COLLAGENASE ACTIVITY IN REGENERATING AND DENERVATION-INDUCED REGRESSING FORELIMBS 0F LARVAL AMBYSTOMA MEXICANUM Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY BARBARA P. JOHNSON . MULLER 1976 IIIIIII II III 31293 II IIIIIIIIIIIIIIIII This is to certify that the thesis entitled COLLAGENASE ACTIVITY IN REGENERATING AND DENERVATION- INDUCED REGRESSING FORELIMBS 0F LARVAL AMBYSTOMA MEXICANUM presented by Barbara P. Johnson-Muller has been accepted towards fulfillment of the requirements for __PIL.1L__degree in M @GM Major professor Date __Noy_._h.,_19_1£_ 0-7639 ' -Afit. UNI I ' 7" I‘m ABSTRACT COLLAGENASE ACTIVITY IN REGENERATING AND DENERVATION-INDUCED REGRESSING FORELIMBS OF LARVAL AMBYSTOMA MEXICANUM by Barbara P. Johnson—Muller Both regenerating and denervation-induced regressing larval urodele limbs undergo a period of histolysis following amputation and wound closure. During this period phagocytic cells remove cellular and extra- cellular debris, extracellular matricies disappear, and dedifferentiated cells are released from limb stump tissues. In regenerating limbs dedif— ferentiated cells form a proliferating blastema, and histolysis of the stump subsides when the blastema begins to differentiate. In amputated, nerveless limbs no blastema forms, and histolysis of the stump continues, resulting in complete resorption of the limb. The factors controlling, and the mechanisms underlying histolysis of regenerating and resorbing larval limbs are not fully understood. However, there is histological evidence that collagen degradation is involved in both processes, and a collagenase has been detected in regenerating adult urodele limbs during the period of limb stump histolysis. The experiments in this investigation were designed 1) to invest- igate collagenase activity in regenerating and denervation—induced re- gressing larval Ambystoma mexicanum forelimbs to assess the phases of regeneration and regression in which collagenase activity is significantly Barbara P. Johnson-Muller above unamputated limb tissue levels, and 2) to partially characterize larval A. mexicanum collagenase. Collagenase activity in larval limb tissues was assayed using the radioactive reconstituted collagen fibril assay at 25°C and 36°C at pH 7.6. This assay was run in conjunction with 1) dialysis of degrada- tion products formed at 36°C to demonstrate that collagen was degraded to small peptides, and 2) disc-gel electrophoresis of degradation pro- ducts formed at 25°C to demonstrate collagenase characteristic cleavage -of collagen to TCA and TCB subunits. Crude collagenase extract was ob- tained by direct extraction of lyophilized tissue with a neutral buffer. In this investigation the pattern of collagenase activity coincides with the pattern of limb stump histolysis in both regenerating and denervation—induced regressing larval limbs. This suggests that colla— gen degradation by collagenase is one of the mechanisms underlying his- tolysis of amputated larval limbs whether or not they subsequently re- generate. The pattern of collagenase activity in regenerating and denervated regressing limbs differs in this study. In both cases, collagenase activity rises rapidly following amputation to maximum levels, However, collagenase activity in regenerating limbs then returns to essentially normal limb tissue levels, while collagenase activity in denervated regressing limbs remains high. Since nerves stimulate proliferation of the blastema, these results indicate that the blastema may be inhib- iting collagenase activity in regenerating limbs, perhaps by producing a collagenase inhibitor. Collagenase activity was not directly related to the protein con- tent of crude enzyme extracts in this study, suggesting that specific Barbara P. Johnson—Muller changes in collagenase activity occur during regeneration and regress— ion of larval urodele limbs. Larval A. mexicanum collagenase in this investigation shares attributes with other vertebrate collagenases. It is active at slight- ly alkaline pH, is inhibited by EDTA and mammalian serum, and is not inhibited by cysteine or soybean trypsin inhibitor. It also degrades collagen at 36°C to dialyzable peptides, and at 25°C to characteristic TC95 and TCB subunits, as well as to several discrete slightly smaller TCA subunits. In addition, crude collagenase extract from regenerating limbs, but not from regressing limbs, contains a neutral protease which converts (3 to 0: subunits, indicating that connective tissue may be degraded differently in regenerating than in denervated regressing larval limbs. COLLAGENASE ACTIVITY IN REGENERATING AND DENERVATION-INDUCED REGRESSING FORELIMBS OF LARVAL AMBYSTOMA MEXICANUM By \- IIQ’ or Barbara P. Johnson-Muller A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1976 TO DAVID ii ACKNOWLEDGMENTS The author wishes to express thanks to Dr. Stephen Bromley for his special encouragement during the final stages of preparation of this thesis. Graditude is also extended to the members of my committee, Dr. Stephen Bromley, Dr. Thomas Connelly, Dr. Evelyn Rivera, and Dr. Charles Tweedle, for their editorial assistance in preparation of the thesis. Special thanks are extended to Dr. Jerome Cross for helpful suggestions concerning the experimentation in this thesis. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . , . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . vi INTRODUCTION . . . . . . . . . . . . . . . . . , . . . . . . . . 1 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . 13 Source and Care of Animals . . . . . . . . . . . . . . . . . 13 Preparation of Regenerating and Regressing Limbs . . . . . . 13 Preparation of Crude Enzyme Extracts . . . . . . . . . . . . 17 Preparation of 14C-Collagen . . . . . . . . . . . . . . 18 Radioactive Reconstituted Collagen Fibril Assay for Collagenase . . . . . . . . . . . . . . . . . . . . 18 Calculation of Limits of Detection of Radioactivity . . . . 20 Assay for Dialyzable Degradation Products of 14C-Collagen . . . . . . . . . . . . . . . . . . 20 Assay for Characteristic TCA Degradation Products . . . . . 21 Histology . . . . . . . . . . . . . . . . . . . . . . . . . 22 Morphological Staging of Regenerates and Regressing Limbs . . . . . . . . . . . . . . . . . . . 22 Statistics . . . . . . . . . . . . . . . . . . . . . . . . 25 Experiments . . . . . . . . . . . . . . . . . . . . . . . . 26 RESULTS 0 O O O O O O O O O . 0 Q C O Q Q . Q . . O O O O O O O 30 Mbrphological Development . . . . . . . . . . . . . . . . . 30 The Pattern of Collagenase Activity in Regenerating and Regressing Limbs . . . . . . . . . . . . . . . . . 3O Characterization of Larval Axolotl Collagenase . . . . . . 40 Collagenase Activity in Lyophilized Newt Regenerates . . . 56 DISCUSSION 9 o o o o o o o o a Q o q g Q ' g Q o g Q o 9 Q Q Q 0 57 SWRY o o o o o '0 o o o a 9 Q o o o o o o o o o 0 0 o 1 1 0 0 70 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . 72 iv LIST OF TABLES Page TABLE 1. COLLAGENOLYTIC ACTIVITY AT DIFFERENT TIMES POST AMPUTATION IN NORMAL REGENERATING, DENERVATED REGRESSING, AND CONTRALATERAL REGENERATING LIMBS OF LARVAL AXOLOTLS USING THE 14c-COLLAGEN FIBRIL ASSAY AT 36°C . . . . . . . . 38 2. REACTION VELOCITY PROGRESS CURVES OF COLLAGENASE FROM 7-DAY AXOLOTL RECENERATES AT THREE CONCENTRATIONS . . . . . 45 3. EFFECT OF COLLAGENASE CONCENTRATION ON REACTION VELOCITY AT DIFFERENT TIMES . . . . . . . . . . . . . . . . 45 4. FORMATION OF DIALYZABLE DEGRADATION PRODUCTS AT 36°C BY CRUDE ENZYME EXTRACT FROM NORMAL RECENERATES AND REGRESSING LIMBS . . . . . . . . . . . . . . . . . . . . . 47 5. EFFECT OF KNOWN COLLAGENASE INHIBITORS ON COLLAGENOLYTIC ACTIVITY OF 8-DAY RECENERATES OF LARVAL AXOLOTLS . . . . . . 53 6. NON-SPECIFIC DEGRADATION OF COLLAGEN BY TRYPSIN USING THE 14C—COLLAGEN FIBRIL ASSAY AT 36 c . . . . . . . . . . . 55 7. COLLAGENOLYTIC ACTIVITY OF LYOPHILIZED NENT RECENERATES USING THE 14C-COLLAGEN FIBRIL ASSAY AT 36 C . . . . . . . . 55 FIGURE LIST OF FIGURES The procedure used to obtain regenerating and regressing larval axolotl limbs to be assayed for collagenase act- ivity. A. Diagram of protocol for obtaining normal regenerates (a). B. Diagram of protocol for obtaining regressing limbs (b) and contralateral regenerates (c). Dashed lines represent amputations when on limbs, and denervations when in the shoulder region. . . . . . . . Disc—gel electrophorectic patterns of radioactive guinea pig collagen and a collagen standard showing characteris- tic o< , . and I ‘bands, and an unknown band, x. (a) the collagen standard, Sigma acid solUble calf skin col- lagen; (b) radioactive guinea pig skin collagen; (c) equal concentrations of the collagen standard plus radio— active guinea pig skin collagen. Each gel contains the same amount of protein. . . . . . . . . . . . . . . . . Light micrographs of larval axolotl limb muscle stained for nerves with Samuel's silver stain, A. Normal innervated limb muscle. Arrows point to several nerve fibers. B. Muscle from a limb which has been denerva- ted and amputated for 6 days. No nerve fibers present. (500x)....................... The morphological development of regenerating and regressing larval axolotl limbs at different times post amputation . . . . . . . . . . . . . . . . . . . . Total Collagenase activity of regenerating and regress- ing larval axolotl forelimbs at different times post amputation using the radioactive reconstituted collagen fibril assay at 36'C. Values at 0 time represent enzyme activity in unamputated limb segments. Standard error of difference between two points on different lines- 179 DPM; standard error of difference between two points on the same line: 22 DPM. o a o o o o o o o 0 Q a o o a o o 0 vi Page 16 24 24 32 35 FIGURE 6. 10. 11. Page Specific Collagenase activity of regenerating and regress- ing larval axolotl forelimbs at different times post amp- utation using the radioactive reconstituted collagen fibril assay at 36’C. Values at 0 time represent enzyme activity in unamputated limb segments. Standard error of difference between two points on different lines= 2431 DPM; standard error of difference between two points on the same line - 304 DPM. . . . . . . . . . . . . . . . . . . . . . . 37 Reaction velocity progress curve of collagenase from 7-day larval axolotl regenerates at three concentrations using the radioactive reconstituted collagen fibril assay at 36°C. (0), 0.05 ml. of crude enzyme extract; (x), 0.10 ml. of crude enzyme extract; (0), 0.20 ml. of crude enzyme extract . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Effect of collagenase concentrations on reaction velocity at different times. Values Obtained from graphs in Figure 7. to, 0 time incubation of reaction mixture; t1, 1 hour incubation of reaction mixture; t , 5 hour incubation of reaction mixture; tlo, 10 hour incubation of reaction mixture; tZO' 20 hour incubation of reaction mixture-ogogooo00.090.000.000090944 Disc-gel electrophoresis patterns of collagen degradation products released by collagenase from 7—day larval axolotl regenerates. Enzyme extract was incubated with guinea pig collagen at 25 C for (a) 0 hrs., (b) 12 hrs., (c) 24 hrs., (d) 36 hrs., (e) 48 hrs., (f) 60 hrs., and (g) 72 hrs. Reaction mixture minus enzyme was incubated at 25'c for (h) 0 hrs., and (i) 72 hrs. Fr, front. . . . . 49 Disc-gel electrophoresis patterns of collagen degradation products released by collagenase from 7-day denervated regressing larval axolotl limbs. Enzyme extract was incubated with guinea pig collagen at 25'C for (a) 0 hrs., (b) 12 hrs., (c) 24 hrs., (d) 36 hrs., (e) 48 hrs., (f) 60 hrs., and (g) 72 hrs. Fr, front . . . . . . . . . . 49 Disc-gel electrophoresis patterns of reaction mixtures containing crude enzyme extracts from larval axolotl limbs incubated without collagen at 25°C. Control guinea pig collagen incubated for 72 hrs. (a); crude enzyme extract from 7-day regenerates incubated for (b) 0 and (c) 72 hrs.; crude enzyme extract from 7-day regressing limbs incubated for (d) 0 and (e) 72 hrs. Fr, front. . . . 49 vii FIGURE Page 12. 13. 14. Disc-gel electrophoresis patterns of collagen incubated with trypsin at 25'C. Guinea pig collagen minus enzyme incubated for (a) 0 and (b) 72 hrs.; guinea pig collagen incubated for 72 hrs. with (c) 0.01% trypsin, and (d) 0.06% trypsin. Fr, front. . . . . . . . . . . . . . . . . . 49 Effect of known collagenase inhibitors on total collagen- ase activity of 8-day larval axolotl regenerates using the radioactive reconstituted collagen fibril assay at 36 C. (A) no inhibitors; (B) plus 2 mM Na4vEDTA; (C) plus 5 mM cysteine; (D) plus 10% fetal calf serum. . . . . . 52 Effect of known collagenase inhibitors on specific coll- agenase activity of 8-day larval axolotl regenerates using the radioactive reconstituted collagen fibril assay at 36°C. (A), no inhibitors; (B) plus 2 mM Na4-EDTA; (C) plus 5 mM cysteine; (D) plus 10% fetal calf serum. . . . 52 viii INTRODUCTION 'Regenerating urodele limbs undergo a period of histolysis following amputation and wound closure which is essential to the regenerative process (Carlson, 1974 for review). During this period phagocytic cells remove cellular and extracellular debris, extracellular matricies disappear, and dedifferentiated cells are released from limb stump tissues. These dedifferentiated cells in turn form a proliferating blastema and develop into a new limb. Hence, histolysis of limb stump tissues allows the emergence of the population of cells which become the regeneration blastema and ultimately the new appendage. The factors leading to and controlling histolysis in regenerating limbs, and the mechanisms involved in the process, however, are not fully understood. Regulation of histolysis, when analyzed at the anatomical or histological level, differs in larval and adult urodeles. In larval limbs, histolysis can be induced by damaging the mesodermal tissues of the limb, and can be prolonged or intensified, or both, by additional experimental manipulations of the limb. Larval limbs which have been amputated and then X—rayed (Butler, 1933; Butler, 1935), UV irradiated (Blum, et al, 1957), treated with colchicine (Thornton, 1943), or denervated (Butler and Schotte, 1941; Schotte and Butler, 1941) undergo extensive histolysis and regression, fail to regenerate, and often are completely resorbed. Similarly, unamputated larval limbs which have been denervated and subjected to mesodermal tissue damage by breaking a skeletal element undergo extensive regression (Thornton and Kraemer, 1951). Histolysis stops in regenerating larval limbs when a blastema forms (Butler and Puckett, 1940), and may be inhibited by the blastema. Hence, denervated, amputated limbs, which ordinarily undergo extensive histolysis and regression (Butler and Schotte, 1941), will not regress if the limb has developed a blastema before it is denervated (Schotte and Butler, 1944). Likewise, if an undifferentiated blastema is transplanted to the stump of a freshly amputated and denervated limb, the limb will not regress (Schotte, et al., 1941). In adult urodeles. on the other hand, the mesodermal tissues of the limb are more stable, and histolysis of limb tissues is difficult to maintain. As in larval urodeles. limb amputation in adults induces a period of histolysis which subsides coincident with blastema formation (Norman and Schmidt, 1967). However, experimental manipulations which cause extensive histolysis in larval limbs, cause histolysis to stop in adult limbs. Amputated adult limbs which have been Xerayed (Schmidt, 1968), or denervated (Rose, 1948; Singer and Craven, 1948) do not undergo extensive histolysis and regression, but instead form scar tissue at the tip of the stump. Despite these differences in its regulation, the process of histolySis itself appears at the histological level to be very similar in larval and adult urodeles. In both larval and adult animals histolysis involves a loss of the formed structures of the limb. In regenerating limbs breakdown of the extracellular matricies of skeleton, dermis, perichondrium, and perineurium has been observed during the regressive phase. Following amputation, the cartilage skeleton of A, maculatum larvae undergoes erosion involving an extensive area in the distal region of the appendicular skeleton, and the matrix in that region gradually disappears (Butler, 1933; Butler and Schotte, 1941; Hay, 1958). Similarly, injured bone in adult N, viridescens undergoes extensive erosion following amputation (Schmidt, 1968). The dermis of adult newts loses its adepidermal reticulum of fibers during the first phase of regeneration. Collagen fibrils fragment and disappear altogether (Norman and Schmidt, 1967). Perichondrium in amputated limbs of A, maculatum larvae becomes free from the distal end of cartilage, which is simultaneously undergoing dissolution, and disappears around the region of dedifferentiated cartilage (Butler, 1933). In both larval A? maculatum (Thornton, 1938), and adult newts (Norman and Schmidt, 1967), a well organized perineurium is lacking around nerve bundles in the region just proximal to the plane of amputation, and in some cases the perineurium is completely lost, and individual nerves separate from each other. Similarly, in experimentally induced regressing larval limbs, continued breakdown of the extracellular matricies of the skeleton and perichondrium has been observed. During the extensive histolysis which accompanies resorption of denervated amputated limbs, extreme vacuolation of skeletal elements continues proximally, with distal portions of skeleton collapsing, until the entire skeleton gradually disappears (Butler and Schotte, 1941). During this process, cartilage was described as "melting away in all directions", and it was suggested that "some type of chemical activity" was primarily responsible for cartilage regression. At the same time in the same experimental limbs, perforations appear in perichondrial envelopes, and the envelopes subsequently fold, breakdown, and finally disappear. Since, as in other vertebrates, collagen is the major structural protein of the extracellular matrices of the limb, and urodele collagen is a typical vertebrate collagen (Mailman, et al., 1974), collagen degradation is presumably one of the mechanisms underlying histolysis of regenerating and regressing limbs. Because native collagen is resistant to general tissue proteases, degradation of collagen fibers in tissues is a complex process which is initiated by the tissue collagenases. To better understand the breakdown of limb collagen, a brief discussion of the properties of collagen and tissue collagenases is appropriate. The information on collagen and tissue collagenases which follows, unless otherwise stated, was obtained from several review articles (Gross, et al., 1963; Seifter and Harper, 1971; Gallop, et al., 1972; Lazarus, 1973; Trelstad, 1973; Harris and Krane, 1974). The collagen molecule, tropocollagen (TC), is composed of three polypeptide chains, called 0: chains, each with a molecular weight of approximately 100,000. The at chains can be covalently cross-linked by telopeptides to form dimers ( fi>components), or trimers (6 components). The overall conformation of TC is a helix with short non-helical regions at both ends. Individual 0: chains are coiled in a left-handed polyglycine or poly-L-proline 11 structure, and the three 0: chains are wound around each other to form a coiled-coil structure, designated the major helix. The helical structure of TC is determined by the repeating gly-x-y sequence of the a: chains, and by the stabilizing influence of proline and hydroxyproline which together make up 20% of the amino acids of TC. Alpha chains represent at least five different gene products, 0: 1(1), 0: 1(II), o: 1(III), o: 1(IV), and o: 2, and so far, four different TC molecules from different combinations of these chains have been identified. These TC molecules include [0:1(I)]2a:2 found primarily in skin, tendon, bone and ligaments (Miller, et al., 1967; Miller, et al., 1971; Trelstad, 1974), [a:1(II)]3 found in cartilage (Miller and Matukas, 1969; Trelstad, et al., 1970; Miller, et al., 1971; Strawich and Nimni, 1971; Linsenmayer, 1974), [o:1(III)]3 found in aorta and fetal human skin (Miller, et al., 1971; Chung and Miller, 1974; Trelstad, 1974), and [oc1(IV)]3 found in basement membranes (Kefalides, 1971; Trelstad, 1974). Once in the extracellular space, tropocollagen subunits aggregate with one another to form fibrils, and the fibrils further organize into an orthogonally oriented lattice work, or are randomly interlaced with each other depending on the tissue and species. While in fibrils, the TC molecules develop cross-links within single molecules and among adjacent molecules, which stabilize the fibrils, and, depending on the degree of cross-linking, cause them to become insoluble. Studies on the physical properties of collagen in solution indicate that the resistance of collagen to proteolytic degradation is due to its helical conformation in the native state. Tropocollagen molecules, which are long rigid helical rods (15 X 3000 X) in the native state, can be denatured over a relatively narrow temperature range to the random coil conformation of gelatin. Native collagen in solution at neutral pH denatures at very close to 37°C; native collagen fibrils at neutral pH do not denature until 550-600C. Gelatin, or denatured collagen, is readily degradedby most tissue proteases, while native collagen, at the neutral pH values characteristic of connective tissues, is largely resistant to attack by most proteases (Gross and Lapiere, 1962; Herb and Reynolds, 1974; Steven, et al., 1975). Some regions of the native TC molecule at neutral pH, however, are more susceptible to general proteolytic attack than others. In high concentrations, trypsin slowly and in small increments degrades as much as 25% of the TC molecule from the carboxy- terminal end. Proteolytic enzymes such as pepsin and chymotrypsin can degrade the short non-helical and telopeptide regions of native collagen, causing the molecule to be more soluble in aqueous solution, but not affecting its helical native form (Drake, et al., 1966). At acid pH values, cathepsin Bl, a lysosomal proteinase, can degrade collagen fibrils and insoluble collagen extensively by multiple cleavages of the helical region (Burleigh, et al., 1974). In general, however, at the neutral pH values of extracellular fluids, initiation of native collagen degradation requires a true collagenase. Collagenases have been defined as "enzymes capable of degrading native collagen fibrils under physiological conditions of temperature and pH, or are enzymes which cleave native collagen molecules in solution through the characteristic helical part of the molecule" (Lazarus, 1973). The mechanism of action of vertebrate collagenase on the native collagen substrate is unique. It cleaves the TC molecule across all three 0: chains at one specific bond in each chain (Gross, et al., 1974), producing a fragment 75% of the original length (T095), including the A (NH2) terminus, and a 252 fragment (TCB), including the B (COOH) terminus (Kang, et al., 1966). The TCA fragments produced include both OCA and [5A fragments. In addition to TCA 7 collagenases (from rat and newt) produce Tc:7 and Tc:2 fragments, although it is not known if the additional fragments are actually 5, some purified produced by the collagenase or by another contaminating protease. Collagenases are neutral metal proteinases (Hartley, 1960). As such, they are most active at neutral to slightly alkaline pH, require Ca++ for activity, and are inhibited by EDTA. In addition, some tissue collagenases are inhibited by reagents containing free sulfhydryl groups, such as cysteine, however, none are inhibited by serine proteinase inhibitors such as soybean trypsin inhibitor. Crude enzyme extracts of collagenase which contain other proteases characteristically degrade native collagen fibrils to small dialyzable peptides at 37°C, and to undialyzable TCA and TCB fragments at 25°C. Vertebrate collagenases, like collagen, are ubiquitous, and have been detected in a number of collagen containing tissues, and in cells involved in collagen breakdown. Among the tissues and cells collagenases have been found in are human skin am and 32.13-32.19. (Eisen, et al., 1968; Eisen, et al., 1971), papillary dermis of skin (Reddick, et al., 1974), culture medium of fibroblast-like cells (Hook, et al., 1973; Reddick, et al., 1974; Werb and Burleigh, 1974; Herb and Reynolds, 1974), basement lamella (Nagai and Hori, 1972), culture medium of granulation tissue and wound epithelium of cutaneous wounds (Grillo and Gross, 1967; Donoff, et al., 1971) culture medium of epithelial cells of metamorphosing tadpole tail fin (Eisen and Gross, 1965), inflamed rheumatoid synovium in zizg (Bauer, et al., 1971), bone in culture (Walker, et al., 1964; Vaes, 1972), culture medium of activated macrophages (Senior, et al., 1972; Wahl, et al., 1974; Bauer, et al., 1975; Wahl, et al., 1975; Herb and Gordon , 1975), Kupffer cells of liver (Fujiwara, et al., 1973), culture medium of giant cells (Salthouse and Matlaga, 1972), polymorphonuclear leukocyte granules (Lazarus, et al., 1968; Robertson, et al., 1972), cultured remodeling tissues of metamorphosing tadpoles (Gross and Lapiere, 1962), and the culture medium of resorbing postpartum rat uterus (Jeffrey and Gross, 1970; Jeffrey, et al., 1971). Collagenase degradation of collagen in 11-29. and 32.11119. is primarily regulated by the amount of enzyme present, the form the enzyme is in, and the presence of inhibitors. Except in polymorphonuclear leukocytes, where it is stored in granules, collagenase is synthesized and secreted without storage. This synthesis and secretion has been shown to be inducible in some collagenase producing cell types. Phagocytosis of and continued intravacuolar storage of indigestible particles stimulate macrophages (Herb and Gordon, 1975) and synovial fibroblast-like cells (Werb and Reynolds, 1974) to produce collagenase in tissue culture. Similarly, products secreted by mitogen or antigen stimulated lymphocytes (lymphokines) induce macrophages to produce collagenase gaugi££g_(Wahl, et al., 1975). Some collagenases are secreted and stored in tissues as an inactive zymogen or procollagenase, which can be converted to the enzymatically active form by a tissue protease activator (Harper, et al., 1971; Harper and Gross, 1972; Vaes, 1972; Lazarus and Goggins, 1974; Bauer, et al., 1975). Inhibitors of collagenase which form stable, inactive complexes with the enzyme, are present in extracellular fluids. Active collagenase has been shown to be inhibited by the mammalian serum anti-proteinase, 0: -macroglobulin 2 (Bauer, et al., 1971; Eisen, et al., 1971; Abe and Nagai, 1973), by a low molecular weight protein in rheumatoid synovial fluid (Harris, et al., 1969; Abe and Nagai, 1973), and by a factor, about the same size as the collagenase itself, in the culture medium of fibroblast-like cells (Bauer, et al., 1975). The role of collagenase in connective tissue degradation is thought to be to facilitate primary collagenolysis. It has been suggested that connective tissue collagen fibrils are degraded in a two stage process. In the first stage, collagenase is secreted into the extracellular matrix, and cleaves collagen molecules in the fibrils into characteristic TCA and TCB fragments. These fragments then disperse in the surrounding extracellular fluids, and denature into gelatin because of their lower denaturation temperature (32°C). In the second phase of degradation, the denatured fragments are broken down by other extracellular neutral tissue proteases into small peptides (Sakai and Gross, 1967; Lazarus, et al., 1968; McCroskery, et al., 1973). Or, alternatively, primary collagenolysis results in small fibril fragments being dissociated from collagen fibrils, and phagocytosed by macrophages. In the second stage of degradation, in this case, collagen is degraded within phagolysosomes at acid pH by proteases such as cathepsin Bl (Burleigh, et al., 1974). Native collagen in solution or as fibrils must be used as the substrate in assays for collagenase. Breakdown of collagen molecules in solution can be followed by measuring the loss of specific viscosity of the solution. Breakdown of collagen fibrils can be followed by measuring the release of radioactive degradation products into solution from radioactive collagen gels, and by demonstrating that the soluble components are smaller molecules than the original TC molecules and not just solubilized TC molecules. Evidence that the collagen molecule has been characteristically cleaved into TCA and TCB fragments can be obtained from SLS crystallites, or from disc-gel electrophoresis of the reaction products. As was discussed earlier, there is considerable histological evidence of breakdown of the collagenous structures of the limb during the early histolytic phase of regeneration, and throughout the continuing histolysis which characterizes experimentally induced regression. The 10 biochemical evidence of collagen degradation during histolysis, however, is contradictory. Studies of collagen breakdown during regeneration, where collagenase activity and collagen metabolism were measured, do not agree with each other. Cultured regenerates from adult newts were assayed for their ability to degrade native collagen fibrils, and were found to have maximum collagenolytic activity 15 days post amputation which gradually diminished by 30-35 days post amputation (Grillo, et al., 1968). In addition, highest collagenase activity was found in the stump tissue immediately adjacent to the blastema, with the blastema next most active. These results coincide temporally and spatially with limb histolysis during regeneration, and support the idea that collagenase is initiating degradation of limb collagen during histolysis. 0n the other hand, a study of collagen metabolism during regeneration of boneless, adult newt limbs, indicates that the amount and synthesis of stump collagen does not change significantly during regeneration (Johnson and Schmidt, 1974). In addition, the same study shows relatiVely high levels of newly synthesized collagen in the developing blastema with no concomitant formation of insoluble collagen fibrils,which together indicate degradation. These results were interpreted to mean that collagen degradation in early regeneration is occurring primarily in the developing blastema, and not in the existing extracellular matrix of the limb stump. Another study of collagen metabolism during regeneration of adult newt limbs, where just regenerates and not stump tissue were considered, shows that collagen turnover is low during formation of the blastema, and that the period of maximum collagen turnover occurs 5-7 weeks post amputation during digit formation (Mailman and Dresden, 1976). 11 Since little collagen turnover is occurring in early blastemata in this study, these results support the conclusion of the collagenase study (Grillo, et al., 1968), in which it was demonstrated that collagenase present during the early stages of regeneration is acting primarily on stump collagen. However, high collagen turnover in digit regenerates in this study, suggests the presence of a tissue collagenase during the period 5-7 weeks post amputation which was not detected in the previous collagenase assay (Grillo, et al., 1968). Part of the reason for the contadictory results regarding when and where collagen is being degraded in regenerating limbs may reside in the experimental procedures employed. In the collagenase study, for instance, cultured blastemata were used to obtain active enzyme (Grillo, et al., 1968). Consequently, it is not clear whether collagenase activity de- tected by the assay actually reflects the endogenous levels of active 'enzyme in the regenerating limbs, or dg_ngzg synthesis of collagenase by the tissue explants in response to culture. Similarly, the use of boneless limbs to study collagen metabolism in the limb stump during regeneration (Johnson and Schmidt, 1974) may yield misleading results, since the bones are a majoe collagen containing structure of the limb and are known to be eroded during histolysis (Schmidt, 1968). It is not clear from the biochemical studies mentioned above if limb stump collagen is being degraded during histolysis in regenerating adult newt limbs. Since part of this uncertainty is due to experimental procedure, and since histolysis can be experimentally manipulated in amputated larval urodele limbs, the experiments in this investigation were designed to study collagenase activity in regenerating and experi- mentally-induced regressing larval Ambystoma mexicanum limbs using a 12 direct extraction technique to obtain active enzyme. Since most cell types capable of producing collagenase do not store it (Harris and Krane, 1974), direct extraction of limb tissues should reflect endogenous levels of collagenase activity. The time courSe of collagenase activity in normal regenerating and denervation-induced regressing larval limbs was determined in order to assess the phases of regeneration and regression in which collagenase activity was significantly above normal limb tissue levels. The pattern of collagenase activity in regenerating limbs which were contralateral to the experimentally—induced regressing limbs was also determined as a control on the pattern of activity in the regressing limbs. In addition, to verify that a collagenase was being worked with in this system, larval A. mexicanum collagenase was partially character- ized in this investigation. MATERIALS AND METHODS Source and Care of Animals Ambystoma mexicanum larvae (axolotls), used as the source of regenerating and regressing limbs in this study, were spawned and raised in the laboratory at 20i20C. Newly hatched larvae were raised in large tanks on live brine shrimp. When the average length of the individuals in a spawning reached 3 cm., all the larvae in that spawning were transferred to individual plastic containers to prevent them from cannibalizing each others limbs, and they were switched to a diet of sliced beef liver. Larvae were used in experiments when they reached a length of 5-6 centimeters. Adult newts, Notophthalmus viridescens, used in this study were ordered from William Lee of Oak Ridge, Tennessee. Young guinea pigs, from which 14C-collagen was prepared, were obtained through The Center for Laboratory Animal Research at Michigan State University. Preparation of Regenerating and RegressinggLimbs Three types of experimental limbs were assayed for collagenase activity in this study: 1) normal regenerating limbs, 2) denervated, amputated regressing limbs, which will simply be called regressing limbs, and 3) contralateral regenerating limbs. The basic protocol for 13 14 producing these limbs is shown in Figure 1. To produce all three types of limbs, larvae were immobilized in an aqueous solution of 1:500 ethyl- m-aminobenzoate methanesulfonate (EAM) (Eastman), and were amputated through both forelimbs midway between the wrist and elbow. Protruding skeletal elements that resulted from amputation were trimmed flush with the amputation surface to facilitate faster and more uniform regeneration. To produce normal regenerates, larvae were returned to individual containers at this point, and allowed to regenerate for the desired length of time. To produce regressing limbs and contralateral regenerates, larvae were denervated at the time of amputation by severing the third, fourth, and fifth spinal nerves in the shoulder region of the right limb. These larvae were then returned to individual containers, and allowed to regress on their right side, and regenerate on their contralateral left side. Denervated limbs were maintained in a nerveless condition by re-denervating them every six days. All experimental animals were maintained at 22i1°C while their limbs were regenerating, or regressing. Regenerating and regressing limbs were collected for homogenization using sterile technique. To prevent bacterial and fungal contamination of limb tissues, all glassware and operating tools were autoclaved before use, and all solutions were sterilized with millipore filters (0.45u pore size). In addition, larvae were surface sterilized with antibiotics (Na-penicillin G, Sigma, 0.2 mg/ml.; streptomycin sulfate, Sigma, 0.5 mg/ ml.; fungizone, GlBCO, 0.02 mg/ml.) four hours before their limb tissues were removed. To collect limb tissues, larvae were immobilized in EAM, and were examined under a dissecting microscope to determine the morphological stage of regenerates, and the regressive progress of FIGURE 1. 15 The procedure used to obtain regenerating and regressing larval axolotl limbs to be assayed for collagenase activity. A. Diagram of protocol for obtaining normal regenerates (a). B. Diagram of protocol for obtaining regressing limbs (b) and contralateral regenerates (c). Dashed lines represent amputations when on limbs, and denervations when in the shoulder region. l7 denervated limbs. After examination, regenerating limbs were amputated at the elbow, and regressing limbs were amputated either at the elbow, or 3 mm from the end of the stump depending on how much regression had occurred. All limb tissues distal to these amputations, which includes stump tissues as well as regenerates and regressing tissues, were included in the crude enzyme extracts. Limb segments from the mid forelimb to the elbow of normal unamputated limbs were collected as controls on the amount of collagenase activity in normal limb tissues. All limb tissues were transferred to sterile plastic petri dishes as soon as they were removed, were frozen at -20°C, and were immediately lyophilized. Regenerates were obtained from adult newts in the same manner as mentioned above. Preparation of Crude Enzyme Extracts Crude enzyme extract was prepared from regenerating, regressing, and control limb tissues by the direct extraction method of Nagai and Hori (1972). These extracts differed from those of Nagai and Hori, in that they were prepared from lyophilized tissue instead of freeze-thawed wet tissue. Using sterile glassware and buffer, a homogenate was prepared of ten lyophilized regenerates in 0.5 ml. of 0.05 M Tris-H01 buffer, pH 7.5, containing 0.2 M NaCl, 5 mM CaC12°2H20, and antibiotics (Na-penicillin G, streptomycin sulfate, and fungizone at the concentrations specified in the preceding section) in a 1 ml. capacity glass tissue homogenizer at 0°C. Such homogenates were incubated at 37°C for 66 hrs. in sterile test tubes to extract collagenase. After incubation the homogenates were 18 centrifuged, and the supernatants collected for use as crude enzyme extracts in the collagenase assay. The protein content of each crude enzyme extract was determined using the Lowry method (Lowry, et al., 1951) at one-tenth the suggested volumes. 14 Prgparation of C-Collagen Radioactive guinea pig skin collagen for the radioactive reconstituted collagen fibril assay was prepared from young growing animals (250-300 gm.) which were injected intraperitoneally with 100 no of glycine-14C-UL six hours before their skins were collected (Gross and Lapiere, 1962). Collagen was extracted from the dermis with 0.5 M acetic acid (Gross and Kirk, 1958), was purified by repeated salt precipitation (Jackson and Fessler, 1955), and was lyophilized. Four grams of purified radioactive guinea pig skin collagen with activity of 600 DPM/mg. were obtained from 5 coriums. The identity and purity of the guinea pig collagen was checked using disc-gel electrophoresis. Guinea pig collagen was compared with acid soluble calf skin collagen (Sigma) (Figure 2) using the gel electrophoresis method of Nagai, et a1. (1964). Both collagen preparations contained characteristic on , [3 , and 8 bands which co-migrated, few contaminant protein bands, and an unidentified faster migrating species, x. Radioactive Reconstituted Collagen Fibril Assay for Collagenase Collagenase activity in urodele limb tissues was measured using the radioactive reconstituted collagen fibril assay (Nagai, et al., 1966). All glassware and solutions used in the assay were sterile. To prepare 19 14C-collagen fibrils, 2 mg./ml. of lyophilized radioactive guinea pig skin collagen were dissolved in 0.5 M acetic acid by stirring gently overnight at 4°C. The resulting collagen solution was dialyzed with stirring against 0.15 M phosphate buffer, pH 7.6, for 24 hrs. at 4°C, and then against an 0.4 M NaCl solution at 4°C for 24 hours. Aliquots of the final collagen solution, 0.25 ml. (300 DPM), were pipetted into 3-ml. test tubes, and were incubated at 36°C for 12 hrs. to gel. To the gel, which consisted of native, reconstituted collagen fibrils, were added: 1) 0.25 ml. of 0.1 M Tris buffer, pH 7.5, containing 1 mM CaClZ'ZH 0, and 2 antibiotics (final reaction mixture concentration: Na-penicillin G, Sigma, 0.2 mg/ml.; streptomycin sulfate, Sigma, 0.5 mg/ml.; fungizone, GlBCO, 0.02 ml/ml.), and 2) 0.1 ml. of the enzyme extract to be assayed. Reaction mixtures were incubated at 36°C for 5-66 hrs. depending on the experiment. At the end of the incubation period reaction mixtures were centrifuged at 25,000 x g for 15 min. to separate undegraded collagen fibrils from peptide breakdown products released into the reaction buffer. The supernatant was decanted from the pellet, and both fractions were saved to be analyzed. The collagen pellet was solubilized in 0.15 ml. of glacial acetic acid (99.7%), was added to 15 ml. of dioxane scintillation cocktail, and was counted in a liquid scintillation spectrometer. The CPM thus obtained were converted to DPM to standardize all results. Total collagenolytic activity, the total number of counts released as breakdown products, was determined by subtracting the DPM of experimental reaction mixtures containing crude enzyme extracts from the DPM of a control reaction mixture containing only enzyme buffer. This method of determining how much degradation product was formed, by 20 measuring the amount of substrate left unreacted, was used because of the low radioactivity of the substrate collagen (600 DPM/mg.). It differed from the method of Nagai, et a1. (1966), who measured the radioactivity of the degradation products in the supernatant. The specific activity of each crude enzyme extract was determined also. Calculation of Limits of Detection of Radioactivity Because of the low radioactivity of the collagen substrate in the collagenase assay (300 DPM per reaction mixture), the precision of the counting setup (least detectable amount of radioactivity) was calculated for all experiments (Brewer, et al., 1974). Using the formula: 1/2 K(2Rb/tb) Precision = c.f. where Rb = background counting rate, tb efficiency of the counting system, and K . proportionality constant for - counting time, c.f. = overall statistical significance, all counts (CPM) in all experiments were found to be higher than the least detectable amount of radioactivity of the system at p - 0.01. 14 Assay for Dialyzable Degradation Products of C-Collagen The supernatants of reaction mixtures run at 36°C were pooled and checked for dialyzable counts to verify that the collagen fibril substrate of the assay was being degraded and not just solubilized during the incubation period. Half of each pooled supernatant was dialyzed against distilled H O at 4°C for 72 hours. Then 0.5 ml. aliquots of 2 dialyzed and undialyzed supernatant were added separately to 15 ml. portions of dioxane scintillation cocktail, and were counted. The 21 dialyzable counts in the supernatant were determined by subtracting the counts remaining in 0.5 ml. of dialyzed supernatant from the counts in 0.5 ml. of undialyzed supernatant. A control was run on the above assay procedure to rule out the possibility that intact solubilized collagen was precipitating on or sticking to the dialysis tubing during dialysis, and thereby removing itself from solution, and mimicing the results that would be expected if dialyzable peptides were in the supernatants. Undegraded 14C-collagen, 3 mg/ml., was dissolved in 0.5 M acetic acid, and half of the resulting solution dialyzed against distilled H20 for 72 hrs. at 4°C. Aliquots, 0.25 ml., of the resulting dialyzed and undialyzed solutions were added to 15 ml. of dioxane scintillation cocktail, and were counted. .After adjusting the DPM of the dialyzed collagen solution for the increase in volume it had undergone during dialysis, the counts in dialyzed and undialyzed solutions were found to be not significantly different from each other at p=0.05 (Table 4), indicating that intact collagen in solution is not being removed from solution during dialysis. Assay for Characteristic TCA Dggradation Products Collagenase characteristic TCA degradation products were detected using disc-gel electrophoresis (Nagai, et al., 1964). Reaction mixtures were prepared in exaCtly the same manner as for the radioactive reconstituted collagen fibril assay, and were incubated at 25°C, so TCA degradation products would not be degraded to small peptides, for 12-72 hours. At the end of the incubation period, 18 ul of glacial acetic acid (99.7%) were added to each reaction mixture to make it 0.5 M acetic acid, and the mixtures were placed at 4°C overnight to dissolve all collagen. 22 An aliquot, 0.1 ml., of each reaction mixture was run on a separate polyacrylamide gel, and the resultant gels were stained, destained, and photographed for observation. Histology Since regressing limbs were only re-denervated every 6 days, a study was conducted to make sure that nerves were not growing back into limbs between denervations. Denervated limbs were checked histologically at the light microscope level for the presence of nerves. The limbs of 25 larval axolotls were amputated and denervated, and five limbs were collected on each day 2-6 days post operation. Upon collection, limbs were fixed in Bouin's fixative for 24 hrs., incubated in Lenois' solution for 3 days to remove picric acid, dehydrated, embedded in paraplast, and sectioned at 7 microns. Sectioned tissues were stained for nerves using Samuel's silver stain (Samuel, 1953). Control innervated limbs were fixed and stained in the same way. .Slides were examined with a light microscope and photographed. It was found that by two days post amputation and denervation no small nerve fibers remained in limb muscle, and by 6 days no new nerve fibers had grown back into the limb muscle (Figure 3), demonstrating that regressing limbs remained nerveless between denervations. Morpholggical Staging of Egggnerates and Regressigg Limbs As a rough guide of how fast larval axolotl limbs were regenerating or regressing, experimental limbs were staged while being collected to be assayed for collagenase activity. Limbs were classified as being one of the following stages: 1) no change- no change in stump length, no FIGURE 2. FIGURE 3. 23 Disc-gel electrophoretic patterns of radioactive guinea pig skin collagen and a collagen standard showing characteristic o<, fl , and 3 bands, and an unknown band, x. (a) the collagen standard, Sigma acid soluble calf skin collagen; (b) radioactive guinea pig skin collagen; (c) equal concentrations of the collagen standard plus radioactive guinea pig skin collagen. Each gel contains the same amount of protein. Light micrographs of larval axolotl limb muscle stained for nerves with Samuel's silver stain. A. Normal innervated limb muscle. Arrows point to several nerve fibers. B. Muscle from a limb which has been denervated and amputated for 6 days. No nerve fibers present. (500x) 24 25 noticable accumulation of tissue beyond the level of amputation, 2) regressing limb- shortening of stump, no noticable accumulation of tissue beyond the level of amputation, 3) bulb blastema- accumulation of tissue beyond the level of amputation, rounded outgrowth, 4) cone blastema- cone shaped outgrowth, larger than bulb, S) paddle blastema- dorsal-ventral flattening of distal outgrowth, same size or larger than cone blastema, 6) 2-digit regenerate- formation of a notch in the distal edge of the paddle, same size or larger than paddle, and 7) 3-and 4-digit regenerate- formation successively of a second and third notch posterior to the first notch in the distal edge of the paddle, larger than the preceding stages, beginning of digit outgrowth. Statistics The patterns of collagenase activity during the period 2-16 days post amputation in normal regenerating, regressing, and contralateral regenerating limbs were analyzed using a split plot analysis of variance. This analysis was performed to demonstrate if generally the trend of collagenase activity over time is the same for the three experimental groups, and to compare specifically 1) collagenolytic activity in two experimental groups on a given day, and 2) collagenolytic activity within an experimental group on any day with the control levels of collagenolytic activity in that group. Collagenolytic activity on the same day in different groups were compared using a one-sided t-test of a comparison; collagenolytic activities in the same group were compared with a control using a One-sided Dunnett's t-test for comparisons with a control. Other statistical comparisons of collagenolytic activity done in this study used the Student's t-test. 26 Experiments The Pattern of Collagenase Activity in Regeneratingeand Rggressing Limbs Series 1: Collagenase Activity in Normal Rggenerating Limbs Normal regenerates were collected daily over the period 2-16 days post amputation, and together with segments from unamputated limbs were assayed for collagenase activity using the reconstituted radioactive collagen fibril assay at 36°C. To produce each crude enzyme extract for the assay, 10 right limbs from 10 larvae were pooled at each time post amputation. The entire experiment was run three times, and the mean (n83) values of collagenase activity at each time post amputation were determined (Table 1). A total of 480 larvae were used in this series. Series 2: Collgggnase Activity in Rggressing_and Contralateral Eggeneratigg;Limbs Denervated regressing limbs and the contralateral regenerates on the same animals were collected on the same time schedule, and assayed for collagenase activity in the same way as the normal regenerates in Series 1. Ten denervated regressing right limbs and 10 regenerating contralateral left limbs from 10 larvae were pooled separately at each time period to produce the crude enzyme extracts for the collagenase assay. As in Series 1, these experiments were run three times, and the mean (n83) values of collagenase activity at each time post amputation were determined (Table l). A total of 450 larvae were used in this series. 27 Characterization of Larval Axolotl Collaggnase Series 3: Kinetics of Larval Axolotl Collagenase The kinetics of collagenase activity from normal 7-day regenerates was investigated using the reconstituted radioactive collagen fibril assay at 36°C. Three crude enzyme extracts were produced by pooling 80 regenerates from 40 larvae for each extract, and each extract was assayed at three concentrations, 0.05 ml., 0.10 ml., and 0.20 ml. Reaction mixtures at each concentration were incubated for 5, 7, 10, 20, 30, 40, 50, 60, and 66 hours. Mean values (n33) of collagenase activity at each time of incubation and concentration were determined, and plotted as progress curves of reaction velocities. From these curves the effect of enzyme concentration on reaction velocities at different times was determined. A total of 120 larvae were used in this series. Series 4: Formation of Dialyzable Deggadation Products at 36°C The supernatants of reaction mixtures of crude enzyme extracts which had been assayed for collagenase activity using the reconstituted radioactive collagen fibril assay at 36°C were used to assay for dialyzable degradation products according to the procedure outlined earlier in this section. Supernatants of reaction mixtures containing enzyme extract from normal 8-day regenerates, and from 8-16 day regressing limbs were obtained from Series 1 and Series 2 respectively. Series 5: Formation of Collagenase Characteristic TCA Degradation Products at 25"C Normal 7-day regenerates and 7-day regressing limbs were assayed for the ability to produce collagenase characteristic TCA degradation 28 products using the reconstituted collagen fibril assay and disc-gel electrophoresis. Crude enzyme extracts of regenerating and regressing limbs were both produced by pooling 40 limbs from 20 larvae. Reaction mixtures containing crude enzyme extract from regenerating and regressing limbs were incubated at 25°C for 0, 12, 24, 36, 48, 60, and 72 hours. The following controls were also run: 1) reaction mixture minus enzyme incubated for 0 and 72 hrs., 2) reaction mixture containing crude enzyme extract from 7-day regenerates minus collagen incubated for 0 and 72 hrs., 3) reaction mixture containing crude enzyme extract from 7-day regressing limbs minus collagen incubated for 0 and 72 hrs., and 4) complete reaction mixtures incubated for 72 hrs. to which 0.01% and 0.06% trypsin (Sigma, 2X crystallized, from Bovine pancreas) solutions had been added, giving final reaction mixture concentrations of 0.002% and 0.01% trypsin. All experiments were run twice. Forty larvae were used in this experiment. Series 6: Effect of Known Collagenase Inhibitors Crude enzyme extract from normal 8-day regenerates was assayed for collagenase activity in the presence of known vertebrate collagenase inhibitors using the reconstituted radioactive collagen fibril assay at 36°C. Reaction mixtures contained the following final concentrations of inhibitors: 2 mM Nah-EDTA. 5 mM cysteine, and 10% fetal calf serum (GIBCO). Three crude enzyme extracts were produced by pooling 20 regenerates from 10 larvae for each extract. This experiment was run three times, and the mean collagen degradation (n83) with each treatment determined. Thirty larvae were used in this series. 29 Series 7: Non-Specific Collagen Degradation berrypsin Using the reconstituted radioactive collagen fibril assay at 36°C, the following solutions were assayed for collagenase activity: 1) 0.01% trypsin, 2) 0.06% trypsin, 3) 0.06% trypsin + 0.01% soybean trypsin inhibitor, 4) crude enzyme extract from normal 7-day regenerates + 0.01% soybean trypsin inhibitor. Three crude enzyme extracts from normal 7-day regenerates, the same extracts used in Series 3, were used in this series, and each extract was incubated for 30, 50, and 66 hours. Each trypsin containing reaction mixture was run three times. The mean (n=3) collagen degradation with each treatment was calculated. Collagenase Activity of Adult Newt Reggnerates Series 8: Lyophilized Regenerates Normal 16-day adult newt regenerates, the stage were maximum collagenolytic activity was obtained in cultures, were lyophilized, directly extracted, and assayed for collagenase activity using the reconstituted radioactive collagen fibril assay at 36°C. Reaction mixtures were incubated for 20, 48, and 72 hours. To produce crude enzyme extracts for the assay, 18 regenerating limb segments were homogenized in 5 ml. of enzyme buffer. The experiment was run twice with two different enzyme homogenates, and mean values (n-2) of enzyme activity were determined. A total of 18 newts were used in this series. RESULTS Morphological Develgpment Normal and contralateral larval axolotl regenerates appear morphologically to be developing at essentially the same rate during the period 2—16 days post amputation (Figure 4). In both cases a bulb blastema appears at 5 days post amputation, a cone at 8 days, a paddle at 9 days, and digits at 12 days post amputation. Denervated amputated limbs, on the other hand, during the same time period do not change morphologically until 8 days post amputation (Figure 4). At that time, the limbs begin to shorten between the amputation surface and the elbow, and continue to shorten for the next 8 days, until, by 16 days post amputation, they have regressed to the elbow. The Pattern of Collaggnase Activity in Regggerating and Regressing;Limbs Like the rate of morphogenesis, the general pattern of collagenase activity in normal and contralateral larval axolotl regenerates is essentially the same (Figures 5 and 6, Table 1). Collagenase activity in both types of regenerating limb rises rapidly after amputation to maximum levels at 6-7 days post amputation, and thereafter returns rapidly to normal tissue levels. Total and specific collagenase activity in normal regenerates are significantly above control tissue levels (p80.05) 3-9 days and 3-8 days post amputation respectively. Similarly, in 30 31 .soaumusmsm umoa moawu usmuommav um mnaaa Huoaoxm Hm>uma wcammmummu pan wsaumuocomou mo unmeaoao>ov HdOfiwoaonmuoa osH .q MMDUHm 32 q mMDuHm :ofluousaa< umom when 3.3.3»? «arses. a m A o m s n N b P by I b b b _ /A .g //¢//////// A AEHA magnumuwom ennui—omen mmm oumuocowom Honouoamuuaoo D oumumaowom HQEHOZ a L . a FE w _ use. I //////M/ .cowom sunsets .aowom semanIM .aowom sawsn-u «Housman savoum maoumofim maoo «Housman Adam sass was Iaoouwom owcmzo 02 38839 33 contralateral regenerates, total and specific collagenase activity are significantly above control tissue levels (p80.05) 3-10 days and 3-9 days post amputation respectively. The general pattern of collagenase activity in regressing limbs differs from that of regenerating limbs (Figures 5 and 6, Table 1). Like regenerating limbs, collagenase activity in regressing limbs rises rapidly after amputation to maximum levels at 8 days post amputation, however, unlike regenerating limbs, collagenase activity then remains high instead of returning to control tissue levels. In regressing limbs total and specific collagenase activity are higher (p=0.05) than control tissue levels 2-16 days and 3-16 days post amputation respectively. The trends in collagenase activity over time in normal regenerating, regressing, and contralateral regenerating limbs were compared to determine if and when the three groups of limb tissues differed from each other in enzyme activity. The F statistics obtained from a split plot analysis of variance for a groups times days interaction, strongly indicates that collagenase activity over time is not the same for the three groups of limbs, either for total activity (p=0.001), or for specific activity (p=0.01). A more specific analysis to determine on which days post amputation collagenase activity is different in the three experimental groups, however, failed to show significant differences below p=0.l. An analysis of collagenase activity in regenerating and regressing limbs at 11-16 days post amputation, the time of greatest difference in enzyme activity among these experimental groups, showed significant differences only at somewhat greater than p=0.l. It is felt, however, that since the F statistic strongly indicates a difference in these experimental groups, and since the greatest difference in enzyme 34 .zma NN ImGHH OBMm saw so muawoa o3u coosuon oucmuommwo mo uouuo wumocmum “Zma owa ummsHH ucouommav so musaom osu cmoauon mucouomwfiv mo uouuo pumpsmum .mucoawom mafia woumusmemcs ca huw>fiuum Oaxaca uaomouaou mafia o um mosam> .ooom um momma Happen commaaoo unusuauusooou o>auopoaomu Ono mafia: coauousaam umoa mosau udopmmwao um mosaamuow auoaoxm am>uma wafimmouwou one waaumumcowou mo huw>wuoo ommaownaaoo Houoa .m mMDuHm 35 m BSDHW soauousmad uoom manna m." ma .3 a «a 5 oh .m m a m m w h N b II IIIIII ”, J . ,, . \ .. / , I, an / \\ .. I, w I \o I x .. \ \ o / \\\.\. // \w.... r... a:.......... .. ....... waflwamuwum 0.00000. ououoaowom HonouuauuusOOIIIII oumuoaowmm Hwauoz manogoaé IIIIIIIIIIIII IIIIIIIIIIIIII IIIIIt—muwuumnam Hoooa aIdmvsl'Siq 99/Z_OI x WHO 36 .Zmo com ImGHH OBMm onu so mucaom 03u somzuon mucoummmwv mo uouuo pumpsmuw “Xmo quu umocfia uamwomwaw so musfiom osu comauon musmu0mmwo no woman vumoamum .muaoamom paHH emumusmemc: ca muw>fiuom mahucm mammoumou 08am 0 up mosam> .ooom um xmmmm kuoam commaaoo ovusuflumaooou m>auum0Homu mam moan: coaumusmsm umom moeHu uaouowwfip um mnaHHmuom Huoaoxm am>uma wafimmmummu new wcaumuocowou mo hua>fiuom ommammeHou ofimwooam .o mMDQHm 37 0H mu ha ha NH AeHA.:.:.:. mcaanouwom 330:0me llll Honoumawuuaoo ounuoaowom Hushoz o mmDQHm . coqumuame< umom mama HH 0H o m n w m c m u b uraaozg 'SWY‘SJH 99/€_01 X WAG 38 TABLE 1 COLLAGENOLYTIC ACTIVITY AT DIFFERENT TIMES POST AMPUTATION IN NORMAL REGENERATING, DENERVATED REGRESSING LIMBS OF LARVAL AXOLOTLS USING THE AND CONTRALATERAL REGENERATINC i4C-COLLAGEN FIBRIL ASSAY AT 36 C rw".1 IDayJV Total Activity- Protein Cbntent Specific ActiVItyJVI Post DPM Substrate of Crude Enzyme DPM substrate Degrad- Amp. Degraded/66 hrs. Extract— mg/ml ed/66 hrs./mg. Protein RunIl Run#ZIRun#3 Mean Run#ltRun#2 Run#3 Run#1 Run#2 Run#3 Mean 0 20 18 8 15 0.64 0.62 0.74 304 290 108 234 INormal Regenerate 2 45 88 90 74 0.96 0.59 0.73 469 1492 1233 1065 3 127 124 189 147 1.04 0.64 0.77 1240 1938 2455 1878 4 177 199 182 186 0.92 0.88 0.78 1924 2261 2333 2173 5 205 139 212 185 0.98 0.79 0.71 2092 1759 2986 2279 6 222 238 221 227 1.04 1.17 0.81 2135 2034 2728 2299 7 227 226 210 221 0.82 1.14 0.87 2768 1982 2414 2388 8 195 176 171 181 1.30 1.03 0.90 1500 1709 1900 1703 9 57 79 120 85 1.18 0.80 0.82 483 988 1463 978 10 0 112 99 70 1.49 1.19 0.90 0 941 1100 680. ll 18 51 26 32 1.49 0.86 0.87 121 593 299 338 12 0 16 0 5 1.30 1.13 0.85 0 142 0 47 13 38 ‘0 0 13 1.42 1.08 0.86 268 0 0 89 14 51 59 12 41 1.54 0.76 1.00 331 776 120 409 15 31 48 0 26 1.76 0.87 1.09 176 552 0 243 16 0 25 0 8 1.80 1.36 1.03 0 184 0 61 Regressing Limb 2 76 120 108 101 0.86 0.83 0.72 884 1446 1500 1277 3 76 175 125 125 0.79 0.80 0.72 962 2188 1736 1629 4 143 209 200 184 0.89 0.79 0.72 1607 2646 2778 2344 5 172 168 189 176 0.80 0.74 0.79 2150 2270 2392 2271 6 166 181 212 186 0.60 0.69 0.74 2767 2623 2865 2752 7 212 168 218 199 0.67 0.83 0.74 3164 2024 2946 2711 8 250 252 221 241 0.82 0.71 0.76 3049 3549 2908 3169 9 261 229 236 242 1.01 0.68 0.70 2584 3368 3371 3108 10 250 208 244 234 0.78 0.70 0.82 3205 2971 2976 3051 11 258 202 238 233 0.81 0.82 0.78 3185 2463 3051 2900 12 222 209 230 220 0.92 0.98 0.92 2413 2133 2500 2349 13 194 215 212 207 0.76 1.02 0.92 2553 2108 2304 2322 14 244 130 245 206 0.88 0.58 0.90 2773 2241 2722 2579 15 240 173 200 204 0.86 0.76 0.78 2791 2276 2564 2544 16 211 181 221 204 1.04 0.59 0.80 .2029 3068 2762 2620 39 TABLE 1 (cont'd.) r - [55y Total Activity- Protein Content Specific Activity— Post DPM Substrate of Crude Enzyme DPM substrate Degrad- Amp. Degraded/66 hrs. Extract- mg/ml ed/66 hrs./mg. protein ‘Fun#l Run#2 Run#3 Mean Run#l Run#2 Run#3 Run#l Run#2 Run#3 Mean Contralateral Regenerate 2 35 99 97 77 0.65 0.74 0.68 538 1338 1426 1101 3 56 180 125 120 0.87 0.81 0.72 644 2222 1736 1534 4 120 167 180 156 1.01 0.68 0.90 1188 2456 2008 1881 5 137 155 199 164 0.80 0.86 0.83 1712 1802 2398 1971 6 166 182 206 185 0.78 0.87 0.80 2128 2092 2575 2265 7 201 188 209 199 0.69 0.86 0.80 2913 2186 2612 2570 8 190 173 161 175 0.82 0.89 0.94 2317 1944 1713 1991 9 135 148 100 128 0.97 0.92 0.77 1392 1609 1299 1433 10 92 107 64 88 0.94 0.89 0.90 979 1202 711 964 11 14 25 34 24 0.98 1.08 1.02 143 231 333 236 12 19 23 17 20 1.01 0.96 0.92 173 240 185 199 13 20 17 11 16 0.92 0.87 0.86 217 195 128 180 14 13 32 10 18 1.04 1.16 0.97 125 276 103 168 15 11 29 21 20 1.00 1.28 1.00 110 227 210 182 16 7 34 20 20 1.15 1.31 1.22 61 260 164 162 40 activity between experimental groups occurs 11—16 days post amputation, that collagenase activity in regenerating and regressing limbs is significantly different during this time period, and that the small sample size (n=3) for each data point is preventing significant differences from being detected. Further, it is felt that since only relatively small differences in enzyme activity occur between normal and contralateral regenerates during the entire time period studied, and between regenerating and regressing limbs 2-8 days post amputation, that 1) the trend in collagenase activity in normal and contralateral regenerates is the same over the time period studied, and 2) the trend in collagenase activity in regressing limbs is the same as that of regenerating limbs 2—8 days post amputation, but differs from regenerating limbs 9-16 days post amputation. Characterization of Larval Axolotl Collagenase Kinetics— The reaction velocity progress curves of collagenase from 7-day axolotl regenerates (Figure 7, Table 2) are of the general form produced by most enzyme reactions, in which velocity falls with time. In most enzyme reactions initial velocities are proportional to the ~ enzyme concentration, and hence a straight line is produced if initial velocity is plotted against enzyme concentration. When apparent velocities at different times from Figure 7 were plotted against the collagenase concentration, the resulting curves become straighter as t approaches 0, indicating that velocity is proportional to enzyme concentration in this system (Figure 8, Table 3). 41 FIGURE 7. Reaction velocity progress curve of collagenase from 7-day larval axolotl regenerates at three concentrations using the radioactive reconstituted collagen fibril assay at 36°C. (0), 0.05 ml. of crude enzyme extract; (x), 0.10 ml. of crude enzyme extract; (0), 0.20 ml. of crude enzyme extract. 42 Ox 0 5710 Au-oa xv nooonwoo somoaaoo-u 3 mo 2mm 66 60 50 40 3O 20 ‘(hrs.) Time FIGURE 7 FIGURE 8. 43 Effect of collagenase concentration on reaction velocity at different times. Values obtained from graphs in Figure 7. t , 0 time incubation of reaction mixture; t , 1 hour incubation of reaction mixture; t , 5 hour incubation of reaction mixture; tl , 10 hour incubation of reaction mixture; t20, 20 hour Incubation of reaction mixture. DPM of 14C-Collagen Transformed/Hr. 25 20 15 10 44 / t‘0 / / / / / / / l / t / 1 // t5 / / // t , lo 1- / / / I t20 0.65 0110 T20 [Collagenase] (m1. crude enzyme extract) FIGURE 8 45 TABLE 2 REACTION VELOCITY PROGRESS CURVES OF COLLAGENASE FROM 7—DAY AXOLOTL RECENERATES AT THREE CONCENTRATIONS Mean DPM of 14C-Collagen Degraded.i Ofi Hrs. 0.05 ml. of Enzyme 0.10 ml. of Enzyme 0.20 ml. of EnzymeI 5 29i2 5212 79t5 7 43t5 69i6 109i8 10 69112 103i11 128112 20 96i6 ll7t5 147*15 30 lZlill 150t17 147t17 40 132t8 151t15 152*20 50 126£15 145t15 150*16 60 138i15 153i27 154t23 66 141t12 158t25 160t24 TABLE 3 EFFECT OF COLLAGENASE CONCENTRATION ON REACTION VELOCITY AT DIFFERENT TIMES Apparent DPM of 14C—Collagen Degraded/Hr. Hrs. 0.05 ml. of Enzyme 0.10 ml. of Enzyme 0.20 ml. of Enzyme 0 6.0 12.0 24.0 1 6.0 11.5 18.0 5 6.0 10.8 16,4 10 5.8 10.3 12.8 20 4.8 7.0 7.3 46 Formation of Dialyzable Degradation Products at 36°C- Crude enzyme extracts from 8-day regenerating and 8-16 day regressing limbs are both able to breakdown collagen to dialyzable peptides at 36°C (Table 4). In reaction mixtures of both extracts, 81% of the counts released into the supernatant were dialyzable. Formation of Characteristic TCA Dggradation Products at 25°C- Crude enzyme extracts from both regenerating and regressing larval axolotl limbs degraded collagen to collagenase characteristic TCA fragments at 25°C. The disc-gel electrophoresis patterns of collagen incubated with crude enzyme extract from 7-day regenerates for 0-72 hrs. (Figure 9a-g) show otA andfl A degradation products in the gels by 12 hrs. of incubation. The {3 A band, however, disappears by 36 hrs. of incubation and does not appear again, even though the /5 band almost completely disappears by 72 hrs. of incubation. The o:A component, on the other hand, is present throughout the incubation period, as are several discrete degradation products of ocA which are the major species accumulating. These results indicate that at least three reactions are occurring in this system: 1) collagenase is degrading TC molecules to T095 degradation product, which results in formation of «$5 and I3 $5, 2) a neutral proteinase is catalyzing hydrolysis of the cross-linked portions of collagen molecules, resulting in conversion of /3 andtg degradation products to on and o: degradation products, and 3) either collagenase or proteases are degrading TCA 75 molecules to several slightly smaller molecules. The disc-gel electrophoresis patterns of collagen incubated with crude enzyme extract from 7-day regressing limbs for 0-72 hrs. 47 TABLE 4 FORMATION OF DIALYZABLE DEGRADATION PRODUCTS AT 36°C BY CRUDE ENZYME EXTRACT FROM NORMAL RECENERATES AND REGRESSING LIMBS ' V. Mean DPM vw % of CountSI in A Control— A. Undialyzed 14C-Collagen Solution B. Dialyzed . 14C—Collagen Solution Normal Regenerates— A. Counts in Supernatant B. Counts in Dialyzed Supernatant C. Dialyzable Counts (A—B) Regressing Limbs- A. Counts in Supernatant B. Counts in Dialyzed Supernatant C. Dialyzable Counts (A-B) 457 452 340 66 274 304 58 246 __v 100 100 100 19 81 100 19 81 48 FIGURE 9. Disc-gel electrophoresis patterns of collagen degradation products released by collagenase from 7-day larval axolotl regenerates. Enz e extract was incubated with guinea pig collagen at 25 C for (a) 0 hrs., (b) 12 hrs., (c) 24 hrs., (d) 36 hrs., (e) 48 hrs., (f) 60 hrs., and (g) 7% hrs. Reaction mixture minus enzyme was incubated at 25 C for (h) 0 hrs., and (i) 72 hrs. Fr, front. FIGURE 10. Disc-gel electrophoresis patterns of collagen degradation products released by collagenase from 7-day denervated regressing larval axolotl limbs. Enzymeo extract was incubated with guinea pig collagen at 250 C for (a) 0 hrs., (b) 12 hrs., (c) 24 hrs., (d) 36 hrs., (e) 48 hrs., (f) 60 hrs., and (g) 72 hrs. Fr, front. FIGURE 11. Disc-gel electrophoresis patterns of reaction mixtures containing crude enzyme extracts from larval axolotl limbs incubated without collagen at 250 C. Control guinea pig collagen incubated for 72 hrs. (a); crude enzyme extract from 7-day regenerates incubated for (b) 0 and (c) 72 hrs.; crude enzyme extract from 7-day regressing limbs incubated for (d) 0 and (e) 72 hrs. Fr, front. FIGURE 12. Disc-gel electrophoresis patterns of collagen incubated with trypsin at 25 C. Guinea pig collagen minus enzyme incubated for (a) 0 and (b) 72 hrs.; Guinea pig collagen incubated for 72 hrs. with (c) 0.01% trypsin, and (d) 0.06% trypsin. Fr, front. 49 FIGURE 10 FIGURE 11 FIGURE 12 50 (Figure 10) are essentially the same as those of Figure 9 except: 1) [3 A and its degradation products accumulate, indicating the absence of a neutral proteinase capable of converting ’3 to 0: subunits in this system, and 2) there appears to be less degradation of the collagen after 72 hrs., even though crude enzyme extract from 7-day regressing limbs contained more protein (1.06 mg./ml.) than crude enzyme extract from 7-day regenerates (0.78 mg./ml.) and the same size aliquot of each was used in reaction mixtures. Disc-gel electrophoresis patterns of control reaction mixtures containing 1) collagen minus enzyme incubated for 0 and 72 hrs. (Figure 9h and i), 2) crude enzyme extracts from 7-day regenerating and 7-day regressing limbs minus collagen incubated for 0 and 72 hrs. (Figure 11), and 3) collagen plus 0.01% and 0.06% trypsin incubated for 72 hrs. (Figure 12) demonstrate respectively that I) collagen in the assay is not being degraded by a collagenase endogenous to the collagen substrate itself (Pardo and Tamayo, 1975), 2) TCA degradation products are not a component of, or a degradation product of the crude enzyme extracts themselves, and 3) TCA degradation products are not the result of non-specific trypsin degradation of collagen. “Effect of Known Collagenase Inhibitors- Both total and specific collagenase activity in reaction mixtures containing crude enzyme extract from normal 8-day regenerates and EDTA or fetal calf serum are significantly lower (p=0.001) than the equivalent reaction mixtures containing no inhibitors. EDTA inhibits 90%, and fetal calf serum 65% of the collagenase activity. 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