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PubMed1978

Identification of the functional ionic groups of papain by pH/rate profile analysis.

The pH dependence of papain catalysis was analyzed by a scheme which evaluates the kinetic contribution of both protonated and unprotonated species of functional groups involved in catalysis. Kinetic measurements were made at constant pH, without buffers, by automatic titration. The rate-determining step for papain-catalyzed hydrolysis of alpha-N-benzoyl-L-arginine ethyl ester, determined by nucleophile competition, changed from acylation below pH 6.5 to mixed acylation-deacylation above pH 6.5. Kinetic analysis indicated that three prototropic groups governed the pH-specificity of alpha-N-benzoyl-L-arginine ethyl ester hydrolysis. These prototropic groups had pKa values of 4.8, 6.5 to 6.7, and 8.7. Theoretical treatment of the kinetics provided an excellent fit with the experimentally found profile when the contribution of all three prototropic groups was considered. Analysis showed that, in acid, the pathways of papain catalysis were functional with either two or three active-site protons. In base, a single functional ionic pathway is associated with an active site with only one proton. Pathways involving an unprotonated active site are catalytically inoperative in both acid and base. These results indicate that papain exhibits several catalytically functional ionic pathways. The results are discussed in terms of pKa assignments, and the mechanism of papain catalysis.

Arginine↗
PubMed1989

The interaction of papain with polycations.

An investigation has been made of the interactions of the enzyme papain with the polycations protamine, polybrene, poly(L-lysine), spermine, spermidine and the neutral polymer polyvinylpyrrolidone (PVP). At low concentrations, each behaves as an inhibitor of the enzyme. As their concentrations increased above a certain level, the activity of the systems increased, and their inhibition of the enzyme appeared to be less pronounced. When acting by themselves in the presence of the substrate haemoglobin, each of the polycations was a weak proteolytic catalyst with a ranking of catalytic effectiveness of protamine greater than polybrene greater than poly(L-lysine) greater than polyvinyl-pyrrolidone greater than spermidine greater than spermine. This effect could explain the anomalous inhibition of papain by these polycations. The interaction of papain with dansyl protamine (DNSP) and the extent of complex formation were studied using a fluorescence polarization technique and the results showed that there was a strong interaction occurred. The strength of binding was assessed by determination of the critical electrolyte concentration (0.2 M, NaNO3). The stoichiometry of the DNSP-papain complex was found to be 63 base moles of DNSP to one mole of papain.

Catalysis↗
PubMed1996

Effects of lung volume on airway resistance during induced constriction in papain-treated rabbits.

It has been reported that both the elasticity of the cartilage and airway-parenchymal interdependence can modify shortening of the airway smooth muscle and airway narrowing during induced constriction. We hypothesized that induced softening of the cartilage could alter airway compliance and/or the forces of mechanical interdependence, resulting in an increased degree of airway narrowing in response to a contractile stimulus. To test this hypothesis, we compared the effects of changing lung volume on airway resistance (Raw) under baseline conditions and during methacholine (MCh)-induced constriction in papain-treated (n = 6) and control rabbits (n = 6). With use of the alveolar capsule technique, Raw was directly measured under baseline conditions at different levels of end-expiratory transpulmonary pressure (Ptp = 4-12 cmH2O). Then aerosolized MCh was delivered (0.2-25 mg/ml) and measurements were performed at different levels of Ptp (4 and 12 cmH2O). From measured tracheal flow and tracheal and alveolar pressure in open-chest animals during mechanical ventilation (tidal volume = 6 ml/kg, breathing frequency = 1 Hz), we calculated Raw by subtracting tissue resistance from lung resistance. Papain treatment significantly increased Raw both under baseline conditions and after induced constriction. We found that increasing Ptp decreased Raw before and after MCh in both groups; however, the effects of changing Ptp on Raw were less in papain-treated animals. These observations suggest that both cartilage elasticity and mechanical interdependence are important determinants of airway smooth muscle shortening. The observation that volume dependence of Raw was less in papain-treated animals is consistent with the hypothesis that papain effects significant changes in the parenchymal attachments.

Airway Resistance↗
PubMed1987

Pulmonary vascular structural and functional changes in papain-induced emphysema in dogs.

We evaluated the pulmonary circulatory effects of emphysema produced by papain in 6 dogs that received 4 weekly treatments of papain administered by aerosolization. A control group of 6 dogs received saline treatments. Hemodynamic measurements were performed during normoxic and hypoxic (10% oxygen) ventilation approximately 3 and 6 months after treatment. Baseline hemodynamic parameters were comparable in both groups and were unchanged at 6 months in the control group. In the papain-treated group at 6 months, there were significant reductions in PaO2 (95 +/- 4 to 79 +/- 3 mm Hg, p less than 0.005) and increases in mean pulmonary artery pressure (12 +/- 1 to 18 +/- 3 mm Hg, p less than 0.05) and pulmonary vascular resistance (2.0 +/- 0.3 to 6.2 +/- 2.1 units, p less than 0.01). Morphometric evaluation demonstrated increased medial thickness in muscular pulmonary arteries less than 50 mu in size. Morphologic evaluation confirmed the presence of panlobular emphysema with papain administration. We conclude that chronic emphysema produced in dogs by aerosol administration of papain results in elevated pulmonary artery pressure, which is characterized pathologically by medial hypertrophy of small pulmonary arteries. This model appears suitable for further study of the pathogenesis of pulmonary vascular disease associated with chronic lung disease.

Animals↗
PubMed1992

The baculovirus Autographa californica nuclear polyhedrosis virus genome includes a papain-like sequence.

The published DNA sequence that includes the gene for the envelope glycoprotein gp67 of the baculovirus Autographa californica nuclear polyhedrosis virus also contains a segment that shows 32% amino acid identity to papain, a cysteine endopeptidase from the papaya plant (Carica papaya). The viral papain-like sequence is apparently affected by a frame-shift mutation, but otherwise appears capable of encoding a functional enzyme. Catalytically essential amino acids appear to be conserved, as do disulphide bridges. The overall structure of the putative protein is similar to that of papain, as judged by hydropathy profile. Secondary structure prediction using a consensus of seven methods indicates that the putative viral enzyme retains the alpha/beta domain structure of papain, including the long helix beginning with cysteine-25. Infecting SF9 cells with the virus did not lead to a detected increase in cysteine endopeptidase activity, and a cysteine endopeptidase inactivator appeared to have no effect on infectivity. Irrespective of whether the sequence encodes a functionally active cysteine endopeptidase, this is the first example of a papain-related sequence in a viral genome.

Amino Acid Sequence↗
PubMed1987

Studies on isolated smooth muscle cells. IX. Application of papain for isolation of single smooth muscle cells from guinea-pig taenia coli.

To prepare single smooth muscle cells from the taenia coli of guinea pig, the application of papain to the enzymatic solution was examined under two conditions: 1) the isolation in a modified Tyrode solution (containing 0.18 mM Ca2+: 0.18 mM Ca2+-Tyrode solution) and 2) the isolation in a high-K+ Tyrode solution (Na+ was replaced by K+, and Ca2+ was not added: high-K+ Tyrode solution). The presence of papain during collagenase digestion reduced contamination of broken cells and cell debris. In the case of the high-K+ Tyrode solution, papain increased the yield of single cells significantly. The cells were contracted in a dose-dependent manner by Ca2+ in the high-K+ Tyrode solution and by carbachol in 0.18 mM Ca2+-Tyrode solution; furthermore, the contractions were antagonized by verapamil and atropine, respectively. Treatment with papain did not affect cell sensitivity to the stimulants. Therefore, our results suggest that the addition of papain is useful for the isolation of single cells to investigate the physiological and pharmacological characteristics of smooth muscle.

Animals↗
PubMed1989

Elastase and papain inhibition by serum of mammals.

The protease-antiprotease imbalance concept has gained wide acceptance in reference to experimental animal models of human pulmonary emphysema. The destructive process may be induced by proteolytic enzymes such as porcine pancreas elastase (E) or papain (P), or by oxidants such as chloramine T (CT) which cause inactivation of alpha-1-antiprotease. The susceptibility to development of severe emphysema has been found to differ among animal species. We investigated the inhibition of elastase and papain by serum of seven species (rat, dog, human, rabbit, sheep, hamster, mini pig) and the effect of CT on serum inhibition. The relative inhibitor concentration of serum was expressed in terms of its inhibitor capacity for elastase and papain (EIC and PIC) defined as number of inhibited units (U) of enzyme catalysed hydrolysis of chromogenic peptide substrate (SAPNA and BAPNA) per unit of serum. The effect of CT on enzyme inhibition was quantified in terms of fractional loss of inhibition relative to control. The serum concentration of inhibitor was highest in the rat (EIC 8642 +/- 989 microU/microliter, PIC 214.2 +/- 110.3 microU/microliter; means +/- SD). Next in order of decreasing EICs were dog, human, rabbit, sheep, hamster, and mini pig exhibiting the lowest EIC (2523 +/- 184 microU/microliter) while sheep had lowest PIC (39.6 +/- 3.5 microU/microliter). The EIC/PIC ratio varied from 20 (mini pig) to about 100. The reduction of elastase inhibition after CT exposure of serum was high (80-100%) in rat, dog, human, and hamster, moderate (40%) in mini pig and rabbit, and low (10%) in sheep. Because papain was directly affected by CT the effect of CT on papain inhibition could not be analysed. The data suggest that the intrinsic antiprotease and antioxidant screen varies among experimental animals. For the purpose of animal models of emphysema, hamster appears to be most susceptible to the progressive destruction of lung parenchyma elicited by experimental burden of proteases or oxidants.

Animals↗
PubMed1975

Effect of papain-induced emphysema on permeability of rat lung to drugs.

To investigate the effect of a papain-induced emphysema-like condition on pulmonary absorption of drugs, rats were exposed to either papain aerosol or distilled water aerosol (control) intermittently for 2 wk, and rates of drug absorption from damaged and control lungs were compared. To measure absorption rates, 0.1 ml of drug solution (0.1-10 mM) was administered through a tracheal cannula to anesthetized animals, and after various times lungs were assayed for unabsorbed compound. In absorption experiments with the lipoid-insoluble compounds, mannitol, p-aminohippuric acid, and procaine amide ethobromide, all three drugs were absorbed from the lungs about twice as rapidly in papain-treated rats as in control. In contrast, procaine amide, a relatively lipoid-soluble drug, was absorbed at the same rate in both control and papain-treated animals. The results suggest that papain-induced lung damage increases the porosity of the pulmonary epithelium.

Aerosols↗
PubMed2000

Concentrations and antioxidative activity of anserine and carnosine in poultry meat extracts treated with demineralization and papain.

Anserine and carnosine found in animal skeletal muscle are capable of inhibiting the catalysis of lipid oxidation by heme and non-heme iron. A demineralization technique and a proteolytic enzyme (papain) were used in this research in order to reduce the levels of proxidants while maintaining high levels of anserine and camosine in poultry (chicken, duck and turkey) meat extracts. Undemineralized poultry meat extracts contained larger amounts of anserine, camosine, heme and non-heme iron (p < 0.05) than did demineralized poultry meat extracts. Both undemineralized and demineralized breast meat extracts of chicken, duck and turkey contained higher concentrations of anserine and camosine, but lower amounts of heme and non-heme iron than did thigh meat extracts. In chicken, duck and turkey meat (breast and thigh) extracts (undemineralized and demineralized), the anserine concentrations were greater (p < 0.05) than the camosine concentrations. The hydrogen-donating ability of undemineralized and demineralized chicken breast meat extracts was not significantly different (p > 0.05): however, demineralized chicken breast meat extracts showed higher (p < 0.05) ferrous chelating ability than did undemineralized meat extracts. The concentrations of anserine, camosine, heme and non-heme iron in chicken breast meat extracts increased (p < 0.05) with the addition of papain (1%) to the meat mixture before extraction. Heme and non-heme iron in the chicken breast meat extracts increased as the reaction time for papain increased from 30 to 120 min, but the concentrations of anserine and camosine were not significantly affected by the longer reaction time for papain. The hydrogen-donating ability and ferrous chelating ability of demineralized chicken breast meat extracts were not significantly affected by papain. The ratios of carnosine/anserine were very specific in the chicken, duck and turkey meat extracts (breast and thigh); and the turkey meat extracts had lower (p < 0.05) camosine/anserine ratios than did the chicken and duck meat extracts. The camosine/anserine ratios of undemineralized and demineralized poultry meat extracts were not significantly different (p > 0.05). This suggests that the carnosine/anserine ratios of undemineralized chicken (0.62 - 0.80), duck (0.75 - 0.77) and turkey (0.15 - 0.16) meat extracts could be used to estimate the single meat species in uncooked or cooked meat products.

Animals↗
PubMed2002

The Arabidopsis xylem peptidase XCP1 is a tracheary element vacuolar protein that may be a papain ortholog.

XCP1 is a xylem-specific papain-like cysteine peptidase in Arabidopsis. To determine whether XCP1 could be involved in tracheary element autolysis, promoter activity and localization of XCP1 were investigated using XCP1 promoter-beta-glucuronidase fusions and immunofluorescence confocal microscopy. A tracheary element expression pattern was detected for XCP1. Results from confocal microscopy and biochemical subcellular fractionation indicated that XCP1 was localized in the vacuole. Ectopic expression of XCP1 resulted in a reduction in plant size in some lines and early leaf senescence, as indicated by early loss of leaf chlorophyll. Reduced plant size was correlated with higher levels of XCP1, as shown by immunoblot and peptidase activity gel analyses. The XCP1 prodomain exhibits exceptionally high similarity (greater than 80%) to the prodomains of papain and other papain-like enzymes isolated from papaya (Carica papaya) laticifers when compared with all other reported papain-like enzymes. The potential for XCP1 and papain to perform common functions as catalysts of autolytic processing following cell death due to programmed suicide or to wounding is discussed.

Arabidopsis↗
PubMed2003

[X-ray microanalysis of the activity of immobilized papain].

The localization of activity of immobilzed papain by covalent-binding was studied by X-ray microanalysis. N alpha-benzoyl-L-arginineamide hydrochloride and FeCl3 served as substrate and capturer respectively. Substrate was catalysed by immobilized papain to produce L-arginine and NH3. NH3 was captured by FeCl3 to form precipitate Fe(OH)3. Precipitation deposited was used to locate active site of immobilized papain. The results show that the macroporous resins carrier of immobilized papain leads to greater enzyme activity, and the distribution of activated enzyme is uniform. Most of activated enzyme was immobilized on the macroporous resins. The optimum condition of localization of activity of immobilized papain was studied.

Arginine↗
PubMed1991

[Analysis of the results of surgery and nucleolysis using papain in 1085 cases of lumbar disk hernias].

1,085 patients have been treated for intervertebral disc herniation with lombo-sciatica: in 751 cases surgical discectomy without laminectomy has been performed with or without microscope; 334 underwent papain nucleolysis. The two clinical groups were separated on clinical and radiographic backgrounds: nucleolysis was performed in patients with no motor disturbance, no stenosis of the lumbar canal, no displaced fragment; the other patients received surgical treatment. The results were evaluated according to MacNab's criteria with a follow-up of 12 to 84 months (mean 17.2 months). The results after surgical treatment were excellent in 53.8% and good in 36.8% of the cases. For papain treatment they were excellent in 32.7% and good in 38.8% of the cases. Papain nucleolysis therefore appears to be an efficient method for the treatment of intervertebral disc herniations associated with lombo-sciatica but, overall, gives poorer results than surgery in spite of a strict selection procedure. In this study, complications due to papain nucleolysis were restricted to chemical spondylodiscitis (5 cases) and to 2 benign allergic syndromes. Statistical analysis of all the cases and multifactorial correlation research pointed out the importance of "workmen's compensation" as prognostic factor. Since this study, 90% of the patients presenting with disc herniation associated with lombo-sciatica recalcitrant to medical treatment undergo surgical discectomy; papain nucleolysis is reserved for sub-ligamentary and non-displaced forms without stenosis of the lumbar canal and represent 10% of our current population.

Adolescent↗
PubMed1991

Processing of the papain precursor. Purification of the zymogen and characterization of its mechanism of processing.

The precursor of the cysteine protease papain has been expressed and secreted as propapain from insect cells infected with a recombinant baculovirus expressing a synthetic gene coding for prepropapain. This 39-kDa secreted propapain zymogen molecule is glycosylated and can be processed in vitro into an enzymatically active authentic papain molecule of 24.5 kDa (Vernet, T., Tessier, D.C., Richardson, C., Laliberté, F., Khouri, H. E., Bell, A. W., Storer, A. C., and Thomas, D. Y. (1990) J. Biol. Chem. 265, 16661-16666). Recombinant propapain was stabilized with Hg2+ and purified to homogeneity using affinity chromatography, gel filtration, and ion-exchange chromatographic procedures. The maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 65 degrees C and under reducing conditions. Precursor processing is inhibited by a variety of reversible and irreversible cysteine protease inhibitors but not by specific inhibitors of serine, metallo or acid proteases. Replacement by site-directed mutagenesis of the active site cysteine with a serine at position 25 also prevents processing. The inhibitor 125I-N-(2S,3S)-3-trans-hydroxycarbonyloxiran-2-carbonyl-L-tyrosine benzyl ester covalently labeled the wild type papain precursor, but not the C25S mutant, indicating that the active site is accessible to the inhibitor and is in a native conformation within the precursor. Based on biochemical and kinetic analyses of the activation and processing of propapain we have shown that the papain precursor is capable of autoproteolytic cleavage (intramolecular). Once free papain is released processing can then occur in trans (intermolecular).

Amino Acid Sequence↗
PubMed1990

Secretion of functional papain precursor from insect cells. Requirement for N-glycosylation of the pro-region.

The synthetic gene coding for the precursor of the cysteine protease papain (EC 3.4.22.2) has been expressed using the baculovirus/insect cell system. The prepropapain gene was cloned into the transfer vector IpDC125 behind the polyhedrin promoter. The recombinant construct was then incorporated by homologous recombination into the Autographa californiaca nuclear polyhedrosis virus genome. The host Spodoptera frugiperda Sf9 cells infected with the recombinant baculovirus secrete an enzymatically inactive N-glycosylated papain precursor. This zymogen could be activated in vitro to yield about 400 nmol of active papain per liter of culture. The recombinant active mature papain was enzymatically indistinguishable from natural papain but the precursor was not processed to the same amino acid residue. The insect cells also accumulated prepropapain and glycosylated propapain intracellularly. This accumulation was an indication that there are rate-limiting steps in the secretion of proteins from insect cells in this expression system. Characterization of mutants of the precursor has shown that entry into the secretory pathway and addition of carbohydrate are prerequisite conditions for the production and secretion of functional propapain.

Amino Acid Sequence↗
PubMed1975

[Pulmonary function in papain induced emphysema in dogs (author's tranls)].

By intratracheal injection of the protease Papain to experimental animals parenchymal changes in the lung can be induced, that resemble human emphysema. Papain (dosage 26 to 112 mg, 1 to 4 injections) was given intratracheally to 8 bastard dogs, (weighing from 12.5 to 20 kg) during light general anesthesia. Pulmonary function was assessed in weekly intervals and related to morphologic changes in the lung. Static compliance of the lung and FRC measured during respiratory arrest were increased after papain, bronchial resistance, measured while artificially ventilated at constant pressure was also increased. Changes of static lung compliance and FRC were seen after the first administration of papain, but further increased with time of observation and after multiple doses of papain. Increase of resistance was not found before 5 weeks. At quiet breathing resistance was not increased at all. No significant changes were found of arterial pO2 and pCO2, pH, standard and actual bicarbonate, diffusion capacity for O2, tidal volume, minute ventilation and ventilatory rate. Morphological findings confirmed the changes described by others. Pulmonary function appears to be pathological at a time when morphology still seems to be normal. The question is discussed to what extent the model of experimental emphysema induced by proteolytic enzymes can contribute to the understanding of human pulmonary emphysema. Lung function in the course of experimental emphysema is compared with function in different clinical types of emphysema.

Airway Resistance↗
PubMed1989

Active and inactive forms of the transition-state analog protease inhibitor leupeptin: explanation of the observed slow binding of leupeptin to cathepsin B and papain.

Leupeptin and similar peptide argininal (arginine aldehyde) transition-state analog protease inhibitors exist in three covalent forms in aqueous solution, the leupeptin hydrate (IH), a cyclic carbinolamine form (IC) generated by the addition of the guanidino epsilon N to the aldehydic carbon, and the free aldehyde form (IA). 1H NMR in D2O show their equilibrium concentrations to be 42, 56, and 2% for IH, IC (R and S enantiomers), and IA. The rates of conversion of (formula; see text) were determined by 1H NMR in D2O by trapping IA with semicarbazide. Application of a deuterium isotope effect of 2.8 led to rate constants in H2O for kC of 0.092 min-1 and kD of 0.73 min-1. The equilibrium concentration of IA and rates for kC and kD are then used to explain the lag phase in the inhibition of cathepsin B and papain by leupeptin. Two circumstances are observed. (i) At micromolar concentrations of leupeptin and papain the binding of leupeptin is biphasic with rate constants identical to kD and kC. (ii) At more dilute nanomolar concentrations of total leupeptin and proteases, the observed lag phase for approach to steady-state inhibition (with rate constant k') is now explained by the low values of the koff rate constants (0.072 min-1 for cathepsin B and 0.024 min-1 for papain) together with the extremely low concentrations of the active inhibitor form IA, with k' = kon[IA] + koff. While kon[IA] is slow, the second-order rate constant kon is found to be quite fast, 1.2 x 10(7) M-1 s-1 for cathepsin B and 1.8 x 10(7) M-1 s-1 for papain. Thus, the binding of leupeptin to cathepsin B and papain may show a lag phase, but this is not due to slow binding.

Algorithms↗
PubMed1985

Studies on the induction and expression of T cell-mediated immunity. XV. Role of non-MHC papain-sensitive target structures and Lyt-2 antigens in allogeneic and xenogeneic lectin-dependent cellular cytotoxicity (LDCC).

The present study examines the role of nonclass I MHC target membrane structures involved in lysis by cytotoxic lymphocytes in the lectin-dependent cellular cytotoxicity (LDCC) system. Cytotoxic cells of rodents and humans have been shown to nonspecifically lyse target cells of different origins and species. The mechanism by which such cytotoxicity takes place is not known, although several hypotheses have been put forth. This study examines the role of class I MHC expression on target cells and Lyt-2 antigens on the cytotoxic cells in both allogeneic and xenogeneic LDCC systems. Human peripheral blood lymphocytes, and murine-allosensitized cytotoxic T lymphocytes (CTL) are used as effector cells, and class I MHC-negative Daudi and class I MHC-positive Raji human target cells are used. The studies reveal that in LDCC, human lymphocytes lyse HLA-negative Daudi targets to the same extent as HLA-positive Raji targets. This was shown by the 51Cr-release assay as well as the single cell assay. Furthermore, allosensitized murine CTL lyse Daudi and Raji in LDCC, demonstrating that lysis of class I negative targets is achieved by xenogeneic CTL. Target cells treated with papain were found to be resistant to lysis in LDCC. However, incubation of papain-treated targets for 1 to 2 hr resulted in recovery of cytotoxicity. In the single cell assay, the papain treatment of targets slightly reduces the frequency of binders but significantly reduces the frequency of killers, revealing that the papain-sensitive structures are essential during the lethal hit stage of lysis. Monoclonal anti-Lyt-2 antibody inhibits lysis of both Daudi and Raji targets by murine CTL. The lack of discrimination between mouse and human targets by murine CTL suggests that the Lyt-2 molecule may have a different role than merely interacting with the target cell. In the LDCC system, we propose that class I MHC molecules are not essential for lysis. Several papain-sensitive and -insensitive target cell structures may participate in the cytolytic mechanism, and we propose that both the lectin and Lyt-2 molecules are involved in the perturbation of cell membrane conformation essential for lysis.

Animals↗
PubMed1984

Human erythrocyte acetylcholinesterase is an amphipathic protein whose short membrane-binding domain is removed by papain digestion.

Human erythrocyte acetylcholinesterase was shown to be an amphipathic protein in which proteases could cleave the hydrophobic domain from the enzymatically active hydrophilic domain. Papain and Pronase cleaved these domains with greatest efficiency, as measured by the disaggregation of purified acetylcholinesterase to disulfide-linked dimers (G2) on sucrose density gradients in the absence of detergent. Nonspecific proteolytic degradation was reduced both by the inclusion of edrophonium chloride, which protected acetylcholinesterase from inactivation, and by covalent attachment of papain to Sepharose CL-4B. In contrast to nondigested control acetylcholinesterase, the papain-disaggregated enzyme did not bind detergent according to hydrodynamic criteria and could not be reconstituted into liposomes. Thus, we conclude that the hydrophobic domain removed by papain digestion is in fact the membrane-binding domain in situ. This domain appeared largely inaccessible to proteases in intact erythrocytes, however, as less than 10% of the enzyme activity was solubilized by protease digestion. The hydrophobic domain removed by papain appeared very small, as nondigested control and disaggregated enzyme were identical in molecular weight and amino acid composition within experimental error. The fully reduced 75-kDa catalytic subunits of nondigested control enzyme appeared about 2 kDa larger than the corresponding subunits of disaggregated enzyme on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, an indication that the hydrophobic domain was cleaved from the COOH or NH2 terminus of the catalytic subunit primary structure. Studies in which the NH-terminal amino acid was labeled by reductive methylation suggested that the hydrophobic domain is at the COOH terminus.

Acetylcholinesterase↗