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PubMed1969

Relationship between the latent form and the active form of the autolytic enzyme of Streptococcus faecalis.

A 10-hr starvation of Streptococcus faecalis ATCC 9790 for the amino acids methionine and threonine results in cells which are resistant to autolysis and which contain greatly reduced quantities of both active and latent (proteinase activable) forms of the autolytic enzyme (an N-acetyl-muramide glycanhydrolase). Cell walls were isolated from cells harvested at various times during the recovery from such starvation and were assayed for active and latent forms of the autolysin. Within 10 min of recovery the latent enzyme began to increase. Only after 30 to 60 min did the active enzyme begin to increase; after a similar lag, the cells' proneness to lysis markedly increased. The intracellular localization of both forms of the autolysin was examined, using as an experimental tool the ability of added cell wall to bind autolysin. (14)C-lysine-labeled, inactivated cell walls were added to exponential-phase cells, which were then disrupted, and the mixed wall population was isolated. Measurement of the (14)C release during wall autolysis indicated that the active enzyme in the cells was not available for binding to the added (14)C-labeled walls and was therefore wall-bound in vivo. In contrast, up to 85% of latent autolysin activity was found to have been efficiently bound to the added (14)C walls. The results obtained suggest (i) cellular autolysis is a reflection of the level of active enzyme and not of latent enzyme, and (ii) autolysin is synthesized and mainly located in the cytoplasm as an inactive latent precursor (proenzyme) which is transported to sites on the cell wall associated with wall biosynthesis, where it becomes activated.

Autolysis↗

PubMed1974

Physiological studies of Bacillus subtilis minicells.

Minicells produced by Bacillus subtilis strains carrying the div IV-B1 mutation, (CU 403 div IV-B1 and CU 403 div IV-B1, tag-1), were purified by a procedure which destroys parental cells with ultrasound, but spares minicells. Such preparations generally contain 10(9) or more minicells/ml and less than 10(4) colony-forming units/ml. Purified minicells are resistant to autolysis in tris(hydroxymethyl)aminomethane buffer, pH 7.5, at 30 C, conditions which result in total lysis of parental cells. Minicells are not completely devoid of autolytic activity, however. The medium in which minicells are produced, the temperature at which purified minicells are incubated, and the genotype of cells from which the minicells are derived all influence the rate of autolysis of purified minicells. These parameters are demonstrated by using minicells obtained from div IV-B1 and div IV-B1, tag-1 strains. Ultrastructural differences have been observed in the products of autolysis of these two minicell strains. Minicells are sensitive to low levels of lysozyme and yield miniprotoplasts when the wall is removed in an osmotically protective environment. Although minicells are unable to grow, they can maintain their integrity over long periods of time, which suggests functional energy metabolism in minicells. Direct measurements of adenosine 5'-triphosphate (ATP) levels by the luciferase assay indicated that minicells can produce ATP. Oxygen consumption, measured by standard respirometry techniques, also indicates functional metabolism in minicells. These findings demonstrate that minicells purified by ultrasound are suitable material for study of physiological processes in anucleate cells.

Adenosine Triphosphate↗

PubMed1968

Autolytic enzyme system of Streptococcus faecalis. IV. Electron microscopic observations of autolysin and lysozyme action.

Cell walls (LOG walls) were isolated from cultures of Streptococcus faecalis ATCC 9790 in the exponential phase of growth. These walls were either allowed to undergo autolytic dissolution (in the presence or absence of trypsin) or wall autolysis was inactivated with sodium dodecylsulfate (SDS walls). Inactivated walls were treated either with lysozyme or with isolated, partially purified S. faecalis autolysin. During wall lysis, samples were removed, negatively stained with phosphotungstate, and examined in the electron microscope. Both lysozyme and isolated autolysin appeared to act over the entire surface of SDS walls. After partial dissolution, a fibrous network over the surface was revealed. Lysozyme digestion revealed the presence of prominent, highly-contrasted equatorial and subequatorial bands around the walls. After trichloroacetic acid extraction, the bands were seen less frequently and less distinctly in the partially lysozyme digested walls, suggesting that the bands contained nonpeptidoglycan polymers. In the absence of trypsin (which activates a latent form of the autolysin), autolysis of LOG walls appeared to start at the equatorial bands and to proceed back towards the apex of the coccus. Ribbons of wall material coming off the wide edge of the nearly hemispherical wall fragments were observed. Activation of latent autolysis resulted in lytic action over the entire wall surface. The results are consistent with the previously postulated location of active autolysin at the areas of new wall synthesis and the random location of latent autolysin in LOG walls.

Autolysis↗

PubMed2001

Effect of postmortem storage on mu-calpain and m-calpain in ovine skeletal muscle.

Casein zymography was used to determine the effect of postmortem storage on the proteolytic activity of mu-calpain and m-calpain in lamb longissimus. Casein zymography assays were conducted on crude muscle extracts (only one centrifugation). Six market weight crossbred lambs were slaughtered and a portion of the longissimus lumborum was removed at death (within 15 min of exsanguination) and after 3, 6, 9, 12, 24, 72, and 360 h postmortem. Muscle samples were snap-frozen in liquid nitrogen and stored at -70 degrees C. Soluble muscle proteins were extracted from muscle samples and analyzed by in-gel casein assay to measure calpain proteolytic activity. There was a gradual decline in mu-calpain activity (P < 0.05) such that after 24 and 72 h postmortem, mu-calpain had lost 42 and 95% of its activity, respectively. After 360 h postmortem, no mu-calpain activity could be detected (under the conditions used in this study). Autolysis of mu-calpain could be detected as early as 3 h postmortem. It was demonstrated that the detectable level of mu-calpain activity is a function of the amount of muscle protein electrophoresed. Hence, the activity data reported are in relative terms, rather than absolute values. Furthermore, it was demonstrated that the activity data also are a function of the assay methods used. Different methods have different lower detection limits. Of the three assays examined, 14C-labeled casein was the most sensitive, then the in-gel casein assay, and the least-sensitive method was the standard casein assay. Unlike mu-calpain, postmortem storage had no effect on m-calpain (P > 0.05). When the calcium concentration of a muscle extract was increased to the level that induces m-calpain autolysis, m-calpain was autolyzed and its autolysis was readily detected by the in-gel casein assay. Collectively, these results demonstrate that calcium concentration in postmortem muscle is only high enough to activate mu-calpain. These results support the widely believed conclusion that mu-calpain-mediated proteolysis of key myofibrillar and cytoskeletal proteins is responsible for postmortem tenderization. Hence, understanding the regulation of mu-calpain in postmortem muscle should be the focus of future studies.

Animals↗

PubMed1999

Outcomes of 4 methods of debridement using a decision analysis methodology.

OBJECTIVE: To compare the clinical efficacy of 4 debridement alternatives in pressure ulcer management. DESIGN: Nonexperimental design combining computer modeling and a decision analysis methodology. Data input into the model were derived from a literature review of the MEDLINE database for the years 1985 to 1995 (indexing terms: pressure ulcer, decubitus, economics, cost-effectiveness, and outcome) and a Delphi consensus process with an independent panel of 9 specialists in geriatric care. SETTING: Long-term care. PATIENTS: Hypothetical elderly female resident with a new full-thickness pressure ulcer (mild odor, minimal draining, no undermining, intact periulcer skin). INTERVENTIONS: The study patient underwent 1 month of therapy with each of 4 debridement methods: autolysis, wet-to-dry dressings, collagenase (Santyl) or fibrinolysin (Elase). MAIN OUTCOME: Specific clinical outcomes (time to clean wound bed, risk of infection) and relative cost-effectiveness of each debridement method were calculated. RESULTS: The likelihood of achieving a clean wound bed at 2 weeks was 70% for collagenase, 57% for fibrinolysin, 50% for autolysis, and 30% for wet-to-dry dressings. The total cost for 1 month of treatment was $610.96 for collagenase, $920.73 for autolysis, $986.38 for fibrinolysin, and $1,008.72 for wet-to-dry dressings. CONCLUSIONS: The study patient was most likely to have a clean wound bed after 2 and 4 weeks of treatment, less likely to need to switch debridement methods, and of equal or less likelihood of experiencing and infection and subsequent hospitalization when her wound was debrided with collagenase. Treatment with collagenase also resulted in the lowest total cost of treatment. Longer term, prospective studies using actual utilization data are needed to refine these estimates of the clinical and cost-effectiveness of each product.

Aged↗

PubMed1985

Triphenyltetrazolium staining of irreversible ischemic injury following coronary artery occlusion in rats.

The objectives of this study of triphenyltetrazolium chloride (TTC) gross histochemical staining of myocardial infarcts were to determine the accuracy of sizing of established infarcts, to determine the time course of development of staining defects during infarct evolution, and to determine the effect of clinically relevant autolysis on the detectability of early infarcts. Transverse sections of excised rat hearts after left coronary artery occlusion were incubated in TTC for 5 minutes at 37 C. After 48 hours, planimetrically determined infarct size (percent left ventricle [LV%]) by TTC and by conventional histologic study (H) correlated well: TTC (LV%) = 1.04 X H(LV%) - 2.94 (r = 0.98; P less than 0.001). Ischemic zone staining defects, detected in 50% of hearts 30 minutes after occlusion, were noted in all hearts occluded 3 hours or longer. Gross staining contrast was not diminished by in situ autolysis for 6 hours at ambient temperature or for 24 or 72 hours at 4 C. In conclusion, TTC reliably permits quantitation of the size of completed infarcts. TTC often detects ischemic injury as early as 30 minutes after coronary occlusion in the rat and in all hearts by 3-6 hours. Infarct demarcation by TTC is preserved during simulated clinical autolysis.

Animals↗

PubMed1986

[Neurons in the hyperchromic state].

The paper summarizes findings concerning the ultrastructure and cytochemistry of the cortical hyperchromic neurons which appear under different experimental conditions (in intact animals, in mechanical traumatization of neuronal tissue, during formation of a local focus of destruction, upon the administration of aminazine, and in the process of autolysis). Characteristics of the development of the hyperchromic status in each of the above experimental situations are specified. The authors consider the questions concerning the life-time development of neuronal hyperchromia, a relationship of hyperchromia with a certain function of the cell and the reversibility of the ultrastructural and cytochemical shifts characteristic of hyperchromia. A conclusion is made about the development of hyperchromia during life (with the exception of autolysis), its correlation with a decreased functional activity of the cell and the possibility of hyperchromia involution. Hyperchromic cells proved to be highly resistant to autolysis.

Animals↗

PubMed1988

Structure and mechanism of formation of recombinant-derived chymosin C.

Chymosin C, an autolysis product of chymosin A, is not formed from chymosin B (Foltmann, B. (1966) C. R. Trav. Lab. Carlsberg 35, 143-231) even though chymosins A and B differ in only a single residue (residue 286 is Asp in chymosin A but is Gly in chymosin B). Autolysis of recombinant-derived chymosin A yielded chymosin C for structural analysis. N-terminal sequencing revealed two peptide chains in chymosin C, one of which begins with Gly43, the N terminus of chymosin A; the other begins with Asp289. C-terminal sequencing, peptide mapping, and amino acid analysis showed that chymosin A undergoes autolytic excision of Asp286--Glu287--Phe288 in yielding chymosin C. As predicted from the established disulfide pattern and verified by size-exclusion chromatography under denaturing conditions, no disulfide bond cross-links the two chymosin A fragments which comprise chymosin C. The N-terminal fragment (243 residues) is termed the C-protein, and the C-terminal fragment (77 residues) is termed the C-peptide. Studies with synthetic peptide analogues of relevant regions of the chymosin sequences suggested that Tyr285--Asp286 is the first chymosin A peptide bond hydrolyzed during chymosin C formation and that Tyr285--Gly286 in chymosin B is not cleaved. Cleavage of Phe288--Asp289, a bond which is present in both chymosins A and B, most likely occurs in the A form as a secondary event following nicking at Tyr285--Asp286. This explains why chymosin A gives rise to chymosin C, but chymosin B does not. Examination of the sequence of a prochymosin gene (Hidaka, M., Sasaki, K., Uozumi, T., and Beppu, T. (1986) Gene (Amst.) 43, 197-203) showed that the autolysis-sensitive region of the polypeptide is encoded by genomic DNA located at the end of one exon and at the beginning of the next. Thus, chymosin C illustrates the correlation of protease-sensitive regions of protein sequences with genomic splice junctions.

Amino Acid Sequence↗

PubMed1977

Factors in the survival of stored corneas.

In studying the survival of stored corneas we wished to know if excising the cornea was the most critical step or if one storage medium was superior to another. Rabbit corneas were excised and stored in various media at 4 degrees C or 23 degrees C for four days. The storage media included M-K medium, balanced salt solution (BSS) and 10(-8)M concentration of hydrocortisone in M-K medium and BSS. Comparisons were made by measuring acid phosphatase as an index of autolysis. Results showed a reduced amount of autolysis at 4 degrees C as compared to 23 degrees C. No statistical difference between M-K medium and BSS was seen but steroid decreased autolysis. M-K medium plus hydrocortisone seemed to be the best of the solutions studied, but removal of the cornea seemed to be the most important factor in its survival.

Acid Phosphatase↗

PubMed1980

Changes in energy transduction efficiency in mitochondria isolated from ischemic myocardium.

The effects of temporary coronary artery occlusion (TCAO) in vivo and ischemia in situ (autolysis) on mitochondrial functions and inner membrane permeability were investigated. It was found that one day after TCAO the permeability of inner membrane to H+ was increased 64% and the maximal activity of the glutamate plus malate oxidase system was reduced 35% with respect to control. The proton-electrochemical gradient and the rate of oxidative phosphorylation were decreased 35% and 55%, respectively. The most significant alterations during autolysis were observed in respiratory control ratio, state IV respiration and in H+ permeability of inner membrane. The H+ permeability of inner membrane increased progressively with time, and after 4 hr of autolysis it was 378% higher than the control. The data suggest that the alterations in energy transduction efficiency of mitochondria isolated from ischemic myocardium depend largely on the increase of inner membrane permeability to H+.

Animals↗

PubMed1993

[A neuropathological study on respirator brain].

A neuropathological study was conducted on 101 respirator brains. The patients comprised 62 men and 39 women, ranging in age from 11 to 84 years, the average being 49.4 years. Of these, 51 cases died of cerebrovascular disease, including 26 cases of subarachnoid hemorrhage. The average duration of mechanical supportive care was 99 hours. The mean weight of the brain at autopsy was 1,460 g. Characteristic pathological findings were brain edema, congestion, herniation and various subarachnoid hemorrhages. Brain consistency was soft despite adequate fixation. Histologically, the cytoplasm of neurons in the cerebral cortex, basal ganglia, cerebellum and brain stem was pale and ghost like. In the white matter, myelin staining was quite pale and nuclei of the glial cells were shrunken and piknotic. Autolysis of the granular layer of the cerebellar cortex and necrosis of anterior parts of the pituitary gland were evident in all of the brains examined. There were no reactive astrocytosis and no infiltration neutrophils in the superficial areas of the cerebrum and brain stem, possibly due to temporary or partial circulation. There exists some correlation between the degree of autolysis and the duration of artificial aeration. But no relationship between the former and the basic disease was found. In the respirator brains, autolysis was most prominent in the cerebral cortex, thalamus, tegmentum of the brain stem, granular layer of the cerebellar cortex and the pituitary gland. It was possible to establish the diagnoses of underlying diseases such as cerebrovascular disease, meningoencephalitis, tumor and degenerative disease in the majority of cases through neuropathological analysis on the respiratory brains.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMedDate not supplied

[Effect of bovine serum albumin on the character of autolytic changes in the lipid component of rat gray and white brain matter in vitro].

The influence of cytoprotecting concentrations of bovine serum albumin (BSA) on the autolytic alterations of rat brain white and grey matters in the physiological solution containing was investigated, In the autolysed grey matter exogenous BSA prevents the decomposition of phospholipids and the gaining the free fatty acids up to 1 hr of incubation and increased the content of cholesterol esters at the 4th and 24th hrs of autolysis BSA increased the phospholipids breakdown at the late stages of autolysis (24 hrs). On the contrary, BSA activated the phospholipids break down in the white matter already at the early periods of incubation (10 mins). It also altered the character of cholesterol and esters pendulous interactions on the opposite one, promoting the significant increase of the latters, especially at the 10th min and 4th hr of the autolysis.

Animals↗

PubMed2003

Degradation of normal and proliferated peroxisomes in rat hepatocytes: regulation of peroxisomes quantity in cells.

Degradation and turnover of peroxisomes is reviewed. First, we describe the historical aspects of peroxisome degradation research and the two major concepts for breakdown of peroxisomes, i.e., autophagy and autolysis. Next, the comprehensive knowledge on autophagy of peroxisomes in mammalian and yeast cells is reviewed. It has been shown that proliferated peroxisomes are degraded by selective autophagy, and studies using yeast cells have been especially helpful in shedding light on the molecular mechanisms of this process. The degradation of extraperoxisomal urate oxidase crystalloid is noted. Overexpressed wild-type urate oxidase in cultured cells has been shown to be degraded through an unknown proteolytic pathway distinct from the lysosomal system including autophagy or the ubiquitin-proteasome system. Finally, peroxisome autolysis mediated by 15-lipoxygenase (15-LOX) is described. 15-LOX is integrated into the peroxisome membrane causing focal membrane disruptions. The content of the peroxisomes is then exposed to cytosol proteases and seems to be digested quickly. In conclusion, the number of peroxisomes appears to be regulated by two selective pathways, autophagy, including macro- and microautophagy, and 15-LOX-mediated autolysis.

Animals↗

PubMed1996

Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans.

The terminal process of xylogenesis, autolysis, is essential for the formulation of a tubular system for conduction of water and solutes throughout the whole plant. Several hydrolase types are implicated in autolysis responsible for the breakdown of cytoplasm. Here, we characterize p48h-17 cDNA from in vitro tracheary elements (TEs) of Zinnia elegans which encodes a preproprotein similar to papain. The putative mature protein, a cysteine protease, has a molecular mass of 22,699 Da with a pI of 5.7. DNA gel blot analysis indicated that p48h-17 is likely encoded by one or two genes. The p48h-17 mRNA accumulated markedly in in vitro differentiating TEs, whereas it appeared not to be induced in response to senescence and wounding in the leaves or H2O2 challenge in the cultured mesophyll cells. In stems, the expression of the p48h-17 gene was preferentially associated with differentiating xylem. Activity gel assays demonstrated that a cysteine and a serine protease, which had apparent molecular masses of 20 kDa and 60 kDa, respectively, were markedly induced during in vitro TE differentiation. The cysteine protease activity was also preferentially present in the xylem of Zinnia stems. Transient expression of the p48h-17 cDNA in tobacco protoplasts resulted in the production of a 20 kDa cysteine protease. Taken together, the results indicate that the p48h-17 gene appears to be preferentially associated with xylogenesis, and both the cysteine and serine proteases might be involved in autolysis during xylogenesis.

Amino Acid Sequence↗

PubMed1988

Peroxidase activity in traumatic skin lesions.

Peroxidase activity was determined in experimental compression-excoriation lesions and incision wounds of rat skin after different periods of vital time. The peroxidase enzyme was extracted from the tissues by homogenization in 0.5% cetyltrimethylammoniumbromide, and the enzyme activity was measured from the supernatant by o-dianisidine-H2O2 assay. In the blood of the rats a mean activity of approx. 5.26 +/- 1.11 U/g dry weight was observed. In the control specimens of the skin the activity was very low and generally below the detection limit of the methods used. In 30-min-old compression-excoriation lesions the mean peroxidase activity was 0.38 +/- 0.21 U/g dry weight. In lesions older than 30 min the activity started to increase rapidly. In 4-h-old compression-excoriation lesions it was 10 times higher than the 30-min level and was 40 times higher in 12-h-old lesions and 70-100 times higher in 1-3-day-old compression-excoriation lesions, respectively. In 30-min-old incision wounds the mean peroxidase activity was 0.65 +/- 0.37 U/g dry weight. The increase of the activity compared with the 30-min level was even faster in the incision wounds: in 4-h-old wounds the mean activity was 50 times higher, in 12-h-old wounds 200 times higher and in those of 1-5 days it was several hundreds of times higher. Compression-excoriation lesions made after death showed activity similar to the control specimens. Postmortem autolysis at +22 degrees C resulted in a loss of the enzyme activity in 1-day-old compression-excoriation lesions so that after 3 days approx. 80% remained, and after 5 and 7 days approx. 40% was present. After 3 days of autolysis at +4 degrees C, nearly 100% of the activity remained and approx. 90% was present after 5 and 7 days of autolysis. Increased peroxidase activity was also detectable in human vital excoriations in the specimens which were taken in autopsies several days postmortem.

Animals↗

PubMed1976

Glial fibrillary acidic protein from bovine and rat brain. Degradation in tissues and homogenates.

Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol wt. polypeptide in bovine tissues incubated at 24 degrees C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000-40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 degrees C. At pH 8.0 PROTEOLYSIS OF The glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.

Animals↗

PubMed1988

Regulation of activity of calcium activated neutral protease.

Various lines of evidence suggest that calcium dependent protease (CANP, calpain) exists in the cytosol as an inactive proenzyme which is converted to an active form by autolysis. During autolysis only the N-terminal regions of both subunits of proCANP are modified. About 20 and 90 residues are removed from the large and small subunits, respectively. The N-terminal region (domain I) of the large subunit modified during autolysis precedes the protease domain and corresponds to the propeptides of various cysteine proteinases. Analyses of the autocatalytic activation of CANP in the presence of plasma membranes reveal that proCANP translocates to the membrane in the presence of microM Ca2+ and is activated at the membrane. The CANP inhibitor and Ca2+ are the most important factors for the regulation of CANP activity. The primary translation product of the mRNA for rabbit liver CANP inhibitor contains four internal repeats. Structural analyses of the liver and erythrocyte inhibitors reveal that they contain four and three repeats, respectively. The repeating unit was identified as the functional unit of the inhibitor and each unit inhibits one mole of CANP. On the basis of these results, an activation mechanism for proCANP at the membrane was proposed. The native enzyme, which has been called CANP or calpain, should now be called proCANP or calpainogen. CANP and calpain should be used for the autolyzed active form.

Animals↗

PubMed1991

Comparison of the autolyzed and unautolyzed forms of mu- and m-calpain from bovine skeletal muscle.

Bovine skeletal muscle mu- and m-calpain autolyze when incubated with Ca2+. During the first 30 to 300 s, autolysis: (1) has little effect on the specific proteolytic activity of either mu- or m-calpain when assayed at 5 mM Ca2+; and (2) produces two new proteolytically active forms of calpain in addition to the original mu- and m-calpain. The four proteolytically active forms of calpain are: (1) autolyzed mu-calpain, having polypeptide subunits of 76 and 18 kDa and requiring 0.60 microM Ca2+ for half-maximal activity; (2) mu-calpain with 80- and 28-kDa subunits and requiring 7.1 microM Ca2+ for half-maximal activity; (3) autolyzed m-calpain with 78- and 18-kDa subunits and requiring 180 microM Ca2+ for half-maximal activity; and (4) m-calpain with 80- and 28-kDa subunits and requiring 1000 microM Ca2+ for half-maximal activity. All four forms of the calpains have similar pH optima (7.4 to 7.6) and almost identical circular dichroism spectra in the far ultraviolet (all four have little secondary structure with 26-30% alpha-helix and less than 10% beta-sheet structure). Autolyzed mu- and unautolyzed mu-calpain are fully activated proteolytically by Mn2+ with activity starting at 125 microM Mn2+. Autolyzed m-calpain is also activated by Mn2+ up to 80% of the maximum proteolytic activity obtained with Ca2+; Mn2+ activation begins at 320 microM Mn2+. Unautolyzed m-calpain has only 6 to 8% as much activity in the presence of Mn2+ as it does in the presence of Ca2+. Autolysis increases the axial ratios of the calpains from 3.5 to 4.6 for mu-calpain and from 3.7 to 5.0 for m-calpain (assuming 20% hydration). The estimated length of the calpain molecules increases by 13% upon autolysis from 73 to 84 A for mu-calpain and from 76 to 90 A for m-calpain (assuming 20% hydration). The autolyzed calpains elute after their unautolyzed counterparts off a DEAE-ion exchange column. Because autolyzed forms of the calpains are not found in DEAE elution profiles of cell extracts, bovine skeletal muscle cells must contain very little (less than 5% of total calpain) or none of the autolyzed form of the calpains.

Animals↗