Concentrations Of Pronase Research Articles

Immunohistochemical localization of glandular kallikrein in the endocrine and exocrine human pancreas.

Fab fragments from two new monospecific anti-human tissue kallikrein sera were examined for their capacity to inhibit the functional activities of purified human urinary kallikrein and purified human pancreatic kallikrein. Fragments from a new anti-urinary kallikrein serum and from an anti-pancreatic kallikrein serum yielded mixed inhibition of kinin-generating activity and minimal inhibition of esterolytic activity. In contrast to the previously described "active site directed" anti-urinary kallikrein, these new antisera demonstrated little specificity for epitopes near the enzymatic site of urinary or pancreatic kallikrein. When used to localize kallikrein antigen in human pancreas obtained at surgery, IgG fractions of the new anti-kallikrein sera yielded moderate acinar and ductal staining in the absence of pretreatment of the tissue with trypsin or pronase. Short incubation with 0.125 mg/ml of either enzyme permitted the discrete localization of islet beta cell kallikrein antigen, while increased pronase concentrations decreased kallikrein antigen in both islets and exocrine tissue and led to islet destruction. Both antibody specificity and tissue preparation influence kallikrein localization in human pancreas.

Binding of beta-bungarotoxin to synaptic membrane fractions of chick brain.

beta-Bungarotoxin (beta-BuTx) is a presynaptically active snake venom phospholipase A2 which, in the chick, is cytotoxic for certain subclasses of central and peripheral neurons. In order to elucidate whether the toxin's specificity is due to the existence of specific neuronal binding sites, the binding of 125I-labeled beta-BuTx to synaptic membrane fractions of chick brain was investigated. These experiments defined a single class of saturable high affinity binding sites (KD = 0.47 +/- 0.14 nM, k+1 = 4.3 x 10(6) M-1 s-1, k-1 = 1.08 x 10(-4) s-1) whose pharmacological properties correlated with that of the cytotoxic action of beta-BuTx on cholinergic neurons in chick retina cultures. The density of 125I-beta-BuTx binding sites in synaptic membrane fractions was low (50 fmol/mg of protein). Also specific toxin binding occurred only to membrane fractions of chick organs known to contain beta-BuTx-sensitive cells or nerve endings, i.e. brain, retina, and muscle, but not liver and heart. The binding of 125I-beta-BuTx to synaptic membrane fractions was dependent on Ca2+; Co2+ and Sr2+, but not Mg2+, could replace Ca2+ in the binding reaction. The membrane binding site for 125I-beta-BuTx was sensitive to heat and high concentrations of pronase, and thus most likely is a protein.

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ( I 50 = 6·10 −7 M ). The concentration dependence of the binding to ghosts reveals two saturable components. [ 14C]Flufenamate binds with high affinity ( K d 1 = 1.2·10 −7 M ) to 8.5·10 5 sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ( K d 2 = 10 −4 M ) to a second set of sites (4.6·10 7 per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [ 14C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [ 14C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [ 14C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [ 14C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.

Differences in the distribution of specific glycoproteins among the regions of a single identified neuron

Neurons are highly differentiated cells whose various regions must differ in macromolecular composition. This is demonstrated in the present study which shows that specific membrane glycoproteins are routed to particular sites in the cell. When [ 3H]fucose or [ 3H]N-acetylgalactosamine are injected into R2, the giant neuron of Aplysia, they are incorporated into relatively few glycoproteins, several of which can be readily identified from cell to cell. Because R2's cell body is so large, its surface membrane can be isolated 94% free of cytosol by manual dissection. The purity of the membrane was assessed by checking the distribution of the enzyme choline acetyltransferase. R2's axon can also be analyzed separately from the cell body. At 24 h after injection, SDS polyacrylamide gel electrophoresis shows that glycoprotein-I (mol. wt. 180,000) is the major labeled component of the external membrane where it is enriched relative to the content of the other cytosolic membranes. In contrast, glycoprotein-V (mol. wt. 90,000) predominates among the membranes of the axon. The disposition of glycoprotein-I in the external membrane was indicated by exposing R2 to low concentrations of pronase in situ 24 h after injection. Labeled glycopeptides were released from R2's surface and gel filtration and high voltage electrophoresis indicated that some of these were derived from glycoprotein-I. Examination of the isolated surface membrane after proteolysis showed a reduced amount of labeled glycoprotein-I. Consistent with these findings, a glycoprotein of similar molecular weight as component-I was labeled when R2 was treated with galactose oxidase and potassium borotritide. These results indicated that the carbohydrate moieties of glycoprotein-I extend from R2's surface.

The three cortical membranes of the gregarines (parasitic protozoa). Characterization of the membrane proteins of Gregarina blaberae

GREGARINES, WHICH ARE PARASITIC PROTOZOA LIVING IN INVERTEBRATES, POSSESS A CORTICAL STRUCTURE SPECIFIC TO THEIR VEGETATIVE STAGE: namely two additional cytomembranes are lying just under the plasma membrane. This cortical complex has been isolated by centrifugation on discontinuous sucrose gradients and characterized chemically. Its integrity was tested by electron microscopy. Ghost proteins were resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. About 30 polypeptides of mol.wt. 15000-300000 were present in this fraction and four glycoproteins were detected after periodate/Schiff staining. Ten major proteins were labelled after lactoperoxidase-catalysed iodination. The GP(2) glycoprotein (41000-49000 apparent mol.wt.) appears to be a major component of the cell surface. Effects of trypsin and Pronase digestion on ghosts and cells were monitored by gel electrophoresis and by electron microscopy. Ghosts treated with low trypsin or Pronase concentrations (10-25mug/ml) became drastically disorganized; many proteins were vigorously attacked in comparison with those of control ghosts. Variations in proteinase-sensitivity of proteins are pointed out. The GP(3) glycoprotein (130000-160000 apparent mol.wt.) seemed to be the only glycoprotein released from the cell surface by trypsin. Whole cells treated under the same conditions or with higher proteinase concentrations (up to 1mg/ml) do not exhibit morphological modifications of the cell surface; furthermore, no discernible cleavage of membrane proteins was indicated by electrophoretograms. It is postulated that cell-surface proteins are protected by the dense carbohydrate cell coat. By using various different methods (change of ionic strength, detergent, denaturing agent, labelling experiment) it was possible to localize several major proteins within the protozoon cortical membranes.

Rosette formation with protein A-coated erythrocytes: A method for detecting both IgG-bearing cells and another subset of human peripheral blood B lymphocytes

Human peripheral blood lymphocytes were tested for ability to form rosettes with human red blood cells (HRBC) coated with staphylococcal protein A (SpA-HRBC). Purified T lymphocytes did not form rosettes, but in T-cell-depleted suspensions the number of cells forming rosettes with SpA-HRBC was significantly greater than in unfractionated suspensions. When non-T-cell suspensions were depleted of Ig-bearing lymphocytes, cells forming rosettes with SpA-HRBC were no longer detectable. The number of cells forming rosettes with SpA-HRBC in either unfractionated or T-cell-depleted suspensions was significantly higher than the number forming rosettes with HRBC coated with anti-human γ chain immunosorbent-purified rabbit antibodies. Membrane components reacting with SpA-HRBC were not passively adsorbed on the cell surface and could be actively synthesized by lymphocytes. The SpA-reacting membrane components present on some B lymphocytes were sensitive to treatment with low concentrations of pronase, but other B cells maintained ability to form rosettes even after treatment with higher pronase concentrations. Incubation of T-cell-depleted lymphocyte suspensions with anti-human γ, and also with anti-human μ or anti-human δ chain F(ab′) 2 fragments induced significant reduction in the number of cells forming rosettes. After incubation of the same cell suspensions with a mixture of anti-γ, anti-μ and anti-δ (F(ab′) 2 fragments, virtually all the lymphocytes lost the ability to form rosettes with SpA-HRBC. These findings suggest that rosette formation with SpA-HRBC may be used as a method for detecting IgG-bearing cells but also to detect a subset of IgM- and/or IgD-bearing human B lymphocytes.

Urea treatment and pronase digestion of antitumor protein antibiotics, auromomycin and neocarzinostatin.

Low molecular weight substances were separated from antitumor protein antibiotics, auromomycin and neocarzinostatin, by Sephadex G50 column chromatography, after denaturation with 8 M urea. The low molecular weight fraction of auromomycin, but not the protein fraction, showed antimicrobial and DNA-cleaving activities. More than 90% of the auromomycin and neocarzinostatin proteins were digested with a high concentration of pronase E. The digested samples of both antibiotics exhibited the same degree of activities as the original drugs in the inhibition of growth and DNA synthesis of mouse lymphoblastoma L5178Y cells and in causing strand scission of isolated PM2 phage DNA. The low molecular weight chromophores were recovered on Sephadex G50 column from the pronase-digested antibiotics. The results suggest that the in vitro biological activity of auromomycin and neocarzinostatin are principally attributed to the non-protein compounds of low molecular weight.

Cell Differentiation in Dictyostelium discoideum

We have shown previously that amoebae of D. discoideum strain V12 M2 starved at low density in the presence of cyclic AMP fail to form either stalk cells or prespore cells; a low molecular weight factor released by cells at high density promotes stalk formation under these conditions but formation of prespore cells requires ‘cell contact’. Here we summarise evidence that: 1. Elevated intracellular cyclic AMP levels are required for all developmental gene expression beyond the preaggregative phase, and ammonia antagonises this expression in some way. However, the action of ammonia is not pathway specific. 2. ‘Cell contact’ is a specific requirement for entry into the prespore pathway of gene expression since isolated cells provided with cyclic AMP synthesise much reduced amounts of the presporespecific enzyme uridine diphosphate (UDP) galactose polysaccharide transferase but normal amounts of the pathway-indifferent enzyme glycogen phosphorylase. 3. The ‘cell contact’ mechanism is uniquely sensitive to low concentrations of pronase. This protease selectively inhibits transferase synthesis and blocks in vitro spore differentiation (in a spore-forming mutant). It does not prevent chemotactic aggregation, stream formation, or stalk cell formation in the presence of cyclic AMP.

Structural polypeptides of the murine coronavirus JHM.

Analysis by SDS-polyacrylamide gel electrophoresis shows that the purified coronavirus JHM contains six polypeptides. The apparent mol. wt. of the polypeptides (GP1, GP2, GP3, VP4, GP5 and VP6) are 170000; 125000; 97500; 60800; 24800 and 22700, respectively. Four polypeptides are glycosylated (GP1, GP2, GP3 and GP5). The analysis of particles obtained after limited proteolysis with pronase suggests that GP2 and GP3 are protruding from the lipid envelope and, together with GP1, form the spike layer. Protein VP6 and a part of GP5 are located within the lipid bilayer. Protein VP4 is susceptible to digestion at a concentration of pronase which changes the morphology of the virus particles making the interior of the virus accessible. Subviral particles produced after treatment with the detergent Nonidet P40 banded at a higher density than the virus and contained only VP4, GP5 and VP6.

Fc-Receptors for IgG and IgM Immunoglobulins on Human T Lymphocytes: Mode of Re-Expression after Proteolysis or Interaction with Immune Complexes

The susceptibility to proteolysis and the mode of re-expression of receptors for IgM or IgG present on two different subpopulations of human T lymphocytes (T.M and T.G cells, respectively) have been investigated. The IgM receptor was highly susceptible to both trypsin and pronase, whereas the IgG receptor was resistant to trypsin and sensitive only to high concentrations of pronase. The receptors have been removed by treating purified human T cells with pronase and their reappearance on the cell surface has been followed in vitro. The IgM receptors on the cell surface were detectable within 2 hr and the resynthesis was completed in 6 hr. IgG receptors were detectable in 4 to 6 hr and the resynthesis completed within 12 hr. When protein synthesis was inhibited by culturing the cells in the presence of cycloheximide for up to 12 hr, only the IgM receptor (which had a higher turnover rate) failed to be expressed. Whereas interaction of IgG immune complex with the IgG receptors was previously shown to induce a modulation of the receptors, contact with antigen-IgM antibody complexes did not alter the mode of expression of IgM receptors.

Effect of hydrolytic enzymes and protein-modifying reagents on gonadotropin receptors in bovine corpus luteum cell membranes

Preincubation of membranes with various concentrations of pronase, trypsin, lipase, phospholipase A from Vipera russelli and from Crotalus durissus terrificus, phospholipase C from Bacillus cereus and from Clostridium welchii, acetic anhydride, 2,4-dinitrofluorobenzene and tetranitromethane resulted in a dose-dependent inhibition of 125I-labeled human choriogonadotropin binding. At the submaximal concentrations of enzymes and at both submaximal and maximal concentrations of protein-modifying reagents, the losses were always greater with 125I-labeled human choriogonadotropin than with 125I-labeled human lutropin. The inhibition of binding was a consequence of changes in the membranes rather than changes in the hormone caused by the agents being carried over to the final incubation. Inhibition of binding was non-competitive and irreversible. In untreated membranes, the 125I-labeled human choriogonadotropin binding was homogenous ( K d = 1.7 · 10 −10 M; N = 60 fmol/mg protein). Treatment of membranes with various enzymes and protein-modifying reagents except tetranitromethane resulted in heterogeneous binding. The number of available high affinity receptors was greatly reduced in every case. However, the affinity of these sites were either unchangedd (trypsin, lipase, phospholipase A from V. russelli, dinitrofluorobenzene and the tetranitromethane) or decreased (pronase and acetic anhydride). the newly appeared second receptor site had a K d which varied from 3.2 · 10 −10 to 7.1 · 10 −9d M depending on the agent used, and the receptor numbers were low in all cases except acetic anhydride. Receptor occupancy conferred the receptors with marked protection against various hydrolytic enzymes, dinitrofluorobenzene and tetranitromethane. These data suggest the inhibition of binding by the above agents was primarily a consequence of changes in the receptor molecules themselves.

Lectin-mediated agglutination of erythrocyte ghost membranes following depletion of membrane protein and intramembrane particles

Profound digestion of unsealed human erythrocyte ghosts with high concentrations of Pronase results in a near complete loss of intramembrane particles while trypsin digestion is less effective. The small vesicles formed by proteolysis are agglutinable by soybean agglutinin (SBA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA), but not concanavalin A (ConA). Densitometer tracings of Pronase-treated vesicles analyzed on SDS-polyacrylamide gels demonstrated no detectable protein or glycoprotein migrating slower than the marking dye. The vesicles showed a loss of 90% Lowry positive material (the remainder may be non-protein chromogens), near depletion of sialyl residues, no significant change in lipid composition, and equal amounts of phospholipid phosphorus compared to an equal volume of ghosts. The lipid material extracted from Pronase-derived vesicles or intact ghosts inhibited hemagglutination with SBA and WGA but not ConA. SBA but not ConA was found to specifically bind to Pronase-derived vesicles while both lectins bound to native ghosts. These observations suggest that neither the integrity of the intramembrane particles nor the presence of membrane glycoprotein appears essential for SBA-, WGA-, and PHA-mediated agglutination. Furthermore, it appears that native membrane glycolipids (and perhaps glycopeptides) can bind SBA, WGA and PHA. The membrane glycolipids may play a larger role than heretofore realized in lectin-mediated agglutination of cells.

Proteolytic dechorionation of annual fish embryos.

Incubation of early embryos of Nothobranchius korthausae in sterile Tris-buffered pronase with added salts and EDTA results in complete dechorionation, after which development proceeds normally and at the same rate as in control embryos. The time required for dechorionation varies according to the age of the embryo and the concentration of pronase employed.

Membrane Ig on human lymphocytes: rate of turnover of IgD and IgM on the surface of human tonsil cells.

The turnover of IgM and IgD molecules present on the membrane of human tonsil cells has been studied using immunofluorescence and peroxidase-catalyzed membrane radioiodination. With the first of the two techniques cells were treated with pronase to remove membrane immunoglobulin (mIg), placed in culture and stained at intervals to check the reappearance of membrane IgD and IgM on the cell membrane. These experiments showed that membrane IgD (in contrast to membrane IgM) are extremely susceptible to proteolysis. Furthermore, cells treated with a concentration of pronase found to be optimal to remove membrane IgM failed to re-express membrane IgD in vitro. The large majority of tonsil lymphocytes has both membrane IgM and IgD. Due to the different behavior of reappearance of the two membrane molecules after treatment with pronase, it was not possible to obtain the simultaneous re-expression of membrane IgM and IgD by the cells "stripped" with pronase. However, the two molecules were re-expressed in vitro by the cells treated with different pronase concentrations with a similar timing, i.e. 50% or more of the cells re-expressed membrane IgD and IgM after 8 h in culture. 131I-radioiodinated membrane IgD and IgM were also released from the cell surface with a similar timing, the half-life of permanence on the cell membrane being about 4 h for both molecules. These findings thus indicate that IgM and IgD molecules have a similar turnover and that a cell is capable of placing two different Ig molecules at a time on its surface.

Release of growth hormone from ox pituitary slices after pronase treatment.

Proteolytic enzymes have been used both to modify properties of the cell membrane and to dissociate cells from many tissues including pituitary (4, 5, 12). Exposure of secretory tissues to pronase can alter their secretory response. Thus incubation of pancreatic islets of Langerhans in the presence of low concentrations of pronase increased the subsequent release of insulin in the presence of stimulatory and nonstimulatory glucose concentrations (7). The purpose of the present investigation was to determine whether low concentrations of pronase have the same stimulatory effect on the release of a pituitary hormone, growth hormone. Such an effect on hormone release could be of some importance in view of the development of dissociated cell systems as models for the study of the control of hormone release (4, 5).

Maximal concanavalin A-specific agglutinability without loss of density-dependent growth control.

Density-inhibited 3T3 mouse fibroblasts were treated for 10 min with nine concentrations of Pronase ranging from 0.25 to 40 mug/ml. Concanavalin A-specific agglutinability increased from control values of about 25% to plateau values of about 80%. None of these Pronase concentrations brought about an increase in cell number within 84 hr. Pronase-treated cells remained responsive to growth stimulation by serum and cortisol. Therefore, the protease-mediated surface change measured by increased concanavalin A-specific agglutinability is not an event sufficient by itself to bring about cell division.

Preformed messenger of quiescent wheat embryos

A subcellular fraction (messenger fraction) which supports in vitro amino acid incorporation, has been isolated from dry wheat embryos. Analyses of the characteristics of messenger fraction directed amino acid incorporation (kinetics, nucleotide requirements, K + and Mg 2+ optima, and response to inhibitors) indicate that a ribosome-messenger attachment reaction is a prerequisite for messenger fraction activity. Direct demonstration of this reaction is provided by experiments in which messenger fraction is allowed to react in vitro with messenger-free ribosomes. Sucrose density gradient analysis of the reaction shows a progressive conversion of monoribosomes to functional polyribosomes. Upon storage at o° the activity of messenger fraction decays with a half-life of 12–13 h. Loss of activity is not due to ribonuclease as shown by complete stability of tobacco mosaic virus (TMV) RNA stored in the presence of messenger fraction. These data as well as experiments demonstrating the sensitivity of messenger fraction to low concentrations of pronase and N-ethyl maleimide suggest that a component other than RNA is necessary for messenger fraction activity and that the functional form of messenger fraction may be a ribonucleoprotein. The low level of template activity of purified messenger fraction RNA is consistent with this conclusion. In vivo studies show a rapid decrease in messenger fraction concomitant with polyribosome formation during early germination. These data suggest an important physiological role for messenger fraction in the initiation of protein synthesis which occurs at the outset of germination.

Partial purification and some properties of the factor in normal and leukaemic human urine stimulating mouse bone marrow colony growth in vitro.

SummaryThe properties of the factor in human urine which stimulates mouse bone marrow colony growth in vitro were examined. The colony stimulating activity of urine was not lost on concentration of urine, exposure to RNA‐ase, DNA‐ase, ether, 8M urea, pH in the range 2–12, or on fractionation by precipitation with ethanol or ammonium sulphate. Activity was lost on heating (90°/30′), exposure to periodate and incubation with high concentrations of Pronase. There was no difference between the behaviour of the colony stimulating factor in normal and leukaemic human urine concentrates on electrophoresis and on gel‐filtration. The colony stimulating factor in urine concentrates moved in the post‐albumin‐albumin region on electrophoresis at pH 8.6 had an apparent molecular weight of approximately 190,000 on gel‐filtration and an apparent sedimentation coefficient (S20, w) of approximately 3.3S (zone sedimentation on sucrose gradients). A procedure was described for the extraction and partial purification of the colony stimulating factor from large quantities of human urine.The data suggest that the colony stimulating factor may be a glycoprotein and that it could belong to a family of tissue‐specific humoral growth regulating macromolecules.

Characterization of the plasma membrane of Mycoplasma laidlawii: V. Effects of selective removal of protein and lipid

Purified plasma membranes of Mycoplasma laidlawii were subjected to proteolytic enzyme digestion and aqueous acetone extraction. Up to 80% membrane protein could be removed by pronase, with a concomitant decrease in density of residual banding material. This membrane residue was still organized in vesicles and showed a decreased thickness and contrast in electron micrographs. Despite degradation of all characteristic membrane proteins (as shown by detergent—polyacrylamide gel electrophoresis) some protein fragments were retained in vesicular residues. The amino acid composition of this material was indistinguishable from that of total membrane protein, arguing against any enrichment in hydrophobic protein regions. Membrane hexosamine was not solubilized by pronase or aqueous acetone extraction, and could be substantially purified by combining these techniques. Extraction with 90% aqueous acetone removed over 95% of labeled membrane lipid but left sedimentable, protein-rich membranous material with unit membrane morphology. Such lipid-depleted membranes banded in sucrose gradients at a density of 1.25 g/cm 3 as a single narrow band, in contrast to the isopycnic density of 1.17–1.18 g/cm 3 for native membranes. At low pronase concentrations, electrophoresis experiments suggested preferential pronase attack on some higher molecular weight membrane proteins; these same electrophoretic components were preferentially released at very low concentrations of sodium dodecyl sulfate,, indicating differences in the organization of proteins within the membrane. Analysis of membranes labeled with [ 14C]glucosamine or [ 14C]oleate by detergent-gel electrophoresis followed by gel fractionation and counting showed that all oleate-containing lipid moved as a single broad band, while glucosamine migrated as two bands, one extremely broad and heterogeneous.