Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human brain and liver mitochondria contain membrane-bound monoamine oxidase of both A and B types. Monamine oxidase-A (MAO-A), either membrane-bound or in detergent-solubilized extracts from these tissues, was selectively inhibited during incubations with trypsin, chymotrypsin, thermolysin, or papain. MAO-A in solubilized, but not in membrane-bound, preparations was also very sensitive to the action of phospholipase A2, while MAO-B was unaffected. Membrane-bound MAO-A of rat brain mitochondria was more sensitive to phospholipases and less sensitive to proteases than was human brain enzyme, indicating that these agents may reveal species differences in MAO properties. Human brain and liver MAO-A, either solubilized or bound in mitochondrial membranes, apparently contains basic and aromatic peptide moieties that are available to proteases. Hydrolysis of these peptide bonds leads to rapid denaturation unless substrate molecules stabilize the active site. Phospholipase A2 may disrupt the phospholipid microenvironment of MAO-A, the integrity of which is essential for MAO-A activity, but not for MAO-B. No interconversion of the two activities was observed. After phospholipase A2 treatment, remaining MAO-A activity was recovered in low-molecular-weight regions of a gel filtration gradient, suggesting that MAO-A subunits were released. Although these experiments argue against the proposal that phospholipids may regulate the ratio of A/B activities of a single enzyme molecule, it is conceivable that endogenous phospholipases or proteases in mitochondrial membranes may influence MAO-A activity independently of MAO-B activity.
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PMID:Selective effects of proteases and phospholipase A2 on monoamine oxidases A and B of human brain and liver. 637 37

The midgut caecal cells from Rhynchosciara americana larvae possess a plasma-membrane-bound beta-D-glucosidase (cellobiase, EC 3.2.1.21), which is recovered (75-95%) in soluble form both after treatment with Triton X-100 and after treatment with papain. The Triton X-100-solubilized beta-D-glucosidase displays Mr106000 and pI 5.4, whereas the papain-released beta-D-glucosidase shows Mr65000 and pI 4.7. Thermal inactivations of the detergent-solubilized and the papain-released forms of beta-D-glucosidase both follow apparent first-order kinetics with similar half-lives. The papain-released beta-D-glucosidase, after being purified by density-gradient centrifugation, hydrolyses beta-D-glucosides, beta-D-galactosides and beta-D-fucosides at the same active site, as inferred from experiments of competition between substrates. The beta-D-glucosidase seems to operate in accordance with rapid-equilibrium kinetics, since the Km (0.61 mM) for the enzyme is constant over a wide range of pH. The hydrolysis of the beta-D-glucosidic bond catalysed by the beta-D-glucosidase occurs without inversion of configuration, delta-gluconolactone is a strong (Ki 0.5 microM) inhibitor of the enzyme and substituents in the substrate aglycone affect the catalytic constant of the reaction. These data support the assumption that the mechanism of the reaction catalysed by the beta-D-glucosidase involves the intermediary formation of a carbonium ion, rather than a glucosyl-enzyme intermediate.
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PMID:Physical and kinetic properties of a plasma-membrane-bound beta-D-glucosidase (cellobiase) from midgut cells of an insect (Rhynchosciara americana larva). 641 80

Bovine band 3 in membrane-bound and solubilized states was digested with chymotrypsin, trypsin, and papain. Bovine band 3 in red blood cells was fragmented by the proteases in a 5 mM NaH2PO4-Na2HPO4 buffer containing 0.3 M glucose, pH 8.0, but not in a 5 mM NaH2PO4-Na2HPO4 buffer containing 0.15 M NaCl, pH 8.0, in which human band 3 is cleaved by chymotrypsin and papain. When compared with the known data for human band 3, however, major fragments of bovine band 3 derived from intact cells, inside-out vesicles and unsealed ghosts were similar to those of human band 3, except that tryptic fragments were formed on the extracellular attack. The results suggest that bovine band 3 adopts a quite similar molecular arrangement in the membrane to in the human case. However, it was strongly suggested by molecular weight evaluation of fragments that the only detectable water-soluble 38,000-39,000 dalton fragment does not account for the entire hydrophilic pole of the band 3 molecule exposed in the cytoplasmic region of the membrane. When isolated band 3 was treated with the enzymes in a 2% solution of nonaethyleneglycol n-dodecyl ether, the major product was indistinguishable on sodium dodecyl sulfate-gel from the water-soluble fragment of the cytoplasmic domain origin of band 3. This fragment lost its resistance to further enzymatic degradation when treated with dimethylmaleic anhydride, thus band 3 oligomers were converted into their monomers. The chymotryptic 38,000 dalton water-soluble fragment obtained in nonaethyleneglycol n-dodecyl ether solution was a subfragment of a 50,000 dalton piece which was produced in a 2% solution of deoxycholate after chymotrypsin treatment of band 3.
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PMID:Proteolytic digestion of band 3 from bovine erythrocyte membranes in membrane-bound and solubilized states. 674 85

The structure of the muscarinic acetylcholine receptor was investigated by comparing polypeptides identified by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis with the size of the intact receptor in cell membranes as determined by target size analysis. Muscarinic receptors from human, dog, and rat brain, rat and dog cardiac muscle, and guinea pig ileum longitudinal smooth muscle labeled with [3H] propylbenzilylcholine mustard, a covalent affinity reagent, appeared as single polypeptides with molecular weights of 80,000 on NaDodSO4-polyacrylamide gels. NaDodSO4-polyacrylamide gels of ileum smooth muscle muscarinic receptor also consistently displayed smaller peptides of 64, 52, 42, 36, 23, and 18 kDa. In order to determine whether the 80-kDa protein represented all or only a portion of the muscarinic receptor, target size analysis was undertaken. Radiation-induced receptor inactivation was measured by loss of [3H]quinuclidinyl benzilate specific binding and by loss of [3H]propylbenzilylcholine mustard-labeled receptor protein on NaDodSO4 gels. Target size analysis of rat and human brain, canine heart, and guinea pig ileum smooth muscle muscarinic receptors all indicated that the intact membrane-bound receptor has an average molecular mass of 80,000 daltons. These data demonstrate that the protein isolated on NaDodSO4 gels represents the intact receptor molecule. The question of whether structurally distinct receptors exist in different tissues and species was answered, in part, by limited proteolysis studies of the 80-kDa protein isolated from the above tissues. Trypsin and papain produce peptides of 64, 52, 42, 36, 23, and 18 kDa from all receptors studied, indicating a lack of major structural diversity and the absence of multiple structural forms of the muscarinic receptor. Limited proteolysis of the membrane-bound receptor produces a major peptide of 42,000 daltons and minor peptides of 36, 23, and 18 kDa, all of which contain the ligand binding site and protrude from the membrane into the extracellular space.
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PMID:Muscarinic cholinergic receptor structure. Receptor size, membrane orientation, and absence of major phylogenetic structural diversity. 683 79

The topography of the intestinal microvillous membrane and its surface components was examined using biochemical and ultrastructural techniques. Microvillous membrane surface glycoproteins were labeled using everted intact intestinal sacs, prepared from rat proximal small intestine. Galactosyl residues were identified by labeling with galactose oxidase/sodium boro[3H]hydride and free amino groups with pyridoxal phosphate/sodium boro[3H]hydride. Membranes were purified, solubilized in sodium dodecyl sulfate, and protein labeling analyzed by acrylamide electrophoresis. Using the intact intestine, only microvillous membrane surface amino groups were labeled. However, when microvillous membrane vesicles were purified first and then labeled, radioligand binding to galactoproteins and free amino groups substantially increased. Ultrastructural and cytochemical studies with ruthenium red revealed the intact intestinal surface to have a strong electronegative charge due to the presence of anionic sites in both the thick mucopolysaccharide surface coat and its underlying glycocalyx. During purification of microvillous membrane, the mucous coat was detached from the membrane surface, leaving the glycocalyx directly exposed to the external environment. Enzymatic probing of microvillous membrane vesicles with papain left the vesicles intact and revealed the membrane to be asymmetric with the majority of its integral proteins located at the outer membrane surface. This orientation of galactosyl and amino groups towards the intestinal lumen plus the overlying thick mucopolysaccharide coat should theoretically afford a greater degree of protection against destruction by luminal proteases and bile acids. Moreover, the shedding of membrane-bound hydrolases into the mucous layer may allow the "membrane surface" phase of digestion to commence before nutrients have diffused completely through the surface coat to reach the enterocyte surface.
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PMID:Biochemical and ultrastructural characterization of the molecular topography of the rat intestinal microvillous membrane. Asymmetric distribution of hydrophilic groups and anionic binding sites. 685 60

Low concentrations of papin rapidly cleave solubilized or membrane-bound acetylcholine receptor (AcChR) from Torpedo californica into a wide range of small fragments. The alpha subunits of the receptor are most resistant to cleavage. After solubilization in sodium dodecyl sulfate solutions the fragments are dissociated, and on electrophoresis the apparent subunit composition is reduced from four types (alpha, beta, gamma, and delta) to only alpha and finally, with large amounts of papain, to fragments even smaller than alpha. Prior to dissociation in sodium dodecyl sulfate, the proteolytic fragments remain physically and functionally associated. Thus, receptor which has been degraded so as to apparently contain only alpha subunits, or even no obvious subunits, still retains antigenic determinants corresponding to each subunit, still retains its characteristic size and doughnut shape when examined electron microscopically, and still sediments as dimers on sucrose gradients. Moreover, proteolytically nicked receptor remains fully functional in carbamylcholine-induced 22Na+ flux. These results demonstrate that inadequate inhibition of proteases during purification of receptor could account for reports from some laboratories that they have purified receptors containing only alpha subunits or fragments of alpha subunits. Also, our results demonstrate the strong noncovalent association between AcChR subunits which has thus far precluded their separation except under denaturing conditions in sodium dodecyl sulfate.
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PMID:Proteolytic nicking of the acetylcholine receptor. 700 Jan 83

Papain treatment of isolated brush border membrane vesicles was carried out to study peptide transport in the absence of hydrolytic events associated with the brush border membrane. Such a treatment allowed a 70% decrease in the activity of membrane-associated oligopeptidases and the study of peptide transport in the complete absence of free amino acids up to 1 min of incubation. A comparison between the time course curves of glycyl-L-phenylalanine uptake by normal and papain-treated vesicles showed that the overshoots seen in the presence of Na+ and K+ gradients (extravesicular greater than intravesicular) when using normal vesicles were no longer evident after papain treatment. This result, together with the demonstration of uptake into an osmotically reactive intravesicular space and the analysis of uptake of free phenylalanine, allowed the conclusion that peptide transport was the result of two complementary mechanisms, uptake of free amino acids following hydrolysis by the membrane-bound oligopeptidases, and intact peptide transport down a concentration gradient by a non-Na+ (and non-K+)-dependent process. These results also showed the non-involvement of gamma-glutamyltransferase and the gamma-glutamyl cycle in peptide absorption. A linear relationship has been established between initial dipeptide uptake and glycyl-L-phenylalanine concentration for the intact peptide transport process. However, this process can be inhibited to various extents by other di- and tripeptides but the inhibition never exceeded 43%. These results are consistent with both passive and facilitated diffusion mechanisms of intact peptide transport, the latter occurring by either a low affinity-high capacity or a high affinity-low capacity system.
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PMID:Characteristics of dipeptide transport in normal and papain-treated brush border membrane vesicles from mouse intestine. I. Uptake of glycyl-L-phenylalanine. 703 91

A simple and rapid procedure involving papain cleavage of the membrane anchor was used to isolate membrane-bound acetylcholinesterase from bovine erythrocytes. The solubilized enzyme was purified 930-fold by ion exchange chromatography and gel filtration. The properties of the papain-cleaved acetylcholinesterase were compared with those of a commercial acetylcholinesterase, solubilized from the erythrocyte membranes by detergents. Cleavage of the membrane anchor eliminated dimer aggregation, caused a pH shift in thermal stability and resulted in increased stability in organic solvents. Bovine serum albumin, used as stabilizer of the commercial enzyme preparation, increased the thermal stability but concomitantly decreased the activity of acetylcholinesterase at pH 6-8. The improved stability of the cleaved acetylcholinesterase, especially in organic solvents, may enhance the biosensor performance of the enzyme.
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PMID:Improved properties of bovine erythrocyte acetylcholinesterase, isolated by papain cleavage. 776 65

Lysis of papain-treated group A and B erythrocytes by human complement was studied by an anti-A (BRIC. 131) and an anti-B (BRIC. 30) IgM monoclonal antibody in 51Cr release assays. The indirect effect of membrane-bound antibody, i.e. its influence on complement binding to sensitized surrounding cells, was examined in a cold target competition test in which sensitized, non-labelled cells are present along with sensitized labelled cells and complement. The mode by which anti-A antibodies indirectly suppressed lysis of sensitized B cells up to 20-fold was studied by following C1q and C3b binding. C1q binding to both types of erythrocytes was not altered in mixed populations of erythrocytes in the presence of both antibodies. Binding of C3b to a mixture of both cell types was, however, suppressed, when both antibodies were present. C3b deposition in mixed cell populations did not reach a significantly higher extent than deposited to one type of erythrocyte alone. This was consistent with the results from competitive lysis and suggests that the anti-A captured most C3b at high anti-A concentrations and deprived the similarly sensitized B erythrocytes of complement. We think that this phenomenon is not due to an uneven removal of complement regulatory proteins from A and B erythrocytes by papain. Instead, the phenomenon might be due to an inherent property of anti-A mAb to better produce nucleation sites for C3 convertases which, upon binding factor B, better compete for the limiting factor D. A mathematical analysis of cold target competition experiment (containing 2430 individual measurements) also shows that the distribution of complement between the competing A and B erythrocyte population is uneven, since it predicts that in any given antibody combination the majority of complement is bound to A erythrocytes. This is consistent with the measured average percentage of lysis.
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PMID:An indirect effect of an antibody on complement deposition and lysis of differently sensitized surrounding cells. 806 73

Regulation of the expression of major histocompatibility complex (MHC) class I heavy chains not associated with beta 2-microglobulin (beta 2m) on freshly isolated and in vitro cultured human B and T leukemia cells was analyzed. These beta 2m-free class I heavy chains originate from surface beta 2m-associated MHC class I molecules and are expressed as integral membrane glycoproteins on activated, but not resting, cells. We found that the levels of beta 2m-free class I heavy chains can be regulated by proteolytic cleavage and release into the medium of soluble molecules containing the extracellular domains. The release is mediated by a Zn(2+)-dependent, membrane-bound metalloprotease that does not cleave HLA-DR, CD4, and CD71 surface receptors and can be activated by phorbol myristate acetate. Specific cleavage by the metalloprotease occurs at a site close to the papain cleavage site in the alpha 3 domain of class I heavy chains. This site is not accessible to the metalloprotease in beta 2m-associated MHC class I molecules. The dissociation of beta 2m-associated MHC class I molecules and subsequent cleavage of beta 2m-free class I heavy chains may be partially responsible for controlling the levels of MHC class I molecules on the surface of activated cells.
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PMID:Soluble beta 2-microglobulin-free class I heavy chains are released from the surface of activated and leukemia cells by a metalloprotease. 812 26


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