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At least 91 records · Page 5Linked to original sources
PubMed1996

Pronase-based assay method for O6-methylguanine-DNA methyltransferase.

A new, simple, and rapid assay method for O6-methylguanine-DNA methyltransferase (MGMT) has been developed. When [methyl-3H] DNA radiolabeled with N-[methyl-3H]-N-nitrosourea was incubated together with tissue homogenate, [methyl-3H] group was transferred to the enzyme, forming S-[methyl-3H]cysteine. In contrast to the previous methods which determined the amount of [methyl-3H] group removed from [methyl-3H] DNA, the present method measured the amount of [methyl-3H] transferred to the enzyme. This has been done by hydrolyzing the radiolabeled enzyme with pronase which is a proteolytic enzyme with a broad substrate specificity. On pronase digestion, [methyl-3H]-labeled enzyme becomes soluble in trichloroacetic acid. The method is very simple and rapid, and the only expensive equipment required is a scintillation counter which is a relatively routine piece of equipment at present. More than a dozen samples can be processed within 4-5 h without any difficulty. This new method has been employed in the studies on organ distribution of MGMT of rat and mouse.

Animals↗
PubMed1984

Effect of pronase treatment on step-down and step-up photophobic responses in Euglena gracilis.

Pronase-treated cells of Euglena gracilis Z show no discernible ultrastructural effects on the photoreceptor apparatus; however, there are physiological effects on swimming speed and on step-up and step-down photophobic responses, especially the latter. Pronase acts differently on the two photophobic responses: the step-down response is completely inhibited after 2 hr., whereas inhibition of the step-up response occurs in only 50% of the cells even after 24 hr. The effects are fully reversible, with step-up recovery quite rapid and step-down recovery considerably slower.

Animals↗
PubMed2006

Pronase digestion of brush border membrane-bound Cry1Aa shows that almost the whole activated Cry1Aa molecule penetrates into the membrane.

Bacillus thuringiensis insecticidal proteins, Cry toxins, following ingestion by insect larvae, induce insecticidal effect by penetrating the brush border membranes (BBM) of midgut epithelial cells. Purified, activated B. thuringiensis Cry1Aa bound to Bombyx mori BBMV or unbound Cry1Aa were vigorously digested with Pronase. Both digests were compared by Western blotting. Free Cry1Aa was digested to alpha-helix and/or to amino acids at 1 mg Pronase/mL within 2.4 h at 37 degrees C. Whereas, BBMV-bound Cry1Aa was very resistant to Pronase digestion and even at 2 mg for 24 h, 7.5 kDa and approximately 30 kDa peptide were detected by alpha-2,3 antiserum, and alpha-4,5 and alpha-6,7 antisera, respectively. Another approximately 30 kDa peptide was also detected by beta-6-11 and domain III antisera. These fragments are believed either to be embedded in or to strongly interact with the BBMV. The 7.5 and former approximately 30 kDa peptides are thought to be derived from alpha-2,3 helix and stretch of alpha-4 to alpha-7 helices. Furthermore the latter approximately 30 kDa was thought to include the stretch of beta-6 to domain III. Moreover, the embedded Cry1Aa molecule appears to be segregated in some areas of beta-1-5 sheets, resulting in the above two approximately 30 kDa peptides. From these digestion patterns, we proposed new membrane insertion model for single Cry1Aa molecule. On the other hand, in digestion of BBMV-bound Cry1Aa, 15 kDa peptide which was recognized only by alpha-4,5 antiserum was observed. This fragment must be dimeric alpha-4,5 helices and we discussed the origin of this peptide.

Animals↗
PubMed2006

Change in shear stress (Deltatau)/hydraulic conductivity (Lp) relationship after pronase treatment of individual capillaries in situ.

A complex glycoprotein meshwork covers the inner wall of blood vessels and is implicated in mechanotransduction of fluid shear stress (tau). A relationship between Deltatau and capillary Lp has been established. The purpose of this study was to evaluate Lp in response to Deltatau after exposing the capillary lumen to a mild, non-specific protease selected to disrupt its inner matrix. We hypothesized that Lp would not correlate with Deltatau after enzyme treatment. Frogs (Rana pipiens, n=69) were pithed and the mesentery was exteriorized. Lp was assessed at 30 cm H2O using the modified Landis technique after an abrupt, square wave Deltatau produced by a physiologically relevant increase in pressure. Perfusate solutions were 10 mg ml-1 BSA/frog Ringer's (Control) or 0.1 mg ml-1 pronase in BSA/Ringer's (1 min) then BSA/Ringer's alone (Test). Mean (+/-SE) control Lp following Deltatau was 2.2+/-0.2 x 10(-7) cm s-1 cm H2O-1 and individual values correlated positively with Deltatau (r=0.85, P<0.0001, n=41). After pronase, mean Test Lp (17.6+/-2.5 x 10(-7) cm s-1 cm H2O-1) was higher compared to control and Deltatau/Lp plots revealed two subsets of capillaries. Lp correlated strongly with Deltatau in capillaries with diameters 15 microm (r=0.96, P=0.0001, n=8). Slopes were 3.9- and 8.7-fold higher, respectively, compared to control. These data suggest a protective role for luminal constituents of intact capillaries. Mechanisms involved in capillary responses to flow-induced, mechanical stimuli may be located in the cellular structures that form capillaries.

Algorithms↗
PubMed1985

Voltage-dependent removal of sodium inactivation by N-bromoacetamide and pronase.

When perfused internally through crayfish giant axons, pronase removed sodium inactivation more than three times as fast at -100 mV as compared with -30 mV. N-bromoacetamide, applied internally, removed sodium inactivation twice as fast at -100 mV as at -30 mV, and the relative rate of removal declined with membrane depolarization in proportion to steady-state sodium inactivation. We conclude that in the closed conformation the sodium inactivation gate is partially protected from destruction by N-bromoacetamide and pronase.

Acetamides↗
PubMed2007

Pronase-immobilized enzyme reactor: an approach for automation in glycoprotein analysis by LC/LC-ESI/MSn.

An automated analytical approach is proposed for simultaneous characterization of glycan and peptide moieties in pronase-generated glycopeptides. The proposed method is based on the use of a new pronase-immobilized enzyme reactor for the on-line rapid digestion of the target glycoprotein. By coupling the bioreactor to a Hypercarb chromatographic trap column, on-line selective glycopeptide enrichment prior to normal-phase liquid chromatography-mass spectrometry was obtained. A detailed study was carried out for integration and automation of each phase of the proposed analytical procedure. On-line digestion allowed extensive cleavage of the model protein (ribonuclease B), yielding to glycopeptides with peptide moieties up to eight amino acids, carrying the Man5-Man9 N-glycans each, selectively resolved on an Amide-80 column. The use of a linear ion trap instrument resulted in efficient ion capture and led to MS3 acquisition times and spectra quality similar to those for MS2, allowing the unambiguous identification of glycan (MS2) and peptide (MS3) sequences. The proposed procedure reduces the glycoprotein analysis time from approximately 3 days, as in most of the traditional off-line methods, to approximately 1 h.

Automation↗
PubMed1976

Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain.

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.

Animals↗
PubMed1982

Amino acid sequence of the Pronase-released heads of neuraminidase subtype N2 from the Asian strain A/Tokyo/3/67 of influenza virus.

The amino acid sequence of the Pronase-released heads of neuraminidase subtype N2 from the A/Tokyo/3/67 strain of influenza virus was determined by a combination of peptide and nucleic acid sequence analysis. The results show that the Pronase-released heads contain 396 amino acid residues and extend from residue 74 in the original protein to the C-terminus at residue 469. The heads contain five potential glycosylation sites at asparagine residues 86, 146, 200, 234 and 402, but only the first four are glycosylated. The sequence homology with the corresponding region of the previously published sequence of the neuraminidase subtype N1 [Fields, Winter & Brownlee (1981) Nature (London) 290, 213-217] is 45%. Detailed evidence for the sequence data has been deposited as Supplementary Publication SUP 50116 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1981) 193, 5.

Amino Acid Sequence↗
PubMed2001

Carcinoembryonic antigen-related cell adhesion molecule 1 is the 85-kilodalton pronase-resistant biliary glycoprotein in the cholesterol crystallization promoting low density protein-lipid complex.

A pronase resistant 85-kd glycoprotein in the Concanavalin A-binding fraction (CABF) of biliary glycoproteins has been reported to act as a promotor of cholesterol crystallization. De Bruijn et al. (Gastroenterology 1996;110:1936-1944) found this protein in a low-density protein-lipid complex (LDP) with potent cholesterol crystallization promoting activity. This study identifies and characterizes this protein. An LDP was prepared from CABF by discontinuous gradient ultracentrifugation. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotting and immunochemical staining with anti-carcinoembryonic antigen, CEA-related adhesion molecule 1 (CEACAM1) cross-reacting antibodies. Biliary concentrations of CEA cross-reacting proteins were determined in patients with and without gallstones. Two isoforms of CEACAM1 (85- and 115-kd bands), CEA and 2 CEA cross-reacting protein bands of 40 and 50 kd were found in human bile. All bands were also present in CABF, but only a subfraction of the 85-kd band found in the LDP was resistant to digestion with pronase. CEACAM1-85 exhibited potent cholesterol crystallization promoting activity in vitro and accounted for most of the activity in CABF. Total CEA cross-reacting protein concentrations were the same in gallbladder biles from patients with cholesterol and pigment gallstones but only half of those in biles from nongallstone subjects. In conclusion, we have identified the protein component of the cholesterol crystallization promoting LDP to be CEACAM1-85.

Adenosine Triphosphatases↗
PubMed1985

Isolation of tyrosine-O-sulfate by Pronase hydrolysis from fibronectin secreted by Fujinami sarcoma virus-infected rat fibroblasts.

In a recent paper, we reported the loss of large amounts of protein-bound tyrosine sulfate after infection of rat fibroblasts by avian sarcoma viruses. The analogy to the reported loss of surface fibronectin on malignant transformation, which contained sulfate of unknown location, called our attention to this compound. In a previous paper, we briefly reported on isolation from the supernatant fraction of rat fibroblasts infected by Fujinami sarcoma virus fibronectin that yielded tyrosine-O-sulfate on Pronase hydrolysis. In this paper, we confirm and enlarge on this observation. Highly purified fibronectin was obtained from the supernatant fraction secreted by Fujinami sarcoma virus infected rat fibroblasts that contained 1.52 residues of sulfated tyrosine per protein molecule after exhaustive Pronase hydrolysis. Assuming some loss during work up, this probably indicates 2 residues of the tyrosine sulfated per fibronectin molecule.

Animals↗
PubMed1991

Failure of dithiothreitol and pronase to reveal a false-positive cryptococcal antigen determination in cerebrospinal fluid.

A patient with squamous cell carcinoma of the lung and a serum rheumatoid factor (RF) of 1:1,280 had a positive cerebrospinal fluid (CSF) latex agglutination test (LAT) for cryptococcal antigen, in culture-negative, India-ink-negative CSF. Pretreatment of the sample of CSF with 2-mercaptoethanol (2-ME) ablated the antigen titer and established the presence of a false-positive LAT, whereas CSF pretreated with dithiothreitol (DTT) and pronase continued to yield a false-positive result. The differing ability of pronase, DTT, and 2-ME to eliminate interfering substances from CSF has not been previously described. Moreover, because RF is unlikely to cross the blood-brain barrier, the authors postulated that malignant disease was responsible for the patient's false-positive LAT in CSF. Hence, the authors report the case to emphasize that false-positive LAT results in CSF are unlikely to be produced by RF and to underscore the benefit of using enzymatic and sulfhydryl-reducing agents when the validity of the initial test results are in doubt. Such a procedure will optimize the chances of accurately identifying false-positive LAT results in CSF.

Aged↗
PubMed1975

Agglutinability of cattle red cells. 4. The effect of antiglobulin in comparison with treatment by pronase.

The average titre scores varied from zero to 24.7 for the cattle red cells (CRC) from different MZ pairs. These CRC were sensitized with different blood-typing reagents and were titre-tested against the double dilution series of an anti-bovine gamma-globulin serum. A significant negative correlation (r = - 0.82; P less than 0.001) was found between the degree of agglutinability and the amount of neuraminic acid of the surface component of CRC cleaved by pronase. After pronase treatment of the CRC it could be demonstrated that (1) the activity of the V and E'3 blood factors became destroyed; (2) three new specific receptors became evolved; (3) the degree of 'direct' agglutinability due to the A2, O3, W, S2 and Z anti-sera did not parallel with the titre scores obtained in the anti-globulin tests.

Absorption↗
PubMed1988

Luteal progesterone synthesis and high-density lipoprotein. Evidence implicating a pronase-sensitive HDL-binding site in the mechanism by which HDL supports luteal progesterone synthesis.

We have shown previously that corpus luteum cells isolated from the superovulated ovaries of rats treated with 4-amino-pyrazolo[3,4-d]pyrimidine constitute a suitable experimental system by which to investigate the mechanism in which plasma high-density lipoprotein (HDL) plays a role in luteal cellular progesterone synthesis. In the present study, the rate of luteal cellular progesterone synthesis was shown to be stimulated by 125I-labelled HDL up to about 70% of the rate achieved in the presence of native HDL. The concentration of HDL needed for half-maximal stimulation of progesterone synthesis in the presence of lutropin was not significantly different irrespective of whether radioiodinated HDL or unlabelled HDL was used. Experimental conditions for studying the binding of 125I-labelled HDL to isolated luteal cells have been defined and cellular binding affinity and binding capacity have been measured. Exposure of the luteal cells to pronase virtually abolished their capacity to bind 125I-HDL and made them unable to respond to added HDL by increasing their rate of progesterone synthesis in the presence of lutropin. Control experiments showed this effect of pronase on cellular progesterone synthesis not to be due to destruction of cellular lutropin receptors, nor to general cellular damage. This evidence supports the view that luteal cellular binding of HDL is part of the mechanism by which HDL acts in luteal progesterone synthesis. Cellular binding capacity and affinity for 125I-labelled HDL were the same irrespective of whether or not lutropin was present during incubation. Furthermore, the binding capacity and affinity of cells from the ovaries of rats not treated with 4-amino-pyrazolo[3,4-d]pyrimidine were the same as in luteal cells isolated from rats that had been treated.

Adenine↗
PubMed2004

Non-specific depolymerization of chitosan by pronase and characterization of the resultant products.

Pronase (type XXV serine protease from Streptomyces griseus) efficiently depolymerizes chitosan, a linear beta-->1,4-linked polysaccharide of 2-amino-deoxyglucose and 2-amino-2-N-acetylamino-D-glucose, to low-molecular weight chitosans (LMWC), chito-oligomers (degree of polymerization, 2-6) and monomer. The maximum depolymerization occurred at pH 3.5 and 37 degrees C, and the reaction obeyed Michaelis-Menten kinetics with a Km of 5.21 mg.mL(-1) and Vmax of 138.55 nmoles.min(-1).mg(-1). The molecular mass of the major product, LMWC, varied between 9.0 +/- 0.5 kDa depending on the reaction time. Scanning electron microscopy of LMWC showed an approximately eightfold decrease in particle size and characterization by infrared spectroscopy, circular dichroism, X-ray diffractometry and 13C-NMR revealed them to possess a lower degree of acetylation, hydration and crystallinity compared to chitosan. Chitosanolysis by pronase is an alternative and inexpensive method to produce a variety of chitosan degradation products that have wide and varied biofunctionalities.

Acetylation↗
PubMed1978

Slow inactivation of the sodium conductance in squid giant axons. Pronase resistance.

1. Squid giant axons internally perfused with CsF have their Na conductance inactivated due to the low value of the resting potential. When hyperpolarized with voltage clamp to normal values of resting potential, the Na conductance recovers with an exponential time course. The time constant of recovery is of the order of 30 sec at a membrane potential of -70 mV and at 5 degrees C. The recovery from slow inactivation has a Q10 of about 3. 2. The development of inactivation during depolarization is also slow. The time constant varies between 10 and 20 sec at 5 degrees C, depending upon the value of the membrane potential. 3. Slow inactivation is also observed in NaF perfused axons and in intact axons with a low resting potential. 4. Although internal perfusion with pronase (or a purified fraction of this enzymic complex) blocks the fast (h) inactivation of the Na conductance, the slow inactivation remains. The recovery is similar before and after the proteolytic treatment. However, slow inactivation appears to develop faster after enzymic perfusion. 5. Slow inactivation develops without any apparent change in distributed or local membrane surface charge. 6. The experiments suggest that slow inactivation is a general property of the Na conductance as in many other conductance channels in excitable membranes. The experiments can be interpreted by proposing that slow inactivation is a phenomenon independent of fast inactivation, and that pronase somehow accelerates the onset of slow inactivation. 7. An alternative model, in which slow inactivation is coupled to fast inactivation, is proposed. This model is consistent with the results presented here and is very similar to one proposed to explain the frequency response of the sodium currents in Myxicola giant axons (Rudy, 1975, 1978).

Animals↗
PubMed1973

Pronase effect on pancreatic beta cell secretion and morphology.

Pronase at low concentration (4 micrograms per milliliter) produces a reversible increase of glucose-stimulated insulin release in isolated islets of Langerhans. Pronase also affects the ultrastructure of the beta cells by inducing extensive development of tight junctions as well as the accumulation of secretory product within the extracellular spaces.

Animals↗
PubMed1972

Rapid extraction method with pronase B for grouping beta-hemolytic streptococci.

The enzyme, Pronase B, was used to extract the C carbohydrates of betahemolytic streptococci for serological grouping. Reference strains of streptococci, groups A through N, were accurately grouped by using this extraction method. Of the beta-hemolytic streptococci isolated from patients' cultures, 1,197 were classified as either group A or not group A by this method. There was 100% correlation with the reference Lancefield method. In contrast, false reactions occurred with the presumptive bacitracin disc method in 4.1% of the group A and 12.7% of the non-group A cultures. The Pronase method proved simple, accurate, and readily adaptable to the clinical laboratory routine.

Bacitracin↗
PubMed1998

Characterization of membrane-associated Clostridium perfringens enterotoxin following pronase treatment.

After binding, Clostridium perfringens enterotoxin (CPE) initially localizes in a small (approximately 90-kDa) complex in plasma membranes. This event is followed by formation of a second membrane complex, referred to as large (160-kDa) complex. Contrary to a previous hypothesis proposing that CPE inserts into intestinal brush border membranes (BBMs) when this toxin is localized in the small complex, this study shows that BBMs do not offer CPE localized in the small complex protection from pronase. However, our experiments indicate that BBMs do substantially protect CPE from pronase when this toxin is localized in large complex. Since the onset of CPE-induced permeability alterations closely coincides with large-complex formation, these new results suggest that CPE-induced alterations in permeability may result from pore formation due to the partial membrane insertion of CPE when this toxin is present in large complex.

Animals↗