Tryptic cleavage and substructure of bovine cardiac myosin subfragment 1

The method of limited tryptic proteolysis has been used to compare and contrast the substructure of bovine cardiac myosin subfragment 1 (S-1) to that of skeletal myosin S-1. While tryptic cleavage of cardiac S-1, like that of skeletal S-1, yields three fragments, the 25K, 50K, and 20K peptides, the digestion of cardiac S-1 proceeds at a 2-fold faster rate. The increased rate of cleavage is due entirely to an order of magnitude faster rate of cleavage at the 25K/50K junction of cardiac S-1 compared to that of skeletal, with approximately equal rates of cleavage at the 50K/20K junctions. Actin inhibits the tryptic attack at this latter junction, but its effect is an order of magnitude smaller for the cardiac than for the skeletal S-1. Furthermore, the tryptic susceptibility of the 50K/20K junction of cardiac S-1 in the acto-S-1 complex is increased in the presence of 2 mM MgADP. This effect is not due to partial dissociation of the cardiac acto-S-1 complex by MgADP. Our results indicate that in analogy to skeletal S-1, the cardiac myosin head is organized into three protease-resistant fragments connected by open linker peptides. However, the much faster rate of tryptic cleavage of the 25K/50K junction and also the greater accessibility of the 50K/20K junction in the cardiac acto-S-1 complex indicate substructural differences between cardiac and skeletal S-1.     

Cross-linking of actin to myosin subfragment 1: course of reaction and stoichiometry of products

The cross-linking of actin to myosin subfragment 1 (S-1) with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide was reexamined by using two cross-linking procedures [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306; Sutoh, K. (1983) Biochemistry 22, 1579-1585] and two independent methods for quantitating the reaction products. In the first approach, the cross-linked acto-S-1 complexes were cleaved with elastase at the 25K/50K and 50K/22K junctions in S-1. This enabled direct measurements of the cross-linked and un-cross-linked fractions of the 50K and 22K fragments of S-1. We found that in all cases actin was preferentially cross-linked to the 22K fragment and that the overall stoichiometry of the main cross-linked products was that of a 1:1 complex of actin and S-1. In the second approach, actin was cross-linked to tryptically cleaved S-1, and the course of these reactions was monitored by measuring the decay of the free 50K and 20K fragments and the formation of cross-linked products. After selecting the optimal cross-linking procedure and conditions, we determined that the rate of actin cross-linking to the 20K fragment of S-1 was 3-fold faster than the reaction with the 50K peptide. The overall rate of cross-linking actin to S-1 corresponded to the sum of the individual reactions of the 50K and 20K fragments, indicating their mutually exclusive cross-linking to actin. Thus, the reactions with tryptically cleaved S-1 were consistent with the 1:1 stoichiometry of actin and S-1 in the main cross-linked products and verified the preferential cross-linking of actin to the 20K fragment of S-1. These results are discussed in the context of the binding of actin to S-1.     

Generation of peptides suitable for sequence analysis by proteolytic cleavage in reversed-phase high-performance liquid chromatography solvents

A methodological study of practical importance to protein sequencing has been carried out. Peptide mapping and sequence analysis of the cleavage products of reduced and carboxymethylated ribonuclease have been applied to the study of the activity and specificity of trypsin, chymotrypsin, elastase, lysyl endopeptidase (Achromobacter protease I), endoproteinase Arg-C (from mouse submaxillary gland), Staphylococcus aureus V8 protease, pepsin, and thermolysin in the presence of 20% methanol, ethanol, 2-propanol, and acetonitrile at 22 and 37 degrees C. The peptide bond specificities were retained, and the activities were generally unaffected or moderately reduced at 22 degrees C and pH 8. At 37 degrees C the activity of chymotrypsin, endoproteinase Arg-C, V8 protease at pH 4, and pepsin was substantially reduced and decreased in the order methanol, ethanol, 2-propanol, and acetonitrile. The activity of thermolysin at 55 degrees C was reduced very little in the presence of 20% organic solvent and 50 mM Ca2+. In low calcium and 20% 2-propanol at 22 degrees C the activity of thermolysin was restricted to the complete and specific cleavage of peptide bonds N-terminally of Phe, Ile, and Leu. The experiments suggest that secondary proteolytic digestions can be carried out directly in reversed-phase-HPLC fractions, and that organic cosolvents can be applied to control the degree of proteolysis. Moreover, the denaturing potential of these solvents might be useful in the degradation of proteins resistant to proteolysis, for example, in studies aimed at identification of disulfide bridges.     

Light chain dependent effects of actin binding on the S-1/S-2 swivel in myosin

The S-1/S-2 swivel in myosin provides a flexible link between the head and tail portions of the molecule. We have investigated the properties of the swivel by employing limited proteolysis methods. Our results indicate that the binding of actin to heavy meromyosin inhibits both the chymotryptic and papain cleavage of the S-1/S-2 swivel, and that this effect is dependent on the presence of intact LC-2 light chains. Actin did not slow digestions carried out using heavy meromyosin previously treated with proteases to nick the LC-2 chains to 17,000 or 14,000 Mr fragments. Although the integrity of the LC-2 light chain appears to be required to transmit the effects of actin binding from the myosin head to the S-1/S-2 swivel, the binding of Ca2+ to the 17,000 Mr LC-2 fragment can still affect the chemical reactivity of SH1 thiol groups. Both chymotryptic and papain digestions of heavy meromyosin containing intact or fragmented LC-2 light chain show substantial temperature sensitivity between 5 degrees C and 35 degrees C. Calculated apparent activation energies for this process indicate that the S-1/S-2 swivel in myosin can undergo temperature-dependent structural changes independently of the state of the LC-2 light chain. Thus, both actin binding and temperature variations can induce structural transitions in the S-1/S-2 swivel.     

Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains.

The protein crystals found in potato (Solanum tuberosum L.) tuber cells consist of a single 85-kD polypeptide. This polypeptide is an inhibitor of papain and other cysteine proteinases and is capable of binding several proteinase molecules simultaneously (P. Rodis, J.E. Hoff [1984] Plant Physiol 74: 907-911). We have characterized this unusual inhibitor in more detail. Titrations of papain activity with the potato papain inhibitor showed that there are eight papain binding sites per inhibitor molecule. The inhibition constant (Ki) value for papain inhibition was 0.1 nM. Treatment of the inhibitor with trypsin resulted in fragmentation of the 85-kD polypeptide into a 32-kD polypeptide and five 10-kD polypeptides. The 32-kD and 10-kD fragments all retained the ability to potently inhibit papain (Ki values against papain were 0.5 and 0.7 nM, respectively) and the molar stoichiometries of papain binding were 2 to 3:1 and 1:1, respectively. Other nonspecific proteinases such as chymotrypsin, subtilisin Carlsberg, thermolysin, and proteinase K also cleaved the 85-kD inhibitor polypeptide into functional 22-kD and several 10-kD fragments. The fragments obtained by digestion of the potato papain inhibitor with trypsin were purified by reverse-phase high-performance liquid chromatography, and the N-terminal amino acid sequence was obtained for each fragment. Comparison of these sequences showed that the fragments shared a high degree of homology but were not identical. The sequences were homologous to the N termini of members of the cystatin superfamily of cysteine proteinase inhibitors. Therefore, the inhibitor appears to comprise eight tandem cystatin domains linked by preteolytically sensitive junctions. We have called the inhibitor potato multicystatin (PMC). By immunoblot analysis and measurement of papain inhibitory activity, PMC was found at high levels in potato leaves (up to 0.6 microgram/g fresh weight tissue), where it accumulated under conditions that induce the accumulation of other proteinase inhibitors linked to plant defense. PMC may have a similar defensive role, for example in protecting the plant from phytophagous insects that utilize cysteine proteinases for dietary protein digestion.     

Structure formation in short designed peptides probed by proteolytic cleavage

The formation of local structure, in short peptides has been probed by examining cleavage patterns and rates of proteolysis of designed sequences with a high tendency to form beta-hairpin structures. Three model sequences which bear fluorescence donor and acceptor groups have been investigated: [see text]. Fluorescence resonance energy transfer (FRET) provides a convenient probe for peptide cleavage. MALDI mass spectrometry has been used to probe sites of cleavage and CD spectroscopy to access the overall backbone conformation using analog sequences, which lack strongly absorbing donor and acceptor groups. The proteases trypsin, subtilisin, collagenase, elastase, proteinase K and thermolysin were used for proteolysis and the rates of cleavage determined. Peptide 3 is the most susceptible to cleavage by all the enzymes except thermolysin, which cleaves all three peptides at comparable rates. Peptides 1 and 2 are completely resistant to the action of trypsin, suggesting that beta-turn formation acts as a deterrent to proteolytic cleavage.     

Tryptic digestion of rabbit skeletal myofibrils: an enzymatic probe of myosin cross-bridges

Tryptic digestion of rabbit skeletal myofibrils under physiological ionic strength and pH conditions was used as a probe of cross-bridge interaction with actin in the presence of nucleotides and pyrophosphate. Under rigor conditions, digestion of myofibrils at 24 degrees C results in the formation of 25K, 110K [heavy meromyosin (HMM)], and light meromyosin (LMM) fragments as the main reaction products. Very little if any 50K peptide is generated in such digestions. In the presence of magnesium pyrophosphate, magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and MgATP, the main cleavage proceeds at two positions, 25K and 75K from the N-terminal portion of myosin, yielding the 25K, 50K, and 150K species. The relative amounts of the 50K, 110K, and 150K peptides and the rates of myosin heavy-chain digestion in the presence of pyrophosphate and AMPPNP indicate partial dissociation of myosin from actin. Direct centrifugation measurements of the binding of HMM and subfragment 1 (S-1) to actin in myofibrils confirm that cross-bridges partition between attached and detached states in the presence of these ligands. In the presence of MgADP, HMM and S-1 remain attached to actin at 24 degrees C. However, tryptic digestion of myofibrils containing MgADP is consistent with the existence of a mixed population of attached and detached cross-bridges, suggesting that only one head on each myosin molecule is attached to actin. As shown by tryptic digestion of myofibrils and the measurements of HMM and S-1 binding to actin, nucleotide- and pyrophosphate-induced dissociation of cross-bridges is more pronounced at 4 than at 24 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coagulation of soy proteins induced by thermolysin and comparison of the coagulation reaction with that induced by subtilisin Carlsberg

It is known that coagula are formed when soy proteins are treated by subtilisin Carlsberg. Time-course of the coagulation can be monitored by measuring the turbidity (OD₆₆₀) of soy protein isolates (SPI) solution [Inouye K, Nagai K, Takita T. Coagulation of soy protein isolates induced by subtilisin Carlsberg. J Agric Food Chem 2002;50:1237–42]. In this study, we examined the coagulation of the digests produced in the digestion of SPI with thermolysin. SPI treated at 80 °C coagulated by hydrolysis with either of thermolysin and subtilisin Carlsberg, but that treated at 37 °C did not. The velocities of the coagulation were almost the same between the digestions with thermolysin and subtilisin Carlsberg, while the amount of the coagula in the former was 50–80% of that in the latter. With increasing the thermolysin concentration from 0.1 to 5 μM, the velocity of the coagulation increased while the amount of the coagula was constant. With increasing the reaction temperature from 37 to 70 °C, the velocity increased while the amount decreased. The digests from 11S soy proteins coagulated drastically compared to those from either of SPI, 7S soy proteins, and the mixture of 7S and 11S soy proteins (1:2 for w/v), suggesting that the coagulation of the digests from 11S soy proteins might be suppressed by those from 7S soy proteins.     

Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1

Homogeneous preparations of actin cleaved into two fragments, the N-terminal 9- and C-terminal 36-kDa peptides, were achieved by proteolysis of G-actin with subtilisin at 23 degrees C at a 1:1000 (w/w) ratio of enzyme to actin. The subtilisin cleavage site was identified by sequence analysis to be between Met-47 and Gly-48. Although under nondenaturing conditions the two fragments remained associated to one another, the cleavage affected macromolecular interactions of actin. The rates of cleaved actin polymerization by MgCl2, KCl, and myosin subfragment 1 (S-1) were slower and the critical concentrations for this process were higher than in intact protein. Intact and cleaved actin formed morphologically indistinguishable filaments and copolymerized in the presence of MgCl2. The affinity of actin for S-1 was decreased by about 10-fold due to subtilisin cleavage, but the S-1 ATPase activity was activated to the same Vmax value by both intact and cleaved actins. DNase I inhibition measurements revealed lower affinity of cleaved actin for DNase I than that of intact protein. These results are discussed in terms of actin's structure.     

Structural aspects of myosin subfragment 1 as studied by subtilisin digestion of trypsin-split S-1 (27 kDa-50 kDa-20 kDa)

Limited subtilisin digestion of myosin subfragment 1 (S-1) was carried out, varying the enzyme: substrate weight ratio from 1:200 to 1:10, and changes in structure, and in the MgATPase activities of S-1 and acto-S-1 after proteolysis, were followed. When the starting material--tryptically-cleaved S-1 (27 kDa-50 kDa-20 kDa) ("split S-1")--was subjected to further subtilisin digestion, it was found that with increasing enzyme concentration, the 50 kDa fragment degraded into an 18 kDa fragment via a 33 kDa peptide (50----33----18 kDa), which was not cross-linked with F-actin. On the other hand, the 27 and 20 kDa fragments were rather stable at lower subtilisin concentrations and started to degrade only at higher subtilisin concentrations. These degradations lowered the MgATPase activities of S-1 and acto-S-1. The losses of MgATPase activities of S-1 and of acto-S-1 were mainly due to the degradations of the 27 and 20 kDa fragments, respectively. Addition of EDTA did not affect the subtilisin cleavage pattern of split S-1 but the breakdown of the 50 kDa fragment was extremely depressed, suggesting that some conformational change of the 50 kDa fragment is induced by the binding of divalent cation. The binding of MgADP to split S-1 accelerated the degradation of the 27 kDa fragment and produced a new cut in the 27 kDa fragment (27----20 kDa), resulting in a further loss of the S-1 MgATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flu virion as a substrate for proteolytic enzymes

The proteolysis of flu virions of the strain A/Puerto Rico/8/34 (subtype H1N1) by enzymes of various classes was studied to develop an approach to the study of the structural organization and interaction of the basic protein components of the virion environment: hemagglutinin (HA), transmembrane homotrimeric glycoprotein, and matrix protein M1 forming a layer under the lipid membrane. Among the tested proteolytic enzymes and enzymic preparations (thermolysin, trypsin, chymotrypsin, subtilisin Carlsberg, pronase, papain, and bromelain), the cysteine proteases bromelain and papain and the enzymic preparation pronase efficiently deleted HA ectodomains, while chymotrypsin, trypsin, and subtilisin Carlsberg deleted only a part of them. An analysis by MALDI TOF mass spectrometry allowed us to locate the sites of HA hydrolysis by various enzymic preparations. Bromelain, papain, trypsin, and pronase split the polypeptide chain after the K177 residue located before the transmembrane domain (HA2 185-211). Subtilisin Carlsberg hydrolyzed the peptide bond at other neighboring points: after L178 (a basic site) or V176. The hydrolytic activity of bromelain measured by a highly specific chromogenic substrate of cysteine proteases Glp-Phe-Ala-pNA was almost three times higher in the presence of 5 mM beta-mercaptoethanol than in the presence of 50 mM. However, the complete removal of exodomains of HA, HA, and low-activity enzyme by the HA high- and low-activity enzyme required identical time intervals. In the absence of the reducing reagent, the removal of HA by bromelain proceeded a little more slowly and was accompanied by significant fragmentation of protein Ml1. The action of trans-epoxysuccinyl-L-leucylamido)butane (E-64), a specific inhibitor of cysteine proteases, and HgCl2 on the hydrolysis of proteins HA and M1 by bromelain was investigated.     

Increased proteolytic resistance of ribonuclease A by protein engineering.

Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2',3'-cCMP and in its thermodynamic stability. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS-PAGE, decreased by two orders of magnitude. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. Ser21-Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation.     

ATP-induced structural change in myosin subfragment-1 revealed by the location of protease cleavage sites on the primary structure

To understand the nature of the ATP-induced structural change in myosin subfragment-1, rabbit and chicken skeletal subfragments-1s were cleaved by various proteolytic enzymes in the absence, and in the presence, of ATP and the exact locations of the cleavage sites that were affected by ATP were determined from the amino end analysis of fragments by the use of a protein sequencer. It was found that subtilisin cleaved a site between Gln27 and Asn28 of rabbit subfragment-1 and between Gln28 and Asn29 of chicken subfragment-1 only in the presence of ATP. Thermolysin cleaved a site between Pro31 and Phe32 of chicken subfragment-1 in the presence of ATP, but the same site of rabbit subfragment-1 was not cleaved. The location of these sites is quite similar to the ATP-induced chymotryptic cleavage site of chicken gizzard heavy meromyosin, between Trp29 and Ser30 as reported by others. It is suggested, therefore, that the structure and the ATP-induced structural change in the regions are similar in these subfragment-1s. ATP also changes the cleavage rate of the 26K-50K junction by many proteases. Exact cleavage sites were determined and the relationship between their location and the suppression or the enhancement by ATP of the cleavage was studied. It was found that the cleavage sites were restricted to a quite narrow region and only the cleavage by thermolysin that attacked the middle of the region was enhanced by ATP. The distribution of the cleavage sites and the effect of ATP suggest that ATP induces drastic structural change at the middle of the 26K-50K junction region. The region attacked easily by many proteases coincided very well with a hydrophilic region indicated by the hydropathy index. The region probably protrudes outside and is, therefore, easily attacked by many proteases.     

Flu virion as a substrate for proteolytic enzymes

The proteolysis of flu virions of the strain A/Puerto Rico/8/34 (subtype H1N1) by enzymes of various classes was studied to develop an approach to the study of the structural organization and interaction of the major protein components of the virion: hemagglutinin (HA), transmembrane homotrimeric glycoprotein, and matrix protein M1 forming a layer under the lipid membrane. Among the tested proteolytic enzymes and enzymic preparations (thermolysin, trypsin, chymotrypsin, subtilisin Carlsberg, pronase, papain, and bromelain), the cysteine proteases bromelain and papain and the enzymic preparation pronase efficiently removed HA ectodomains, while chymotrypsin, trypsin, and subtilisin Carlsberg deleted only a part of them. An analysis by MALDI TOF mass spectrometry allowed us to locate the sites of HA hydrolysis by various enzymic preparations. Bromelain, papain, trypsin, and pronase split the polypeptide chain after the K177 residue located before the transmembrane domain (HA₂ 185–211). Subtilisin Carlsberg hydrolyzed the peptide bond at other neighboring points: after L178 (a major site) or V176. The hydrolytic activity of bromelain measured by a highly specific chromogenic substrate of cysteine proteases Glp-Phe-Ala-pNA was almost three times higher in the presence of 5 mM β-mercaptoethanol than in the presence of 50 mM. However, the complete removal of ectodomains of HA by the high-and low-activity enzyme required identical time intervals. In the absence of the reducing reagent, the removal of HA by bromelain proceeded a little more slowly and was accompanied by significant fragmentation of protein M1. The action of trans-epoxysuccinyl-L-leucylamido)(4-guanidino)butane (E-64), a specific inhibitor of cysteine proteases, and HgCl₂ On the hydrolysis of proteins HA and M1 by bromelain was investigated.     

The primary structure of Pseudomonas cytochrome c peroxidase

The primary structure of Pseudomonas cytochrome c peroxidase is presented. The intact protein was fragmented with cyanogen bromide into five fragments; partial cleavage was observed at a Met-His bond of the protein. The primary structure was established partly by automatic Edman degradations, partly by manual sequencing of peptides obtained with trypsin, thermolysin, chymotrypsin, pepsin, subtilisin and Staphylococcus aureus V8 endopeptidase. The order of the cyanogen bromide fragments was further confirmed by overlapping peptides obtained by specific cleavage of the whole protein. Pseudomonas cytochrome c peroxidase consists of 302 amino acid residues giving a calculated M_r of 33 690.     

Activation of rat liver guanylate cyclase by proteolysis.

Guanylate cyclase activity (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.), measured in purified rat liver plasma membranes, was markedly increased by treatment with various purified proteases. The effect was maximal with trypsin, alpha-chymotrypsin, papain, and thermolysin (6- to 8-fold increase with 5 to 20 microgram of protease/ml) and lower with subtilisin and elastase (3- to 4-fold increase). The activation was due to an increase in the maximal velocity of the cyclizing reaction. No modification was observed either in the apparent affinity for the substrate MnGTP or in the cooperative behavior of the enzyme kinetics which displayed Hill coefficients of 1.6 for both basal and activated states. The Triton X-100-dispersed guanylate cyclase remained sensitive to papain, which suggests that the action of proteases was not restricted to an indirect action upon the membranous environment of the guanylate cyclase. In contrast, the cytosolic soluble guanylate cyclase, assayed in the presence or absence of sodium azide, was absolutely insensitive to papain. Thus, proteolysis represents a previously undescribed mechanism for activating membranous guanylate cyclase systems, which might be of importance in the physiological regulation of this enzyme.     

Inhibition of serine proteinases plasmin,trypsin, subtilisin A,cathepsin G,and elastase by LEKTI: a kinetic analysis

The human LEKTI gene encodes a putative 15-domain serine proteinase inhibitor and has been linked to the inherited disorder known as Netherton syndrome. In this study, human recombinant LEKTI (rLEKTI) was purified using a baculovirus/insect cell expression system, and the inhibitory profile of the full-length rLEKTI protein was examined. Expression of LEKTI in Sf9 cells showed the presence of disulfide bonds, suggesting the maintenance of the tertiary protein structure. rLEKTI inhibited the serine proteinases plasmin, subtilisin A, cathepsin G, human neutrophil elastase, and trypsin, but not chymotrypsin. Moreover, rLEKTI did not inhibit the cysteine proteinase papain or cathepsin K, L, or S. Further, rLEKTI inhibitory activity was inactivated by treatment with 20 mM DTT, suggesting that disulfide bonds are important to LEKTI function. The inhibition of plasmin, subtilisin A, cathepsin G, elastase, and trypsin by rLEKTI occurred through a noncompetitive-type mechanism, with inhibitory constants (K(i)) of 27 +/- 5, 49 +/- 3, 67 +/- 6, 317 +/-36, and 849 +/- 55 nM, respectively. Thus, LEKTI is likely to be a major physiological inhibitor of multiple serine proteinases.     

Proteolytic specificity of hemorrhagic toxin b from Crotalus atrox (western diamondback rattlesnake) venom

In our effort to identify the proteolytic specificity of various hemorrhagic toxins isolated from western diamondback rattlesnake venom, hemorrhagic toxin b was isolated in homogeneous form by previously published methods. Hemorrhagic toxin b hydrolyzed glucagon, producing six fragments. The proteolytic sites were identified as Thr(5)-Phe(6), Thr(10)-Ser(11), Asp(15)-Ser(16), Asp(21)-Phe(22) and Try(25)-Leu(26). When oxidized insulin B chain was used, proteolysis occurred at four sites: Asn(3)-Gln(4), His(10)-Leu(11), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The proteolytic specificity of hemorrhagic toxin b is quite different from those of the nonvenom proteases such as thermomycolin, aspergillopeptidase c, alkaline protease from Aspergillus flavus, elastase, subtilisin and papain.     

Correlation between the papain digestibility and the conformation of 10s-myosin from chicken gizzard

In our previous reports, ATP was shown to induce a drastic change in the conformation of gizzard myosin molecules. For example, the sedimentation constant of unphosphorylated myosin (UM) increased from 6S to 10S although an ATP-induced change in the sedimentation constant did not occur with phosphorylated myosin (Suzuki et al. (1978) J. Biochem. 84, 1529). We now report the finding that the ATP-induced formation of 10S-myosin is associated with a drastic change in the papain digestibility of gizzard UM. With 10S-myosin, the cleavage by papain was strongly inhibited at two regions on heavy chains and at one region on light chains; that is, the junction between the 72K dalton and 22K dalton fragments (i.e., a cleavable site in myosin head), the one between the 22K dalton and 130K dalton fragments (i.e., a head-tail junction), and the one between the 3K dalton and 17K dalton fragments of 20K dalton light chains. An even more intimate correlation between the myosin conformation and the papain digestibility of myosin was demonstrated by using thiophosphorylated myosin (thioPM); the cleavages by papain at the 72K-22K dalton junction and the 22K-130K dalton junction were not inhibited when thioPM was digested.     

Limited proteolysis of thermolysin by subtilisin: isolation and characterization of a partially active enzyme derivative

Incubation of the neutral metalloendopeptidase thermolysin at pH 9-10 in the presence of 10 mM CaCl2 for 2 days at room temperature with subtilisin at a 50:1 molar ratio leads to a derivative possessing lower (approximately 3%) but intrinsic catalytic activity. This derivative, called thermolysin S, was isolated by gel filtration in approximately 80% yield and then separated from some residual intact thermolysin by an affinity chromatographic step on Sepharose-Gly-D-Phe. It was found that thermolysin S results from a tight association of two polypeptide fragments of apparent Mr of 24000 and 10000. Dissociation of the complex was achieved under strong denaturing conditions, such as gel filtration on a column equilibrated and eluted with 5 M guanidine hydrochloride. The positions of the clip sites were defined by amino acid analysis, end-group determination, and amino acid sequencing of the isolated fragments and shown to lie between Thr-4 and Ser-5, between Thr-224 and Gln-225, and also between Gln-225 and Asp-226. Thermolysin S, which is therefore a stable complex of fragments 5-224(225) and 225(226)-316, shows a shift in optimum pH of about 1 unit toward the acid range with respect to intact thermolysin and a Km essentially unchanged, with furylacryloyl-Gly-Leu-NH2 as substrate. Inhibitors of thermolysin such as ethoxyformic anhydride and Zn2+ ions inactivate also the nicked enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)