Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Immunoglobulin M (IgM, 19S macroglobulin) is a high molecular weight antibody molecule currently thought to be composed of five identical 7S subunits linked by disulfide bonds, perhaps in a cyclic conformation. Reported molecular weights of IgM vary from 8 x 10(5) to 10(6). Proteolytic cleavage of this molecule under standard conditions with several enzymes results in the formation of ten antigen-binding (Fab) fragments per mole, each Fab having a molecular weight of 40,000. The remainder of the molecule, designated Fc by analogy with the structure of immunoglobulin G (IgG), is highly susceptible to further enzyme cleavage and breaks down to dialyzable peptides. However, under carefully controlled conditions, an intact Fc-like fragment in low yield can be isolated from short-term papain digests of IgM at 37 degrees C. Availability of the Fc portion of the molecule is important not only for structural studies but because it may determine certain important biological properties of the molecule such as complement fixation and placental permeability. We here report that trypsin cleavage of IgM at temperatures exceeding 50 degrees C results in the production of excellent yields of an intact Fc fragment, whereas no detectable Fc fragment is produced by trypsin cleavage at 37 degrees C. Fab fragments are obtained under both temperature conditions, and an Fab dimer has been identified in high temperature digests. This unusual difference in the products of enzyme digestion with temperature is unexplained but may be related to steric changes induced in the IgM molecule by heat. Fc fragment isolated from high temperature digests contains two-thirds of the carbohydrate of the intact molecule, and has a molecular weight of 342,000. The molecular weight falls to 67,300 after treatment with disulfide reducing agents, thus supporting the concept of a pentameric structure for IgM.
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PMID:Immunoglobulin M: pentameric Fcmu fragments released by trypsin at higher temperatures. 498 34

A new subclass of mouse IgG for which we propose the name IgG3 has been shown to have a mol wt of 150,000 consistent with an L(2)H(2) structure, and is present in normal mouse serum at a concentration of 0.1-0.2 mg/ml. Its molecular weight, low carbohydrate content, glycopeptide analysis, and C-terminal analysis are all typical of the IgG class. The intact protein had a strong tendency to form noncovalent aggregates with itself which were dissociable in acid. Upon papain digestion an Fab fragment of 47,000 mole wt was generated along with an Fc fragment which was insoluble at neutral pH. As for its biology, the protein did not fix complement, was not cytophilic for gammaG2 receptor sites on macrophages, and did not show passive cutaneous anaphylaxis. It was very efficiently transported across the placenta so that its concentration in the newborn was twice that in the serum of the mother, compared to the concentration of IgG1 and IgG2 proteins which were only present at one-third the concentration of that found in the serum of the mother. The Fc fragment of this protein reacted with and was solubilized by the staphylococcal A protein which also precipitated the intact immunoglobulin. In addition, the myeloma protein which was the prototype for this gammaG subclass exhibited binding activity for levan which was localized to the Fab fragment.
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PMID:A new mouse immunoglobulin: IgG3. 513 63

A protein-protein complex from human serum was isolated and characterized as a two-component system, containing albumin and IgG in a 1:1 mole ratio. The observations reported here suggest that the albumin-IgG complex may have formed through intermolecular disulfide bonds between albumin and IgG. Immunoelectrophoretic analysis of papain-digested fragments of albumin-IgG complex revealed that the combining sites for albumin may be located in the hinge region between the Fab and the Fc fragments of IgG.
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PMID:Disulfide linking of albumin to the hinge region of immunoglobulin G in normal human serum. 663 56

Anti 5-methyl-cytidine antibodies might be useful agents for the detection and localization of 5-methyl-cytidine of nucleic acids, but only if the antibodies recognize this nucleoside with sufficient specificity. A conjugate containing 18 moles of 5-methyl-cytidine per mole of BSA was prepared and antibodies directed against this nucleoside hapten were produced by immunization of rabbits (as determined by gel diffusion in agar containing excessive amounts of the carrier). A slight crossreaction of cytidine-BSA was eliminated by adsorption on the cross-reacting antigen. Further purification of the antibodies was effected by chromatography on DEAE-Sephadex A-50 and a method for the rapid quantitation of the antibodies showed that 12.7% of the IgG protein are monospecific against 5-methyl-cytidine-BSA. Hydrolysis of antibodies with insolubilized papain produced monovalent Fab fragments which were identified by SDS-Disk-electrophoresis. A two stage method for cross linking the immunoproteins to ferritin by glutaraldehyde was used. The isolation of immunoferritin conjugates by Bio-Gel A 1.5 m column chromatography is described. The identification of the effluents was made by glycerin density gradient ultracentrifugation. The results were visualized by electron microscopy after the treatment of immunoferritin conjugates with (methylated and unmethylated) denaturated DNA, fractionation on the glycerine density gradient, and the spreading by a modification of drop technique.
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PMID:Monospecific antibody against 5-methyl-cytidine for the structural analysis of nucleic acids. 711 47

The X-ray structure of chymopapain, a cysteine proteinase isolated from the latex of the fruits of Carica papaya L., has been determined by molecular replacement methods and refined to a conventional R factor of 0.19 for all observed reflections in the range from 9.5 to 1.7 A resolution. The crystals used in this study contained a unique molecular species of chymopapain with two moles of thiomethyl attached to the two free cysteines per mole of enzyme. A comparison is made with the other known papaya proteinase X-ray structures: papain, caricain, and glycyl endopeptidase. Their backbone conformations are extremely similar except for two loop regions. Both regions are located at the surface of the protein and far away of the active site cleft. In each X-ray structure the same water network was found at the interface between the two domains of the enzyme. A close examination of the active site groove showed that the specificity restrictions dictated by the S2 subsite did not differ significantly among the four proteinases.
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PMID:Structure of chymopapain at 1.7 A resolution. 897 3

Here, we report the biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP) (EC 2.4.2. 30). PARP was effectively mono(ADP-ribosyl)ated both in solution and via an activity gel assay following SDS-PAGE with 20 microM or lower concentrations of [32P]-3'-dNAD+ as the ADP-ribosylation substrate. We observed the exclusive formation of [32P]-3'-dAMP and no polymeric ADP-ribose molecules following chemical release of enzyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrophoresis. The reaction in solution (i) was time-dependent, (ii) was activated by nicked dsDNA, and (iii) increased with the square of the enzyme concentration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribosyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin indicated that the amino acid acceptor sites for chain initiation with 3'-dNAD+ as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-terminal catalytic fragment became ADP-ribosylated with [32P]-3'-dNAD+ as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.
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PMID:Biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase. 1019 6

A multidomain cystatin was purified from the leaves of mature and seedling tomato plants (Lycopersicon esculentum, cv Bonnie Best) that had been sprayed with methyl jasmonate. For seedlings, cystatin purification was accomplished using EDTA washing, KCI extraction, 70 degrees C heat treatment, ammonium sulfate fractionation and gel filtration chromatography. For mature plants, DEAE chromatography was also needed to separate a protease, hydrolysis products of cystatin and serine proteinase inhibitors from the intact cystatin. Purified tomato cystatin has a molecular weight (Mr) of 88 kDa, eight papain binding domains, is a non-competitive inhibitor of papain with K1 of 1.4 nM and is not a glycoprotein. Tryptic peptides (Mr 26, 13 kDa) and most chymotryptic peptides (Mr 33, 13 kDa) of tomato cystatin retain inhibitor activity. Amino acid analysis revealed no Cys; Asx, Glx, Gly, Ser accounted for almost half the residues and there was some homology with potato multicystatin. Activity is stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at > 60 degrees C (Ea = 92.5 kJ/mole). Extracts of mature plants treated with methyl jasmonate contain lower Mr cystatins that appear to result from the action of an endogenous 26 kDa protease on the 88 kDa inhibitor.
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PMID:Purification and characterization of a cystatin from the leaves of methyl jasmonate treated tomato plants. 1108 31

An aqueous solution of ionic liquid BMIM.BF(4) (1-butyl-3-methylimidazolium tetrafluoroborate) was used as the reaction medium for the asymmetric hydrolysis of D,L-p-hydroxyphenylglycine methyl ester to enantiopure L-HPG (L-p-hydroxyphenylglycine) for the first time. Papain was screened out from five proteases and two lipases tested because of its high activity and enantioselectivity for this reaction. A comparative study demonstrated that papain is more active, enantioselective and stable in the sodium phosphate buffer solution of BMIM.BF(4) than in aqueous buffer and in the buffer solutions of several typical organic solvents examined. L-HPG with an e.e. (enantiomeric excess) of 95.6% and the yield of 47.4% (on a mole basis) could be obtained when the reaction was performed in a buffer solution of 12.5% (v/v) BMIM.BF(4) (pH 7.0). The aqueous ionic liquid co-solvent increased papain's half-life by 3.1-, 3.8-, 4.7- and 16.3-fold when compared with aqueous buffer, aqueous propan-2-ol, aqueous acetonitrile and aqueous 1,4-dioxan co-solvent respectively at 40 degrees C. When hydrolysis was conducted under reduced pressure, the yield, the e.e. of the L-HPG and the e.e. of remaining D-hydroxyphenylglycine methyl ester achieved were as high as 49.8, 98.1 and >99.0% respectively.
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PMID:Enzymic asymmetric hydrolysis of D,L-p-hydroxyphenylglycine methyl ester in aqueous ionic liquid co-solvent mixtures. 1530 45

Competitive inhibitors can activate proteases (papain, trypsin, and cathepsin S) to catalyze the synthesis of peptide bonds and accelerate the hydrolysis of poor substrates (from 1 to 99%). Reaction mixtures contained intermediate molecules that were formed by the coupling of the inhibitor with the poor substrate. This and other findings suggest the following chain of events. Part of the binding energy of formation of the enzyme-inhibitor complex was used to activate the inhibitor, i.e., to form acyl-enzyme species with a high-energy bond (e.g., a thioester bond in the case of papain) required for coupling the inhibitor with the substrate to form the intermediate molecule. The latter was subjected to successive reactions which led to a stepwise degradation of the substrate, as well as to the regeneration of the inhibitor. One mole of the inhibitor could catalyze rapid hydrolysis of at least 53 mol of substrate. The intermediate molecules were the species undergoing rapid hydrolysis. Therefore, 1 mol of inhibitor was involved in the synthesis of 53 mol of intermediate molecules; i.e., the inhibitor functioned as a cofactor that catalyzed the synthesis of peptides. Thus, the binding energy of formation of the enzyme-inhibitor complex can be utilized to catalyze the synthesis of peptide bonds in the absence of an exogenous energy source (e.g., ATP).
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PMID:Inhibitors can activate proteases to catalyze the synthesis and hydrolysis of peptides. 1714 50

If a chemical reaction is constrained to occur within an asymmetric structure, e.g. by the presence of bound or otherwise trapped enzyme, coupling of the reaction to the flow of one or more solutes, or to the flow of electric current, becomes possible. Such systems can serve as models in which transport is "driven" by chemical reaction. In this respect the processes involved are analogous to active transport, though the molecular mechanisms may be quite different from those in nature. A simple arrangement of this kind has been studied: a composite membrane consisting of two ion exchange membranes of opposite fixed charge, separated by an intermediate layer of solution containing papain. An uncharged substrate of low molecular weight acts as "fuel" for the system, N-acetyl-L-glutamic acid diamide. This material (not previously described) hydrolyzes in the presence of papain to ammonium N-acetyl-L-glutamine. The composite membrane gives rise to an electromotive force, ultimately reaching a stationary state, when clamped between two identical solutions in which the affinity of the reaction has been fixed. Onsager's reciprocity relation has not hitherto been tested in a case of coupling between chemical reaction and a vectorial flow (here electric current); its validity for this system, in which stationary-state coupling occurs, was established over the experimental range of affinities (up to 3 kcal/mole).
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PMID:The coupling of an enzymatic reaction to transmembrane flow of electric current in a synthetic "active transport" system. 1921 Sep 96


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