Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The temperature-dependence of local melting within the alpha-helical, coiled-coil structure of rabbit myosin rod has been investigated by following changes in the rate constants of proteolytic digestion. The kinetics of fragmentation of the rod by three different enzymes (alpha-chymotrypsin, trypsin and papain) over the temperature range 5 to 40 degrees C (pH 7, I = 0.5) has been monitored by electrophoresis of the digestion products on sodium dodecyl sulfate/polyacrylamide gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzyme by comparison with model substrates. Results from the three enzyme-probes are similar in showing that local melting within the rod occurs in two distinct stages. At temperatures between 5 and 25 degrees C, melting is confined to a restricted segment of the rod structure near the light meromyosin/heavy meromyosin junction. At temperatures between 25 and 40 degrees C, a wider segment of the rod lysing between the junction and the short subfragment-2 segment (the hinge domain) appears to be melting, judging from the broad spectrum of cleavage sites observed in this region. Results are compared with those from other physicochemical methods that measure the hinging or opening of the coiled-coil structure of the rod.
J Mol Biol 1984 Feb 15
PMID:An enzyme-probe method to detect structural changes in the myosin rod. 636 39

The temperature-dependence of local melting within the subfragment-2 region of rabbit skeletal muscle myosin has been investigated using an enzyme-probe technique. Rate constants of fragmentation of two long subfragment-2 particles (61,000 Mr and 53,000 Mr per polypeptide chain) and a short subfragment-2 particle (34,000 Mr per polypeptide chain) by three different enzymes (alpha-chymotrypsin, trypsin and papain) have been determined over the temperature range 5 to 40 degrees C. We followed the time-course of digestion at specific sites at high (I = 0.50, pH 7.3) and low (physiological, I = 0.15, pH 7.3) ionic strengths by electrophoresis of the digestion products on sodium dodecyl sulfate-containing gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzymes by comparison with model substrates. Normalized rate constant versus temperature profiles for the three enzyme-probes are similar in showing that local melting in long subfragment-2 (61,000 Mr) occurs in two distinct stages as was observed earlier for the intact myosin rod. Over the temperature range 5 to 25 degrees C a restricted region at Mr = 53,000 to 50,000 from the N terminus of the rod (the light meromyosin/heavy meromyosin junction) shows the highest susceptibility to proteolytic cleavage. At temperatures above 25 degrees C local melting was detected by all three enzymes at several specific sites within the hinge domain (Mr = 53,000 to 34,000). Activation energies for cleavage at the susceptible sites were similar for the three enzyme probes. They suggest that this region of the myosin rod has significantly lower thermal stability than the flanking light meromyosin and short subfragment-2 segments. These results, together with other physico-chemical studies, point to the hinge domain of the myosin cross-bridge as an important functional element in the mechanism of force generation in muscle.
J Mol Biol 1984 Dec 15
PMID:An enzyme-probe study of motile domains in the subfragment-2 region of myosin. 639 18

Papain is a sulfhydryl protease from the latex of the papaya fruit. Its molecules consist of one polypeptide chain with 212 amino acid residues. The chain is folded into two domains with the active site in a groove between the domains. We have refined the crystal structure of papain, in which the sulfhydryl group was oxidized, by a restrained least-squares procedure at 1.65 A to an R-factor of 16.1%. The estimated accuracy in the atomic co-ordinates is 0.1 A, except for disordered atoms. All phi/psi angles for non-glycine residues are found within the outer limit boundary of a Ramachandran plot and this provides another check on the quality of the model. In the alpha-helical parts of the structure, the C = O bonds are directed more away from the helix axis than in a classical alpha-helix, leading to somewhat longer hydrogen bonds, 2.98 A, compared to 2.89 A. The hydrogen-bonding parameters and conformational angles in the anti-parallel beta-sheet structure show a large diversity. Hydrogen bonds in the core of the sheet are generally shorter than those at the more twisted ends. The average value is 2.91 A. The hydrogen bond distance Ni+3-Oi in turns is relatively long and the geometry is far from linear. Hydrogen bond formation, therefore, is perhaps not an essential prerequisite for turn formation. Although the crystallization medium is 62% (w/w) methanol in water, only 29 out of 224 solvent molecules can be regarded with any certainty as methanol molecules. The water molecules play an important role in maintaining structural stability. This is specially true for internal water. Twenty-one water molecules are located in contact areas between adjacent papain molecules. It seems as if the enzyme is trapped in a grid of water molecules with only a limited number of direct interactions between the protein molecules. The residues in the active site cleft belong to the most static parts of the structure. In general, disorder in atomic positions increases when going from the interior of the protein molecule to its surface. This behavior was quantified and it was found that the point of minimum disorder is near the molecular centroid.
J Mol Biol 1984 Oct 25
PMID:Structure of papain refined at 1.65 A resolution. 650 13

Two rat monoclonal antibodies (both IgG2a isotype and having closely related specificities) and a pool of rhesus immune IgG, all of which inhibit Plasmodium knowlesi merozoite invasion of rhesus erythrocytes, have been studied before and after proteolytic digestion. The F(ab')2 and Fab fragments of both rat monoclonal antibodies show considerably enhanced inhibition of merozoite invasion as compared with the intact IgG. Inhibition by monovalent fragments indicates that these antibodies are not dependent upon merozoite agglutination and may act by blocking merozoite attachment to the specific red cell receptor. The fact that the inhibitory activities of F(ab')2 and Fab are equally enhanced on a weight basis, as compared with IgG, suggests that the removal of Fc may reduce electrostatic repulsion between antibody and merozoite surface, both of which are negatively charged at neutral pH. By contrast, papain digestion of polyclonal IgG derived from an immunised rhesus pool markedly reduces its inhibitory activity. This suggests that much of the inhibition mediated by polyclonal IgG results from merozoite agglutination and that the specificity of the rat inhibitory monoclonal antibodies is poorly represented in the immune pool. The P. knowlesi antigen reactive with the inhibitory monoclonal antibodies is known to be synthesized as a minor 66 kDa polypeptide during the last 1.5 h. of schizont development and is processed to smaller products (44 and 42 kDa) present on the merozoite surface. The present results suggest that this antigen may have particular interest as a vaccine against P. knowlesi malaria.
Mol Biochem Parasitol 1984 Oct
PMID:The Fab fragments of monoclonal IgG to a merozoite surface antigen inhibit Plasmodium knowlesi invasion of erythrocytes. 651 92

The H-2Kk molecule was purified by immunoprecipitation from the glycoprotein fraction of Nonidet P-40 extracts of RDM 4 mouse tumor cells. Cyanogen bromide cleavage of the major papain fragment yielded three peptides, the largest of which consisted of three disulfide-linked peptides which could be separated after reduction and alkylation. These peptides were readily aligned by their homology to similar fragments derived from other H-2 class I molecules. Amino acid sequence analyses of the two nondisulfide-linked peptides, peptide E (residues 1-52) and peptide D (53-98), yielded the following NH2-terminal sequence for the H-2Kk molecule: [sequence in text]. Comparison of this sequence with those of other H-2 class I molecules revealed that: (1) Lys-19, Val-55, Glu-56, Asn-63 and Ile-73 are unique to the H-2Kk molecule; and (2) H-2Kk shares 79-83% homology in this region with other mouse class I molecules. Partial NH2-terminal amino acid sequences are also reported for the three disulfide-linked peptides. Several discrepancies from previously reported partial sequences of the H-2Kk molecule were detected.
Mol Immunol 1984 Mar
PMID:Amino acid sequence analysis of the H-2Kk alloantigen: complete sequence of residues 1-98 and partial sequence from 99 to 263. 671 43

A variety of structural mutations that alter functional properties of regulatory subunit (R) of type I cyclic AMP-dependent protein kinase are available in the cultured S49 mouse lymphoma cell system. Many of these mutations also alter the electrostatic charge of R by about 1 or 2 units. By a novel peptide mapping procedure, a number of these "charge-shift" structural mutations were localized to small regions within the R polypeptide. The procedure employed two-dimensional polyacrylamide gel electrophoresis to separate large overlapping fragments generated from denatured, affinity-purified R by limited digestion with papain. Mutations were mapped to intervals between the endpoints of these fragments. The position of one mutation was confirmed by mapping a new site for cleavage by Staphylococcus aureus V8 protease. Six different Ka mutations, which increase the concentrations of cyclic AMP required for kinase activation, mapped to three clusters in the carboxy-terminal half of R. Second-site mutations that cause phenotypic reversion of a single Ka mutant strain mapped to either side of the original mutation. By using charge-shift mutations for calibration, a map of charge density distribution was constructed for the R polypeptide. This map allowed tentative assignment of mutational lesions to portions of the R amino acid sequence implicated in cyclic AMP binding.
Mol Cell Biol 1984 Jun
PMID:Fine-structure mapping of charge-shift mutations in regulatory subunit of type I cyclic AMP-dependent protein kinase. 673 32

The three-dimensional structure of the sulfhydryl protease calotropin DI from the madar plant, Calotropis gigantea, has been determined at 3.2 A resolution using the multiple isomorphous replacement method with five heavy atom derivatives. A Fourier synthesis based on protein phases with a mean figure of merit of 0.857 was used for model building. The polypeptide backbone of calotropin DI is folded to form two distinct lobes, one of which is comprised mainly of alpha-helices, while the other is characterized by a system of all antiparallel pleated sheets. The overall molecular architecture closely resembles those found in the sulfhydryl proteases papain and actinidin. Despite the unknown amino acid sequence of calotropin DI a number of residues around its active center could be identified. These amino acid side-chains were found in a similar arrangement as the corresponding ones in papain and actinidin. The polypeptide chain between residues 1 and 18 of calotropin DI folds in a unique manner, providing a possible explanation for the unusual inability of calotropin DI to hydrolyze those synthetic substrates that papain and actinidin act upon.
J Mol Biol 1982 Nov 15
PMID:Crystal and molecular structure of the sulfhydryl protease calotropin DI at 3.2 A resolution. 675 64

An IgGl (lambda) protein which showed a unique susceptibility towards papain digestion was isolated from the serum of a patient (Mot) with multiple myeloma. The Fab fragments of this protein were degraded rapidly into smaller peptides via an Fb fragment [Gall & D'Eustachio (1972), Biochemistry 11, 4621-4628], which corresponded to the constant domains (Cl-Chl). Structural analysis of the isolated Fab fragment, which consisted of the intact L-chain, a 17,000 and a 5000 mol. wt peptide fragment, indicated that the initial cleavage site was located in the vicinity of the second hypervariable region of the Fd fragment. Examination of the partial amino acid sequences of the Mot H-chain suggested that the variable region of the H-chain may be a hitherto unknown hybrid of subgroups I and III. This particular structure seems to have made the Fab fragment highly susceptible to papain. In the course of the present study, we also found in the papain digests of several human IgG proteins an 'intermediate' 5S fragment, which had previously been reported exclusively for the papain digest of rabbit IgG.
Mol Immunol 1982 Sep
PMID:An unusual papain cleavage of a human IgG1 (lambda) myeloma protein (Mot). 681 81

The primary structure of the carboxy-terminal portion of the H-2Kd murine major histocompatibility antigen has been determined using radiosequencing methodology. The two peptides encompassing the entire cytoplasmic portion of the H-2Kd molecule were isolated from cyanogen bromide digests of the detergent solubilized molecule. These two peptides are not present in CNBr digests of papain-solubilized H-2Kd. Alignment of the two CNBr peptides was deduced from tryptic overlap peptides derived from the whole molecule. Alignment with the corresponding region of the H-2Kb antigen shows 90% homology and supports the assignment of this segment of H-2Kd as the C-terminal. The sequence obtained in this study is (Met)-Arg-Arg-Asn-Thr-Gly-Gly-Lys-Gly-Val-Asn-Tyr-Ala-Leu-Ala-Pro-Gly-Ser-Gln- Thr-Ser-Asp-Leu-Ser-Leu-Pro-Asp-Pro-Lys-Val-Met-Val-His-Asp-Pro-His-Ser-Leu- Ala. These data allow extensive comparisons with the protein sequences deduced from the 3' ends of H-2d haplotype cDNA and genomic clones as well as with the homologous regions of H-2Kb and H-2Db.
Mol Immunol 1983 Jan
PMID:Amino acid sequence of the cytoplasmic segment of the h-2Kd (H2.31) murine major histocompatibility antigen. 685 75

Rats pretreated with xylene or phenobarbital, and then exposed to n-hexane, exhibited a markedly increased peak serum concentration of the neurotoxic metabolite 2,5-hexanedione. In order to elucidate the mechanism underlying this synergistic effect, the major liver microsomal cytochrome P-450 isozymes induced by xylene and phenobarbital, respectively, were purified. In a reconstituted system both isozymes showed a high enzymatic activity with n-hexane as the substrate. Turnover numbers for the formation of 2-hexanol were 24 and 27 for the xylene- and phenobarbital-induced isozyme, respectively. The turnover numbers for 7-ethoxycoumarin, benzo[a]pyrene, and 1,1,2,2-tetrachloroethane were also in the same range for the two cytochrome P-450 preparations. The isozyme induced by xylene had an amino acid composition very similar to that of the phenobarbital-induced isozyme, and the purified proteins had identical electrophoretic mobilities on polyacrylamide gels in the presence of sodium dodecyl sulfate. Furthermore, similar peptide maps were obtained following digestion with alpha-chymotrypsin and papain, and each isozyme yielded a single immunoprecipitin band upon reaction with the immunoglobulin G fraction from rabbits immunized with the phenobarbital-induced enzyme. We conclude that xylene induces a rat liver microsomal cytochrome P-450 isozyme very similar to the major isozyme induced by phenobarbital and that this induction is the probable explanation for the enhanced formation of 2,5-hexanedione from n-hexane in vivo.
Mol Pharmacol 1983 Jan
PMID:Xylene induces a cytochrome P-450 isozyme in rat liver similar to the major isozyme induced by phenobarbital. 686 1


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