23.

Canonico, P. G. and J.W.C. Bird. 1970. J. Cell. Biol. 45(2):321. de be, C. 1959. In; Lysosomes, a new group of cytoplasmic particles, T. Hayashi, ed. New Yorkr Ronald Press, pp. 128-159. de Duve, C. 1963. In: Lysosomes, A.V.S, de Reuck and M. P. Cameron, eds. Boston: Little Brown and Co., pp. 1-31, de Duve, C. 1967. Protoplasna 63(1-3);35. de Duve, C., B. C, PTesmnan, R. Gianetto, R. Wattiaux and F. Appelmans, 1955. Biochern. J. 60r604. de Duve, C. and R. Wattiaux. 1966. E.&. Physiol. 281435.

Dingle, J. T. and A. J. Ehmett. 1968. Biochem. J. 109(3):19P.

J. Cell, Biol. 41 11, hwcett, D. W, and N, S, McNutt. 1969. Koenig, H. 1963. J. Histochern. Cytochem. 111120.

KO~,R. R. 1965. &. J. Pathol. 471315, Kahn, R. R, 1969. E.Invest, 20,202. 95 Koszalka, T. R. and I. L. Miller. 1960. ---J. Biol. Chem. 2351665. Maier, D. M. and H. Zaiman. 1965. 2. Histochen. Cytochem. 14151396, Milanesi, A, A, and J .W .C , Bird. 1971. Comp, Biochem. Physiol. In Press. Novikoff, A. B. 1962. In: The Cell, Vol. 11. J, Brachet and A. E. Mirsky, eds. New Yorkr Academic Press. pp. 423-488.

Pollack, M. S. and J.W.C. Bird. 1968. Am. J. Physiol. 2151716. ROUS, P. 1925, J. Exp. Med. 41 1399. Smith, Barbara, 1965. Res. Muscular J&strophx Proc, 3rd Symp. 133.

Straus, W. 1967. Inr Enzyme Cytology, D. E. Roodyn, ed. New Yorkr Academic PTess, Inc. pp. 239-319. Swift, H. and Z. Hruban. 1964. Fed. Proc. 23r1026. Tappel, A. L. 1966. In; The Physiology and Biochemistry of 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 Fleet, J. F,, B. V. Hall and J. Simon, 1968. her, J, Pathol, 52(5)11067.

Willermot, J., M. Loulanne and L, Berlinguet. 1969. Arch. Biochem. Biophs 133 1350. D. E. GOLL: Thank you very much, Dr. Bird, for that most interesting and up-to-date summary of muscle lysosomes. It has real implications to the meat industry and shows some possibilities we want to explore in the discussion period. The next speaker on this afternoon's program is Rr. Fred Famish from the Department of Animal Science at Iowa State University. I promised Fred I would forego any long introductions and stories about some of his antics at Iowa State, Fred received his B.S. and M.S. and Ph.D. degrees from the University of Missouri and came to Iowa State in January of 1965 and even though we greeted him with 25 below zero temperatures, he has stayed and maintained a continuing interest in his Ph.D. problem which was the role of lysosomes in meat quality. The title of Red's talk this afternoon is Extent and Role of Proteolysis in Post-mortem Muscle,

###At#