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At least 73 records · Page 4Linked to original sources
PubMed1979

The use of pronase for electron microscopy of protein-complexed DNA.

A method is described which demonstrates the posibility of visualizing protein-complexed DNA by using pronase for both deproteinization and spreading of DNA for electron microscopy. The results show that proteolytic digestion is complete even under conditions of short formaldehyde fixation and pronase is an excellent substitute for cytochrome c for spreading of DNA. The pronase method is successfully applied to virions, native and partially denatured chromatin. The procedure is highly advantageous because it does not require preliminary isolation of DNA, can be applied to microamounts of material and permits the visualization of partial denaturation of DNA in chromatin.

Animals↗
PubMed2002

Improved specificity and sensitivity when using pronase-digested lymphocytes to perform flow-cytometric crossmatch prior to renal transplantation.

Several laboratories have resorted to flow-cytometric crossmatch (FCXM) in an effort to prevent hyperacute and accelerated renal allograft rejections. The currently employed FCXM has problems with both false-positive and -negative reactions, largely as a result of irrelevant IgG binding to Fc IgG receptors. In 1980, we circumvented this problem by digesting Fc IgG receptors with pronase, and demonstrated that, with immunofluorescence microscopy (IF), detection of IgG anti-HLA antibodies was highly sensitive and specific. In 1995, we introduced the pronase technique to FCXM and showed that this enzyme did not decrease HLA expression. We present herein a prospective study at our institution to determine whether FCXM using pronase-digested (PD) lymphocytes is as sensitive and more specific than FCXM with undigested (UD) lymphocytes when compared with the highly sensitive and specific IF assay. In analyzing the 186 donor-specific pre-renal-transplant crossmatches, we found that PD FCXM was as sensitive and specific as IF and was able to detect weak IgG anti-HLA antibodies that bound to B cells. Fourteen of these patients would have been denied transplants if one were to have relied on UD FCXM. The data clearly indicate that PD FCXM can reliably be used to detect weak IgG anti-HLA antibodies before renal transplantation.

Antibodies↗
PubMed1985

Pronase and proteinase K digestion of human immunoglobulin M.

Human 19S IgM was digested with pronase and proteinase K. Proteolysis was relatively fast, producing Fab2 mu-like fragments (approx. mol. wt 114,000) and Fab mu-like fragments (approx. mol. wt 46,500) as major products. Immunochemical analysis indicated that the fragments produced by either enzyme are very similar and that they are produced by cleavage at the C mu 2-C mu 3 and C mu 1-C mu 2 domain junctions respectively. An intermediate species of mol. wt 74,300, immunologically identical to F(ab)2 mu, was also identified. This is thought to represent an F(ab)2 mu fragment with one Fab mu fragment removed. Fc mu-related fragments were not identified in the digestion mixture with either enzyme. Covalently linked and non-covalently linked 7S human IgM (IgMs and IgMr respectively) were digested with pronase and proteinase K. IgMs was degraded very rapidly by either enzyme, producing relatively stable F(ab)2 mu- and Fab mu-like fragments. These fragments were similar in mol. wt and immunochemical properties to those produced from 19S IgM. IgMr was also degraded rapidly by either enzyme, in this case producing Fab mu-like fragments with no detectable F(ab)2 mu-like fragments. The kinetics of digestion and nature of the products suggest that cleavage of 19S IgM by pronase or proteinase K proceeds via an initial attack at the C mu 2-C mu 3 junction, followed by further degradation at the Cmu 1-C mu 2 junction. The results obtained using 7S IgM show that the intersubunit disulphide bonds, and the associated pentameric structure, are responsible for the relative resistance of 19S IgM to proteolysis. The inter-heavy-chain disulphide bonds, in particular the bond at cys 337, are responsible for the limited susceptibility of F(ab)2 mu-like fragments to proteolysis.

Chemical Phenomena↗
PubMed1988

Slow regeneration of immune complex binding sites on the macrophage surface after previous digestion with pronase.

Peroxidase-antiperoxidase (PAP) binding sites on the rat peritoneal macrophage surface were damaged by pronase digestion and the reappearance of functionally intact receptors was investigated morphologically and spectrophotometrically. Pronase digestion decreased the PAP binding ability of macrophages to about 40% of the original value. Removing the pronase the regeneration of ligand binding was very protracted with only about 60% intact receptors even after 30 min. From these findings we conclude that the recycling of internalized Fc receptors greatly contribute to the replenishment of receptors on the cell surface.

Animals↗
PubMed2000

Peptide sequence information derived by pronase digestion and ammonium sulfate in-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We present the use of Pronase digestion and in-source decay in the presence of ammonium sulfate as complementary techniques to confirm the amino acid sequence of a peptide. Pronase, a commercial preparation from Streptomyces griseus, is a combination of proteolytic enzymes. It produces carboxypeptidase and aminopeptidase ladders using a single Pronase digestion and represents an inexpensive, nonspecific, and fast supplement to traditional sequencing enzymes. However, N-terminal peptidase activity appears dependent on the terminal amino acid residue. We also introduce the use of saturated ammonium sulfate as an "on-slide" sample additive to promote in-source fragmentation of peptides. Use of saturated ammonium sulfate resulted in a simple way to increase peptide backbone fragmentation and essentially produced either a cn or yn ion series. Together these techniques provide useful supplements to existing methods for peptide sequence information.

Adrenocorticotropic Hormone↗
PubMed1999

Changes in substance P and calcitonin gene-related peptide binding in the dorsal horn of rat spinal cord following pronase-induced deafferentation.

Quantitative receptor autoradiography was used to evaluate potential alterations in substance P (SP) and calcitonin gene-related peptide (CGRP) binding in the L4 spinal segment of rats following unilateral poisoning of the sciatic nerve with pronase. Ten days after pronase-induced deafferentation there was a significant increase in SP and CGRP binding in the superficial (I-II) and deeper (II-IV) laminae of the dorsal horn ipsilaterally. Densitometric measurements revealed a 50% return towards normal values for SP binding by 90 days postpronase injection in all laminae examined, while the density of CGRP binding showed a partial return towards normal values for laminae III-IV only. These differential responses may be indicative of the mechanisms underlying pronase-induced peripheral neuropathy.

Animals↗
PubMed1975

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).

Animals↗
PubMed1991

Partial pronase digestion of rat gastric mucosa isolates cells undergoing replicative DNA synthesis.

A pronase digestion procedure for the isolation of gastric mucosal cells was evaluated for its usefulness in measuring unscheduled DNA synthesis (UDS). The method has been claimed to be suited for assessing the genotoxicity potential of compounds. Compounds were given orally to rats. After 13 h [3H]thymidine was injected and after another hour the animals were killed. The dissected stomachs were treated with pronase for 45 min and the incorporation of radioactivity into DNA was determined. Autoradiography was also performed both on the mucosa and on the isolated cell suspension. The cell suspensions were found to include cells undergoing normal replicative (S phase) DNA synthesis. Of all cells isolated, 4.8 +/- 0.6% consisted of S phase cells. The total amount of DNA recovered (corresponding to the number of cells isolated) was variable and ranged from 20 to 671 micrograms DNA, i.e. approximately 30-fold variation. Neither omeprazole (10, 30 and 80 mg/kg) nor ranitidine (215 mg/kg) had any effect on [3H]thymidine incorporation. The known carcinogen 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) increased incorporation. Refeeding of fasted animals increased incorporation of [3H]thymidine almost 4-fold, showing that animal feeding status influences incorporation. Hydroxyurea, the selective inhibitor of S phase DNA synthesis, inhibited [3H]thymidine incorporation in control and omeprazole-treated animals as well as that induced by MNNG by 92-98%. The results clearly show that the pronase digestion procedure used releases cells undergoing normal, replicative DNA synthesis and can therefore neither be used for measurements of UDS nor for the assessment of the genotoxic potential of drugs.

Administration, Oral↗
PubMed1975

Alterations in reactivity of the blood factors of cattle red cells after pronase treatment.

Pronase treatment of cattle red cells produced various effects: (a) an increase in reactivity of the J factor and evolution of a specific cryptoantigen; (b) decrease in the A-B, G2, K, I1, O2, O3, P, Q, T1, Y2, A', B', E'2, E'3, I', K', O'-L'-V-L and M2 factors, but (c) no change in the specifity or in the titre of the remaining 16 different blood factors. Most of the pronase-affectable blood factors were destroyed in a rather narrow but characteristic range of pronase treatment intensities. However, at like intensities, variations were demonstrable due to the fact that the blood factor occurred (a) in red cells from different individuals, and (b) in different phenogroups or subgroups of the B locus.

Animals↗
PubMedDate not supplied

Pronase digestion of carious dentin.

The aim of this study was to investigate the individual capabilities of the proteolytic enzyme preparation Pronase, the enzyme collagenase and sodium hypochlorite to disintegrate and solubilize carious dentin. Samples of carious dentin, and samples of sound dentin for comparison, were extracted 4 times in succession for 24 h with buffered solutions of Pronase. Separate carious dentin samples were extracted in the same manner with buffered solutions of collagenase or with aqueous sodium hypochlorite. The extracts, the solid residues left over after the extractions and untreated samples of carious and sound dentin were digested with sulfuric acid-H(2)O(2) and then analyzed for nitrogen content by a special adaptation of the Berthelot color reaction. Although Pronase did not attack sound dentin, it solubilized more than 90% of the nitrogen present in carious dentin. Collagenase solubilized approximately 66% of the nitrogen, whereas sodium hypochlorite released only 12-20% of the nitrogen of carious dentin. In clinical dentistry, chemical disintegration of carious dentin may reduce the need for mechanical removal of sound tooth structure.

Collagenases↗
PubMed1977

Nephritogenic glycoprotein. V. Immunochemical studies on nephritogenic activity of collagenase and pronase-digests prepared from various rat organs.

A new immunochemical procedure was introduced to estimate the nephritogenic activity of collagenase and pronase digests of various rat organs. A glycoprotein isolated from collagenase digests of various rat organs showed the nephritogenic activity as well as the antigenic activity that induces nephrotoxic antibody, which were nearly identical to those of a glycoprotein isolated from trypsin digests of the rat organ concerned. A glycoprotein isolated from pronase digests of various rat organs was proved to have no antigenic activity that induces nephrotoxic antibody, but the existence of nephritogenic activity was proved in this glycoprotein. These results were supported firmly by the assay experiments for nephritogenicity. Ouchterlony gel diffusion test and the assay experiments demonstrated the existence of a common nephritogenic substance among the glycoproteins isolated from trypsin, collagenase and pronase digests of rat organs.

Animals↗
PubMed1985

[Method for isolating single cardiac cells in the guinea pig using pronase. The role of calcium ions].

A method for isolation of guinea-pig cardiomyocytes with pronase has been developed. The method has been assessed in hearts perfused with solutions containing pronase (1 U/ml) and 200 microM Ca2+. Eighty per cent of the cells released were rod-shaped and 1.2 mM Ca2+ tolerant. Enriched medium 199 was used for all solutions. Sodium and slow inward currents recorded from cells dispersed with pronase were similar to those recorded from cells isolated after prolonged exposure to collagenase. Two principal factors are to be marked: (a) presence of high enough amounts of Ca2+ in enzyme solution (up to 200 microM); (b) use of the enriched medium in all the stages of the procedure.

Animals↗
PubMedDate not supplied

[Lysis of the cell walls of streptococcus group A by Streptomyces griseus pronase].

The effect of Streptomyces griseus pronase on Streptococcus group A cell walls was studied. Cell walls were shown to be lysed by pronase, the lysis level being dependent on the molarity of the potassium-phosphate buffer used. With an increase in the buffer molarity from 0.005 M to 0.05 M lysis of cell walls decreased from 70-80% to 30%. By DEAE-cellulose chromatography lysates were separated into two fractions the first of which contained a group specific polysaccharide. A preparative method of obtaining a group specific polysaccharide of Streptococcus group A using Streptomyces griseus pronase under mild conditions is described.

Antigens, Bacterial↗
PubMed1982

The human FcR. III. Effects of pronase on soluble immune complex binding by polymorphonuclear leucocytes, monocytes and pulmonary macrophages.

The effect of Pronase incubation on the Fc receptors of human polymorphonuclear leucocytes (PMN), monocytes and pulmonary alveolar macrophages was evaluated by Scatchard analysis of the binding of soluble immune complexes at equilibrium. All three cell types, when preincubated with Pronase, demonstrated a significant increase in Fc receptor affinity. Maximum binding (which measures the number of Fc receptors) on polymorphonuclear leucocytes was reduced 45%-50% but was unchanged on monocytes and pulmonary macrophages. The changes in Fc receptor affinity and maximum binding of the PMN were reversible in short-term culture, an effect which was prevented by cycloheximide. These studies indicate that the affinity of the Fc receptor of human phagocytic cells may change significantly independent of changes in receptor number and that this effect can be caused by extracellular proteases. In addition, the human polymorphonuclear leucocyte demonstrates a subpopulation of Fc receptors which is decreased by Pronase and which recovers, in vitro, by a process requiring protein synthesis.

Antigen-Antibody Complex↗
PubMed1982

Continued expression of vinca alkaloid resistance by CCRF-CEM cells after treatment with tunicamycin or pronase.

We have shown that a glycoprotein(s) with a molecular weight of approximately 180,000 exists on the surface of cultured lymphoblasts selected for resistance to vinblastine (VLB) (CEM/VLB100). The amount of this glycoprotein, which is barely detectable on the VLB-sensitive cells (CEM), appears to be related in part to the degree of resistance, up to approximately 270-fold. Exposure of the cells to Pronase for 45 to 60 min or growth of the cells for 2 days in tunicamycin, an inhibitor of glycoprotein synthesis, resulted in the absence of the resistance-associated glycoproteins, as determined by polyacrylamide gel electrophoresis of these treated cells after labeling te cells either with sodium [3H]-borohydride or with [14C]- or [3H]glucosamine. Uptake studies with [3H]VLB revealed that CEM cells normally accumulated and retained more drug than did the CEM/VLB100 cells. While the tunicamycin or Pronase treatments slightly increased the uptake of drug by CEM cells, there was no enhanced uptake of [3H]VLB by the tunicamycin- or pronase-treated CEM/VLB100 cells, when compared with untreated controls, indicating that the loss of external surface glycoproteins did not render the resistant cells more "leaky" to drug influx. Additionally, diminished drug retention by the CEM/VLB100 cells was unaffected by these treatments. Moreover, when CEM/VLB100 cells were grown grown for 2 days in the presence of tunicamycin and several concentrations of VLB, no enhanced toxicity of VLB was noted. Other experiments with [14C]glucosamine and [3H]leucine revealed that treatment with tunicamycin did not affect the distribution of proteins in these cells. Taken together, these results permit the suggestion that the carbohydrate moiety of the cell surface resistance-associated glycoproteins does not mediate resistance to the alkaloid per se; however, a role for plasma membrane proteins cannot be ruled out at this time.

Animals↗
PubMed1980

Pronase action on the sheath surrounding ventral nerve cord ganglia in Bombyx mori L.

1. Pronase action on ventral nerve cord ganglia in Bombyx mori L. larvae has been studied from different points of view. 2. Microelectrode penetration in the nervous cell is facilitated by exposure of the tissue to the enzyme. 3. Ultrastructural observations show that pronase action affects mainly the fibrous sheath of the tissue, leading to a partial desheathing of the ganglion. 4. Pronase treatment does not affect significantly the ionic concentrations in the nerve cord.

Animals↗
PubMed1978

Somatostatin-containing neurons in the rat brain: widespread distribution revealed by immunocytochemistry after pretreatment with pronase.

Immunocytochemical staining after controlled proteolytic treatment of the sections with pronase revealed widespread distribution of neuronal cell bodies with somatostatin-like immunoreactivity (SLI) in the rat forebrain. SLI-positive neurons were found in regions of the neocortex, the pyriform cortex, the cingulate cortex, the striatum, the olfactory tract and tubercle, the nucleus accumbens, the septum, and the hypothalamus. These results are consistent with previous radioimmunoassay findings and suggest the presence of large somatostatin-like (possibly precursor) molecules in the neurons stained for SLI after pronase treatment.

Animals↗
PubMed1989

Deafferentation-induced expansion of saphenous terminal field labelling in the adult rat dorsal horn following pronase injection of the sciatic nerve.

We have examined the effect of the degeneration of sciatic nerve afferents on the distribution of saphenous terminals in the adult rat dorsal horn. Deafferentation was produced by injection into the sciatic nerve of pronase, a combination of proteolytic enzymes, which causes death of ganglion cells and degeneration of their terminal fields. The saphenous terminal fields were labelled by exposing the cut nerve to a combination of horseradish peroxidase (HRP) and wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Terminals were mainly found in the superficial dorsal horn, indicating that small-diameter afferents were heavily labelled. In one group of control animals, the normal sciatic and normal saphenous terminal fields were shown to be bilaterally symmetrical. In the experimental group, the initial injection of one sciatic nerve with pronase was followed 4 months later by bilateral HRP/WGA-HRP labelling of both saphenous nerves. In each animal, the terminal field of the saphenous nerve on the lesioned side was expanded in the medial, lateral, and caudal directions. Medially and laterally, the expanded terminal field overlapped more of the sciatic territory than normal; caudally, saphenous terminals were found in the rostral portion of the L5 segment, in an area normally filled by sciatic terminals and devoid of saphenous terminals. The expansion resulted in a total saphenous area 26% larger than the control side. Electron microscopy demonstrated that the label in both the normal and expanded territories was primarily contained in axons and terminals, with minor transneuronal labelling. Labelled terminals in the expanded areas were both simple terminals with round, clear vesicles, and glomerular terminals with multiple synaptic contacts; these terminal types resemble those previously described for primary afferents in the superficial dorsal horn. Although the preexistence of "silent" synaptic terminals in the expanded areas cannot be disproven, the data support the hypothesis that primary afferents in the adult have the potential to sprout and establish synapses when the conditions of the deafferentation are favorable.

Afferent Pathways↗