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PubMed1979

Viral and non-viral induced fusion of pronase-digested human erythrocyte ghosts.

Human erythrocyte ghosts but was able to fuse only iso-human erythrocyte ghosts. Iso- and hypo-human erythrocyte ghosts were incubated with the proteolytic enzyme pronase under isotonic (iso-human erythrocyte ghosts) or hypotonic (hypo-human erythrocyte ghosts) conditions. Gel electrophoresis and electron microscope (freeze-etching) studies revealed that most of the erythrocyte membrane polypeptides were hydrolyzed by pronase under hypotonic conditions. Sendai virus readily agglutinated both pronase-digested iso-human erythrocyte ghosts and hypo-human erythrocyte ghosts were fused by the non-viral fusogenic agent glyceromonooleate. Freeze-etching studies revealed that during fusion the membranes of pronase-digested human erythrocyte ghosts are intermixed.

Erythrocyte Membrane↗

PubMed1995

Alzheimer paired helical filaments, untreated and pronase digested, studied by vertical platinum-carbon replication and high resolution transmission electron microscopy.

Untreated paired helical filaments (PHF) and pronase treated PHF filaments have been stereoscopically imaged with a freeze-drying vertical platinum-carbon replication preparation method for TEM. The untreated PHF have an average wide region, W = 22.8 +/- 2.4 nm, a narrow region width, T = 10.6 +/- 1.7 nm, and a helical turn period, L = 78.6 +/- 13.4. The widths of the pronase treated PHF were significantly reduced and had average measurements of W = 14.8 +/- 1.2 nm, T = 5.7 +/- 1.0 nm, with the helical period unchanged, L = 75.4 +/- 17 nm. The surfaces of the untreated PHF contained approximately 1.0 and approximately 0.4 nm strands, the size of normal and denatured tau monomer. The pronase treated PHF contained approximately 1.0 and approximately 0.4 nm strands as well as approximately 2.0 nm strands. The stereoscopic images of the untreated and the pronase digested PHF do not support a double helical morphology for the PHF. The PHF appear to be long helical ribbons. The approximately 1.0 and approximately 2 nm substructure has been organized both parallel and orthogonal to the PHF-core axis for distances less than 80 nm. The most frequent structural appearance is of a disorganized PHF core. The surfaces of the untreated PHF also have a similar disorganized appearance.

Alzheimer Disease↗

PubMed1996

Probing the structure-function relationship of alpha-latrotoxin-formed channels with antibodies and pronase.

The major toxic component of black widow spider (Latrodectus mactans tredecimguttatus) venom, alpha-latrotoxin, is known to form ionic channels in different membranes. In order to probe the extramembrane domains of alpha-latrotoxin molecule, alpha-latrotoxin channels in planar lipid membrane were treated with antibodies to latrotoxin or with pronase added to different sides of the membrane. It was found that antibody addition to the same side as the toxin (cis) decreased channel conductance only at positive potentials across the membrane. In contrast, trans side addition of antibodies changed the channel conductance at both positive and negative potentials: at positive potential conductance first slightly increased then decreased by more then 50%; at negative potential it decreased much more quickly, to only about 20% of the initial value. No dependence on membrane potential was found for pronase treatment of incorporated channels. For both cis and trans application of pronase, channel selectivity for Ca2+, Mg2+, Ba2+ and K+, Na+, Li+ ions did not change significantly but Cd2+ block was decreased. Trans pronase treatment also resulted in some rectification of I/V curves and an increase in channel conductance. We interpret these findings as evidence that alpha-latrotoxin channel has protruding parts on both sides of the membrane and that its conformation in the membrane depends on membrane potential.

Animals↗

PubMed2004

Pronase treatment facilitates alloantibody flow cytometric and cytotoxic crossmatching in the presence of rituximab.

Rituximab (RIT), a murine/human chimeric monoclonal antibody directed against human CD20 is under investigation for its role in transplantation. RIT causes B-cell crossmatches to appear positive. Pronase, a proteolytic enzyme that targets F(c) receptors removes CD20 from B cells. After CD20 is removed, RIT should not bind, making it possible to detect class I or class II antibodies on treated B cells. In this study, we incubated RIT with normal human serum (NHS, negative control) or pooled sera from highly sensitized (>50% panel reactive antibody, HLA+) subjects awaiting renal transplantation (positive control) and then performed B-cell flow cytometric crossmatches using untreated or pronase treated B cells as targets. We observed that untreated B cells incubated with RIT-spiked NHS displayed a significant increase in surface fluorescence compared with NHS without RIT, similar to the fluorescence that occurs with a positive crossmatch. In contrast, when CD20 was cleaved from the B cells with pronase, B cells displayed a negative crossmatch with the RIT-spiked NHS. In addition, there was no change in the crossmatches of pooled high panel reactive antibody (PRA) sera after pronase treatment. RIT could be used without worry about losing the ability to perform transplant immunologic monitoring.

Antibodies, Monoclonal↗

PubMed1978

Local anesthetic block of sodium channels in normal and pronase-treated squid giant axons.

The inhibition of sodium currents by local anesthetics and other blocking compounds was studied in perfused, voltage-clamped segments of squid giant axon. When applied internally, each of the eight compounds studied results in accumulating "use-depnedent" block of sodium currents upon repetitive pulsing. Recovery from block occurs over a time scale of many seconds. In axons treated with pronase to completely eliminate sodium inactivation, six of the compounds induce a time- and voltage-dependent decline of sodium currents after activation during a maintained depolarization. Four of the time-dependent blocking compounds--procaine, 9-aminoacridine, N-methylstrychnine, and QX572--also induce altered sodium tail currents by hindering closure of the activation gating mechanism. Treatment of the axon with pronase abolishes use-dependent block completely by QX222, QX314, 9-aminoacridine, and N-methylstrychnine, but only partially be tetracaine and etidocaine. Two pulse experiments reveal that recovery from block by 9-aminoacridine or N-methyl-strychnine is greatly accelerated after pronase treatment. Pronase treatment abolishes both use-dependent and voltage-dependent block by QX222 and QX314. These results provide support for a direct role of the inactivation gating mechanism in producing the long-lasting use-dependent inhibition brought about by local anesthetic compounds.

Acridines↗

PubMed2005

Characterization of chito-oligosaccharides prepared by chitosanolysis with the aid of papain and Pronase, and their bactericidal action against Bacillus cereus and Escherichia coli.

Papain (from papaya latex; EC 3.4.22.2) and Pronase (from Streptomyces griseus; EC 3.4.24.31) caused optimum depolymerization of chitosan at pH 3.5 and 37 degrees C, resulting in LMMC (low molecular mass chitosan) and chito-oligomeric-monomeric mixture. The yield of the latter was 14-16% and 14-19% respectively for papain- and Pronase-catalysed reactions, depending on the reaction time (1-5 h). HPLC revealed the presence of monomer(s) and oligomers of DP (degree of polymerization) 2-6, which was also confirmed by matrix-assisted laser-desorption ionization-time-of-flight MS. Along with the chito-oligomers, the appearance of only GlcNAc (N-acetylglucosamine) in Pronase-catalysed chitosanolysis was indicative of its different action pattern compared with papain. Fourier-transform infrared, liquid-state 13C-NMR spectra and CD analyses of chito-oligomeric-monomeric mixture indicated the release of GlcNAc/GlcNAc-rich oligomers. The monomeric sequence at the non-reducing ends of chito-oligomers was elucidated using N-acetylglucosaminidase. The chito-oligomeric-monomeric mixture showed better growth inhibitory activity towards Bacillus cereus and Escherichia coli compared with native chitosan. Optimum growth inhibition was observed with chito-oligomers of higher DP having low degree of acetylation. The latter caused pore formation and permeabilization of the cell wall of B. cereus, whereas blockage of nutrient flow due to the aggregation of chito-oligomers-monomers was responsible for the growth inhibition and lysis of E. coli, which were evidenced by scanning electron microscopy analysis. The spillage of cytoplasmic enzymes and native PAGE of the cell-free supernatant of B. cereus treated with chito-oligomeric-monomeric mixture further confirmed bactericidal activity of the latter. Use of papain and Pronase, which are inexpensive and easily available, for chitosanolysis, is of commercial importance, as the products released are of considerable biomedical value.

Anti-Bacterial Agents↗

PubMed1993

Limited proteolysis of (1,3)-beta-glucan (callose) synthase from Beta vulgaris L: topology of protease-sensitive sites and polypeptide identification using Pronase E.

Plasma membrane (PM) vesicles of defined sidedness were obtained from Beta vulgaris L. and subjected to limited proteolysis to investigate the topology and subunit composition of UDP-glucose: (1,3)-beta-glucan (callose) synthase (CalS). Latency experiments demonstrated that protease-sensitive sites on the CalS complex are located primarily at the cytoplasmic face of the PM, with little or no CalS inactivation occurring as the result of proteolysis at the apoplastic face. In the PM-bound form, CalS activity was resistant to inactivation by Pronase E, however at least four polypeptides previously implicated as possible CalS components (92, 83, 57 and 43 kDa) were extensively hydrolyzed. Polypeptides of 31, 29 and 27 kDa resisted Pronase E hydrolysis and were also enriched in CalS fractions purified by glycerol gradient centrifugation and product entrapment. In contrast to PM-bound CalS, purified CalS was rapidly hydrolyzed by Pronase E, indicating that most Pronase E-sensitive sites are deeply embedded within the PM. This study provides direct biochemical evidence that hydrophobic integral membrane proteins oriented primarily towards the cytoplasmic face of the PM are important for callose biosynthesis in Beta. Furthermore, these results form the basis of a biochemically derived working model largely consistent with morphologically derived models proposed for intramembrane PM-bound, microfibril-synthesizing complexes in higher plants.

Carbohydrate Sequence↗

PubMed1982

Variation in porcine zona pellucida morphology following pronase treatment.

The effects of cell stage and day of embryo collection on the pronase-induced zona pellucida morphology in pig embryos were investigated. Three hundred and seventy-two two- to eight-cell embryos were treated for 3.0 min in 5.0% pronase in Whitten's Medium. Responses in zona pellucida morphology observed after pronase treatment were: (1) absent, (2) stretched and (3) intact. Each pig was coded as to the type of zona pellucida morphology its embryos possessed after enzyme treatment according to the following scale: (1)--only intact, (2)--intact and stretched, (3)--only stretched, (4)--stretched and absent and (5)--only absent. Also, the number of embryos of a particular cell stage was expressed as a fraction of the total number of embryos collected from each pig. The incidence of intact zonae pellucidae was greatest (P less than .05) among two- to three-cell embryos and the incidence of absent zonae pellucidae was greatest (P less than .05) among six-to eight-cell embryos. A significant correlation (r = .79) was observed between day of embryo collection and coded zona pellucida morphology. The results suggest that the pronase-induced zona pellucida morphology observed is dependent on cell stage and day of embryo collection.

Animals↗

PubMed1981

Products of digestion of tryptic M and N blood group glycopeptides with pronase.

Digestion of tryptic M and N blood group glycopeptides with pronase generated a heterogeneous mixture of smaller glycopeptides. Two NH2-terminal glycopeptides, composed of 26--28 and 18--19 amino acid residues, and three fractions containing several glycopeptides deriving from the COOH-terminal portion of tryptic glycopeptides were obtained. The characterization of pronase glycopeptides showed that most of the peptide bonds hydrolyzed by pronase were susceptible to proteolysis only in part of molecules of tryptic glycopeptides, most probably due to a microheterogeneity in their glycosylation. The similar sets of pronase glycopeptides were obtained from tryptic M and N glycopeptides, indicating that the possible heterogeneity in their glycosylation is independent of the blood group type. Only the NH2-terminal glycopeptides showed M and N blood group activity, and only the NH2-terminal glycopeptides obtained from tryptic N glycopeptide reacted with Vicia graminea anti-N lectin.

Epitopes↗

PubMed1983

Inactivation of the scrapie agent by pronase.

Scrapie agent derived from infected mouse brain was partially purified by agarose-polyacrylamide gel electrophoresis. The preparation was subjected to treatment with RNase A, DNase I, pronase, sodium dodecyl sulfate and sodium dodecyl sulfate-pronase combination. Almost total inactivation of scrapie infectivity resulted when the partially purified scrapie agent was treated with sodium dodecyl sulfate-pronase combination whereas the treatment with sodium dodecyl sulfate or pronase alone reduced the infectivity by about 98%. No significant reduction of scrapie infectivity was observed with either RNase A or DNase I treatments. These results confirm that the transmission of scrapie cannot be achieved in the absence of a protein component.

Animals↗

PubMedDate not supplied

Some characteristics of the residue obtained after pronase treatment of sheep erythrocyte membranes. I. Protein and phospholipid patterns.

In this note some data regarding analysis of sheep erythrocyte stromata after pronase treatment are reported. The membrane material obtained after pronase treatment was analyzed by density gradient ultracentrifugation, gel filtration, acrylamide gel electrophoresis and for phospholipid content. With all techniques employed two major lipo or glycolipopeptides can be observed. The whole body of phospholipids is not lost after pronase treatment, therefore lipid-lipid interactions are maintained after pronase treatment, moreover sodium dodecyl sulphate is not efficient in the breakdown of some protein-lipid interactions. The results are discussed in view of the correspondence of the two fractions obtained with portions of the major glycoproteins of the erythrocyte membranes.

Animals↗

PubMedDate not supplied

Some characteristics of the residue obtained after pronase treatment of sheep erythrocyte membranes. II. Carbohydrate patterns.

The composition of single carbohydrate classes of intact and pronase treated sheep erythrocyte membranes has been studied. In comparison with the data obtained from untreated stromata after proteolytic digestion the amount of each class of sugars is decreased. A high disappearance of sialic acids and hexosamines can be observed. Nevertheless if the total sugar content is referred to the residual protein content (16% of the native proteins, an enrichment in carbohydrates, specially neutral hexoses, can be observed. The results indicate that most of the carbohydrate fractions solubilized by pronase treatment are sialic acids and hexosamines. Different molar ratios obtained for the single carbohydrate classes in comparison with NANA before and after pronase treatment suggest a microheterogeneity of the glycoprotein structure of sheep erythrocyte membranes. By the use of several analytical methods the residue obtained after pronase treatment shows two major fractions containing proteins, lipids and carbohydrates. The data are discussed in view of the possible arrangement of lipo-glycoproteins in sheep erythrocyte membrane.

Animals↗

PubMed1968

Hydrolysis of 125-I-labelled thyroglobulin by pancreatin, pronase and pepsin.

1. Pancreatin or Pronase hydrolysis of partially purified heat-denatured (125)I-labelled thyroglobulin does not release all the iodoamino acids from peptide linkage. 2. The addition of thyroidal proteases is essential to obtain maximum hydrolysis of thyroglobulin. 3. Pronase plus a fresh non-radioactive thyroid homogenate gives the best hydrolysis results, but peptide ;mapping' shows that about 10% of the radioactivity remains in the form of peptides. 4. These peptides have approximately the same R(F) as the iodotyrosines on one-dimensional chromatograms developed in butan-1-ol-2n-acetic acid (1:1, v/v). 5. Peptic hydrolysis of thyroglobulin releases virtually all of the iodothyronines but little of the iodotyrosines, in contrast with the action of pancreatin. 6. Hydrolysis of thyroglobulin with pepsin followed by Pronase plus a source of thyroidal proteases is satisfactory, but the results are not superior to those obtained by using Pronase plus a source of thyroidal proteases.

Amino Acids↗

PubMed1970

Antigenicity of proteinases from Streptomyces griseus (pronase).

The five pronase fractions, A(1), A(2), B, C (trypsin-like), and D (elastolytic), obtained by ion-exchange chromatography, were found to be antigenically distinct. Antibodies to pronase inhibited the enzymic activity of each of the enzyme fractions. Pronase trypsin and bovine trypsin, although resembling each other in enzymic activity and in amino acid sequence around their active sites, did not cross-react antigenically with, nor was their enzymic activity inhibited by, the respective homologous antibodies. Inactivation of pronase trypsin by complexing with soya-bean inhibitor AA, was not associated with a decrease in capacity to precipitate with its antibody. It is assumed that the antigenic sites are located far enough from the catalytic site of the enzyme to allow it to precipitate immunologically even when the catalytic site was blocked.

Amino Acid Sequence↗

PubMed1985

Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane : Studies of Photosystem II using Pronase Digestion and Chemical Labeling.

The accessibility of various Photosystem II (PSII)-associated polypeptides to the protease pronase and the chemical modifier trinitrobenzene-sulfonic acid (TNBS) has been investigated. Three polypeptides with apparent molecular weight of 32, 21, and 16 kilodaltons, known to be associated with O(2) evolution, are all resistant to pronase digestion and TNBS labeling in intact thylakoids. All the polypeptides in the isolated PSII preparation were labeled with TNBS while a different pattern of labeling was observed when the PSII complex was isolated from TNBS-modified thylakoids. Attempts to prepare PSII particles from pronase-treated thylakoids using the Triton X-100 solubilization method were unsuccessful. Pronase-treated thylakoids were probed with antisera against the chlorophyll proteins of PSII using immunoblotting techniques. This allowed for a positive identification of proteolytic fragments from the respective proteins. The results are discussed in relation to the transmembrane organization of PSII in spinach thylakoids.

Journal Article↗

PubMed1993

Deafferentation-induced terminal field expansion of myelinated saphenous afferents in the adult rat dorsal horn and the nucleus gracilis following pronase injection of the sciatic nerve.

We have previously demonstrated sprouting of small diameter saphenous afferents, labelled with wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) and (HRP), into the sciatic territory of the adult rat superficial dorsal horn following destruction of sciatic afferents by injection of the sciatic nerve with pronase (a combination of proteolytic enzymes). In the present experiments, we examined the response of myelinated saphenous axons, which terminate in lamina I and the deep dorsal horn (laminae III-V) under the same conditions, with the tracer B subunit of cholera toxin conjugated to HRP (B-HRP) which specifically labels myelinated primary afferents when injected into a peripheral somatic nerve. We also examined changes in the nucleus gracilis, another site of sciatic degeneration and a target of saphenous afferents. Four months after injection of the pronase, the area of label determined by measurement of the width of the saphenous territory in lamina III was expanded by 24% on the pronase side. Since there was also expansion throughout the deep dorsal horn, the area measured by tracing the labelled region in transverse sections was actually twice that of the control side, and the intensity of labelling within the traced area increased by 18%. There was no change in grey matter area due to the lesion. The traced area of labelling in the nucleus gracilis increased by 40%, and increased in intensity by 17%. The substantia gelatinosa is not normally supplied by B-HRP-labelled afferents, and there was no expansion of these sprouted saphenous afferents into the gelatinosa. These results indicate that myelinated afferents can sprout as vigorously in lamina I and the deep dorsal horn as the small diameter afferents do in the substantia gelatinosa; that there is no invasion of the substantia gelatinosa by the myelinated afferents at least as long as the small diameter afferents also have the opportunity to sprout; and that primary afferents have the potential to sprout at more than one site of termination, i.e., both the dorsal horn and the dorsal column nuclei.

Afferent Pathways↗

PubMed1989

Pronase treatment increases the staining intensity of GABA-immunoreactive structures in the paravertebral sympathetic ganglia.

A novel tissue preparation technique for improving gamma-aminobutyric acid (GABA) immunocytochemistry has been developed. The influence of the glutaraldehyde concentration in the fixative and the effect of pronase treatment on the GABA immunostaining were tested. This method includes fixation with a high concentration of glutaraldehyde, gelatin embedding and treatment of the sections with pronase. In sympathetic (paravertebral) ganglia and their connectives, the most intense and specific immunoreaction was obtained with the following procedure: immersion fixation in 5% glutaraldehyde, infiltration and embedding in 15% gelatin, secondary fixation of the samples with 4% formaldehyde, floating frozen sections and digestion with 0.1% pronase for 15-20 min. With this technique, the GABA-containing structures (cells and nerve fibers with varicosities forming basket-like networks around some principal neurons) were selectively labeled. The data presented suggest that (1) a high concentration (5%) of glutaraldehyde in the primary fixative is necessary to preserve a large proportion of the GABA content; (2) this glutaraldehyde fixation partly masks the GABA immunoreactivity; and (3) this masking may be overcome by a proteolytic treatment preceding the immunostaining. This method has been extensively tested for the light microscopic visualization of GABA-containing tissue components in the sympathetic ganglion chain, but it may probably also be used for the immunocytochemical detection of other small molecules in other parts of the nervous system.

Animals↗

PubMed1995

The use of pronase-digested human leukocytes to improve specificity of the flow cytometric crossmatch.

Two-color fluorescence cytometry (FCXM) has recently been introduced to improve the detection of anti-HLA antibodies that react to donor cells, especially in recipients receiving kidney allografts. Although this assay system is highly sensitive, it lacks specificity. Between 70% and 90% of potential kidney recipients with a positive FCXM would have been denied transplant if such an assay had been used alone to detect antidonor antibodies. Lack of specificity is principally due to normal or irrelevant IgG in aggregates or immune complexes binding to Fc gamma R receptors on lymphocytes including B cells and a significant subset of T cells. To circumvent this problem, we digested Fc gamma R receptors on lymphocytes with pronase. We present data demonstrating that pronase digestion of lymphocytes does not alter HLA antigenicity. In addition, pronased lymphocytes allow one to use either single- or two-color FCXM. With single-color FCXM, one can quantitate antibody reactivity to lymphocytes via a cursor (on the fluorescence histogram) that separates lymphocytes that do not bind to antibodies. We present data demonstrating that this modification renders FCXM highly sensitive and specific. In addition, one can discriminate between IgG and IgM antibodies that react to lymphocytes.

Flow Cytometry↗