Low Concentrations Of Papain Research Articles

Effects of protein hydrolysis on pasting properties of wheat flour

AbstractProtein hydrolysis is used widely in the food industry for the purpose of improvement of food or industrial quality, and the processing exists concurrently or subsequently with starch pasting. The effects of protein hydrolysis using papain, pepsin, trypsin, and dithiothreitol (DTT) on flour pasting properties were here investigated at five set concentrations. There were notable decreases in the peak, the trough viscosity, and the integral area of the viscosity curve as the concentrations of papain, trypsin, and DTT increased. DDT and pepsin concentration did not have any effect on final viscosity. Both breakdown and setback showed significant downtrends as the concentration of papain increased but no change as DDT concentration varied. Low concentrations of papain and pepsin prolonged peak time slightly, but higher concentrations shortened it significantly. For trypsin and DTT, low concentrations increased peak time notably, but high concentrations decreased it significantly. Pasting time and temperature increased in the presence of papain, pepsin, and trypsin, more so at higher concentrations. These results supply a basis for the utilization of hydrolyzed protein in the food industry and for the further studies on the interactions between protein and starch during processing.

Detection and localization of a cytoplasmic domain on the beta-subunit of Na+,K+-ATPase. A monoclonal antibody study.

A monoclonal antibody directed against the beta-subunit of dog kidney Na+,K+-ATPase was generated. Immunoblots demonstrate that monoclonal antibody III 18A binds exclusively to the denaturated beta-subunit. Binding experiments with membranes and whole cells reveal that III 18A binds to membranes but not to whole cells, indicating that the antibody binds to a cytoplasmic domain on the native beta-subunit. To localize the antibody-binding epitope, purified membrane-bound enzyme was fragmented by protease treatment. Tryptic digestion yields a 30-kDa fragment of the beta-subunit, which still retains the binding capacity for the antibody. Thus III 18A probably does not bind to the NH2-terminal segment of the protein. On the other hand, fragmentation of the beta-subunit with low concentrations of papain, which is known to yield a 40-kDa NH2-terminal and a 16-kDa COOH-terminal fragment, results in a complete loss of III 18A binding. These results suggest that the antibody-binding epitope is localized at or near a papain cleavage site on the COOH-terminal part of the beta-subunit. This is inconsistent with a structure model of the beta-subunit containing only a single transmembrane hydrophobic segment with a cytoplasmic NH2-terminal portion, but agrees quite well with a hypothetical structure with four intramembrane segments.

Proteolytic substructure of brain myosin.

Individual bovine brain myosin molecules visualized by electron microscopy consist of two globular heads and a fibrous tail, like myosin molecules from other sources. Brain myosin, however, showed much lower solubility at moderate to high ionic strength (0.2 to 0.4 M KCl) than gizzard myosin, and the filaments formed at low ionic strength in the presence of Mg2+ were fairly resistant to low concentrations of ATP, by which gizzard myosin filaments were completely solubilized. Brain myosin was digested with low concentrations of papain, alpha-chymotrypsin, or trypsin, and the fragmentation patterns were analyzed by means of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, sedimentation at low ionic strength, and electron microscopy of the fragments produced. The results indicate that all of the proteases cleave the myosin molecule primarily at sites located in the neck or in the head close to the neck, suggesting that the brain myosin molecule contains a hinge region or an open peptide stretch around these sites. The differences as well as the similarities between the proteolytic fragmentation patterns of brain myosin and other myosins are discussed.

Effect of proteolytic digestion on the Ca2+-ATPase activity and subunits of latent and thiol-activated chloroplast coupling factor 1.

The activation by proteases of the Ca2+-dependent ATPase of chloroplast coupling factor 1 (CF1) has been investigated. Using low concentrations of papain and trypsin, the increase in ATPase activity and the degradation of the five subunits of CF1 were compared. Sodium dodecyl sulfate-gel electrophoresis of protease-treated CF1 revealed that the delta subunit was very rapidly degraded and that the alpha and beta subunits were clipped. The gamma and epsilon subunits were more resistant to digestion. The modification of the alpha subunit of latent CF1 most closely correlated with the activation of Ca2+-ATPase activity. Trypsin treatment of dithiothreitol-activated CF1 resulted in a very rapid increase in Ca2+-ATPase activity and a corresponding rapid cleavage of the gamma subunit to a 25,000-dalton species. With more prolonged treatment, the 25,000-dalton species was cleaved to fragments of 14,000 and 11,000-daltons. Dithiothreitol treatment did not alter the rate of attack on the other subunits. The gamma subunit of heat-activated CF1 was also more susceptible to protease digestion. The increased protease sensitivity of the gamma subunit of soluble CF1 after treatment with dithiothreitol or heat mimics the increased protease sensitivity of the gamma subunit of bound CF1 when thylakoids are treated with trypsin during illumination (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5915-5920). These results suggest that the conformational changes that occur when purified CF1 is exposed to dithiothreitol are similar to those that CF1 bound to thylakoid membranes undergoes under illumination.

Studies on the Induction and Expression of T Cell-Mediated Immunity. III. Amplification by Papain of the Generation of Cytotoxic Lymphocytes to Allogeneic Cells

Abstract We have demonstrated in a recent study (1) that mild proteolytic treatment of immune cytotoxic lymphocytes (CL)2 leads to a 3- to 5-fold increase in the in vitro cytolytic activity upon target cells bearing the sensitizing antigens. In the present report we describe the potentiation of the generation of CL after treatment of normal lymphoid cells with low concentrations of papain. Thus, BALB/c (H-2d) splenocytes treated with papain and subsequently sensitized in vitro or in vivo against allogeneic C57BL/6 (H-2b) lymphoid cells exhibit an enhanced specific cytotoxic capacity as compared with untreated cells. Furthermore, the induction of CL with papain-treated cells occurs under conditions in which non-treated cells show little or no reactivity. Materials and Methods. The preparation of spleen cells and enzymatic treatment with papain were described previously (1).

Substructure of the myosin molecule: I. Subfragments of myosin by enzymic degradation

The formation of light meromyosin and heavy meromyosin by trypsin first demonstrated that myosin had regions of high α-helix content which could be separated from the ATPase and actin-binding centers of the molecule. Here we report the action of papain on myosin. Under suitable experimental conditions, low concentrations of papain can split the enzymic globular regions of myosin from the structural α-helical rod, leaving the latter intact along its length. Further digestion cleaves the rod along two-thirds of its length to form light meromyosin and the rod portion of heavy meromyosin. The size and shape of these proteolytic fragments as deduced from hydrodynamic measurements are compared to the structure seen in electron micrographs of shadow-cast preparations. From these studies we conclude that the myosin molecule consists of two globular units, each about 70 Å in diameter, attached to a double-stranded α-helical rod about 1350 Å long. The effect of Ca- and Mg-actin on the ATPase activity of the cleaved isolated globules is similar to the effect of these cations and actin on the globules in the native or attached state existing in heavy meromyosin. The rod portion of myosin is insoluble at low ionic strength and is therefore responsible for filament formation under physiological conditions. However, only the light meromyosin end of the rod is insoluble in water at neutral pH; the remainder of the rod is soluble at all ionic strengths. There is no evidence for an extensive amorphous region connecting these two subfragments. The intact rod has an α-helical content consistent with a fully helical molecule, and no proline residues could be detected by amino acid analysis.

Studies on the organization of the brush border in intestinal epithelial cells: V. Subfractionation of enzymatic activities of the microvillus membrane

Isolated brush border membranes of the intestinal epithelial cell have been partially digested by treatment with low concentrations of papain. A multienzyme particle containing maltase, sucrase and isomaltase activities is released. Further treatment with papain releases leucyl naphthylamidase activity in particulate form. Trehalase activity remains with the residue of the membrane. The disaccharidase-containing particles can be separated from those containing leucyl naphthylamidase on Sephadex G-200 columns. Alkaline phosphate activity is rapidly destroyed by papain treatment. Leucyl glycine-splitting activity can be separated from leucyl naphthylamide-splitting activity by papain treatment. Thus there is now evidence for two brush-border enzymes involved in the digestion of peptides. The association of membrane enzymes with particles containing multiple activity is in accord with the view of a mosaic substructure of the membrane.

Studies on cellular inhibitors of ribonuclease II. Some properties of the inhibitor from rat liver

Both the rat-liver RNAase inhibitor and heparin inhibited bovine pancreatic RNAase (at pH 7.8 and 5.8), but did not affect the activity of RNAase T 1, an alkaline enzyme of fungal origin, or of several acid RNAases derived from plant tissue. These inhibitors differed in their activity towards the enzymes from rat liver. The acid RNAase was inhibited by heparin, but not by the rat-liver inhibitor. The two alkaline RNAase preparations were inhibited by the rat-liver inhibitor, but only slightly affected by heparin. The purified rat-liver inhibitor was inactivated by low concentrations of papain (of several enzymes tested), periodate, sulfhydryl reactants and protamine, and by high salt concentrations. Inactivation by PCMB and Pb 2+ was partially reversible. Inhibition of RNAase activity by heparin was more sensitive to protamine and to salt concentration than was inhibition by the rat-liver inhibitor. These results indicate the protein nature of the rat-liver inhibitor, but suggest carbohydrate may also be an essential component. The rat-liver inhibitor may be classed as a polyanionic inhibitor, but one which forms a comparatively strong complex with RNAase.

Studies on cellular inhibitors of ribonuclease II. Some properties of the inhibitor from rat liver

Both the rat-liver RNAase inhibitor and heparin inhibited bovine pancreatic RNAase (at pH 7.8 and 5.8), but did not affect the activity of RNAase T 1 , an alkaline enzyme of fungal origin, or of several acid RNAases derived from plant tissue. These inhibitors differed in their activity towards the enzymes from rat liver. The acid RNAase was inhibited by heparin, but not by the rat-liver inhibitor. The two alkaline RNAase preparations were inhibited by the rat-liver inhibitor, but only slightly affected by heparin. The purified rat-liver inhibitor was inactivated by low concentrations of papain (of several enzymes tested), periodate, sulfhydryl reactants and protamine, and by high salt concentrations. Inactivation by PCMB and Pb 2+ was partially reversible. Inhibition of RNAase activity by heparin was more sensitive to protamine and to salt concentration than was inhibition by the rat-liver inhibitor. These results indicate the protein nature of the rat-liver inhibitor, but suggest carbohydrate may also be an essential component. The rat-liver inhibitor may be classed as a polyanionic inhibitor, but one which forms a comparatively strong complex with RNAase.