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At least 127 records · Page 7Linked to original sources
PubMed1986

Mannose incorporation and lectin recognition of pronase-sensitive components in Micrococcus lysodeikticus (M. luteus) membranes.

Isolated cytoplasmic membranes from Micrococcus lysodeikticus were able to incorporate [14C]mannose from GDP-[14C]mannose. Labelled mannose remained in the membrane fraction after its repeated washing and lipid extraction. Sodium dodecyl sulfate gel electrophoresis in 12% acrylamide showed a set of bands with molecular weights ranging from 230 000 to 19 000 which stained for protein and carbohydrate, and incorporated [14C]mannose. Some of these bands reacted with different lectins (concanavalin A, wheat germ agglutinin and ricin). Furthermore, the mannose was incorporated via a glycosylation pathway similar to that followed in eukaryotic system as shown by the preliminary identification of a lipid intermediate transferring the sugar to proteins and by the differential sensitivity to bacitracin and tunicamycin. These complex membrane components were sensitive to digestion with pronase. All the results presented suggest their glycoprotein nature.

Bacitracin↗
PubMed1988

Schistosomin: a pronase-sensitive agent in the hemolymph of Trichobilharzia ocellata-infected Lymnaea stagnalis inhibits the activity of albumen glands in vitro.

The schistosome parasite, Trichobilharzia ocellata, nearly completely inhibits the reproductive activity of its intermediate host, Lymnaea stagnalis. The synthetic activity of albumen glands of infected snails at day 35 postinfection (p.i.) is only 1% of the control value. The parasite acts by humoral means. We tested the hypothesis that (a) specific humoral agent(s) is (are) involved and refer to this (these) agent(s) as schistosomin. The presence of schistosomin in the hemolymph of infected snails was investigated by using galactogen synthesis in albumen glands as an in vitro bioassay. The synthetic activity of albumen glands of noninfected snails decreased by about 50% during a 1-h incubation in the hemolymph of infected snails. This inhibition is attributed to schistosomin. Based on these results, with the present bioassay schistosomin appears in the hemolymph between days 28-36 p.i. onwards. Schistosomin is heat-stable (100 degrees C) and pronase-sensitive, and therefore it might have a peptide nature. Schistosomin suppresses the stimulating action of the female, gonadotrophic dorsal body hormone at relatively low doses, which suggests that it may compete with this hormone for the same receptors. The development of two other bioassays for schistosomin in our laboratory is discussed.

Animals↗
PubMed1989

Cycle- and function-related changes in lectin binding to human endometrium: a histochemical study with pronase treatment.

Forty-eight endometrial biopsy specimens were obtained during a normal menstrual cycle, during pregnancy and from patients with dysfunctional bleeding. The specimens were examined for Peanut (PNA), Soybean (SBA), Vicia villosa (VVA), Phytohem- (PHA), Lens culinaris (LCA) and Concanavalin (succ. Con A) agglutinin binding. The study was performed on paraffin sections using the pronase digestion and either the peroxidase-antiperoxidase or the avidin-biotin-peroxidase method. Cycle-related changes of the PNA, SBA, VVA, and to some degree PHA binding, were based on the transfer of the cytoplasmic reaction toward the glandular lumena. PNA + and SBA + material moved to the cell surface at the transition of the follicular and luteal phase and before the basal vacuolization appeared. Functional disturbances mainly influenced the intensity of the reaction. It was true only for those lectins, whose binding pattern showed cycle-related changes. In curettings from patients with a prolonged menstrual cycle the lectin binding seen in normal late secretory endometrium was absent or significantly diminished. Lectin binding to the endometrial surface epithelium was variable; PHA was the only lectin, the binding pattern of which followed cyclic changes in the glycocalyx, detected previously by means of PAS and alcian blue methods.

Adult↗
PubMed1977

The pharmacological action of pronase-digested egg albumin upon cerebral hypoxia.

The purification of a substance which protects mitochondrial activity against its decay in association with various cerebral pathologies, and the effect of this substance in vitro and in vivo have been mentioned. Defatted egg albumin was hydrolyzed with pronase, and diafiltrated through mesh, to obtain fractions of molecular weight less than 5,000. The diafiltrate was further fractionated using Sephadex G-25 column chromatography, and a fraction which had a protective action against the decay of mitochondrial DNP-ATPase activity was gathered. This digested egg albumin (DEA 5,000 S) showed no immunological reaction and seemingly penetrated well through the cell membrane. DEA 5,000 S prevented the decay of DNP-ATPase activity and swelling of brain mitochondria during aging in vitro. Also, the administration of DEA 5,000 S in vivo shortened the duration of unconsciousness and reduced EEG abnormalities in rats subjected to hypoxia in a special chamber filled with N2 gas. It is suggested that this Digested Egg Albumin has a marked action in restoring the function of metabolically impaired brain.

Adenosine Triphosphatases↗
PubMed1997

Hyperacute renal allograft rejection from anti-HLA class 1 antibody to B cells--antibody detection by two color FCXM was possible only after using pronase-digested donor lymphocytes.

We present a report of a transplant recipient who lost her renal allograft from hyperacute rejection. This was secondary to a weak IgG anti-HLA class I antibody that was only reactive to donor B lymphocytes. This antibody was not detected in her pretransplant serum by the conventional complement-dependent cytotoxicity assays using donor blood lymphocytes. Pretransplant sera were analyzed retrospectively by two-color flow cytometric crossmatching (FCXM). It was difficult to determine if the recipient's serum contained an IgG antibody specific for HLA on donor B cells since IgG from control AB sera and pretransplant sera bound equally well to CD19 B cells. However, when donor lymphocytes were pretreated with pronase to digest the membrane receptor for Fc domain of IgG (Fc gamma R) on non-T-cells, control IgG in AB serum did not bind to B cells and, hence, it was easy to detect binding of IgG (in pretransplant sera) to HLA on B cells. This case underscores the importance of identifying weak anti-HLA class I antibodies reactive only to B cells. Moreover, it shows that the currently used two-color FCXM lacks the specificity to detect such antibodies.

Adult↗
PubMed2005

Quality of pronase dissociation of mature inferior colliculus neurons.

One major advantage of acutely dissociated inferior colliculus (IC) neurons in electrophysiological investigations is their complete isolation from the surrounding cellular network. In this way, patch-clamp recordings can be performed under controlled conditions to study membrane properties of IC neurons in more detail. The aim of the present study was to adapt a dissociation method for immature IC neurons to the highly sensitive, fragile and vulnerable mature IC neurons of mammals (mice). The modification of a pronase-based dissociation protocol with respect to concentration, incubation time and handling (trituration) of the cells yielded intact, live IC neurons with a clean cell surface so that they were well suited for further electrophysiological investigations in our study. The largely modified dissociation protocol is described in detail and critically discussed.

Humans↗
PubMed1984

Kinetic analysis of sodium channel block by internal methylene blue in pronased crayfish giant axons.

The cationic dye methylene blue (MB+) blocks INa in a voltage and time-dependent manner and exhibits no frequency dependent block at 1 Hz when internally perfused in normal or pronase-treated crayfish axons. Peak INa decreases with increasing MB+ concentrations in the range 50 microM to 5 mM, but the blocking time constant approaches an asymptote at concentrations above 500 microM. IgON is not noticeably affected by internal MB+ at concentrations of 500 microM or below, in the absence of external tetrodotoxin (TTX). However, 5 mM MB+ produces a visible suppression of IgON that is reversible following washout. A pseudo-first-order analysis of MB+ blocking kinetics suggests a drug binding site deep in the transmembrane voltage field (dz = 0.85, KD = 11 microM at 0 mV). The voltage sensitivity of the individual rate constants is highly asymmetric, suggesting that the major energy barrier for MB+ is very close to the axoplasmic margin of the voltage field. Reversing the Na+ gradient and direction of INa has little effect on the kinetics of MB+ block. The kinetic properties of state-dependent vs. state-independent blocking schemes are investigated and compared with our observations of MB+ block. Analysis of hooked sodium tail currents following depolarization to various test potentials demonstrates quantitatively that MB+ binds in a state-dependent manner to open sodium channels. The appropriateness of first-order kinetic analysis of drug block is then considered in light of these observations.

Animals↗
PubMed1987

Structure of acidic oligosaccharides isolated from pronase-treated glycoprotein of bonnet-monkey (Macaca radiata) cervical mucus.

The major glycoprotein component of cervical mucus of bonnet monkey was treated with Pronase, and the enzyme-resistant glycoprotein purified by gel filtration on Sepharose 4B followed by DEAE-cellulose chromatography. Alkaline-borohydride cleavage of the carbohydrate chains gave a mixture of neutral and acidic oligosaccharides. Seven acidic oligosaccharides were characterized by chemical and enzymic procedures; their proposed structures are: alpha NeuAc(2----3)-[beta GalNAc(1----4)]beta Gal(1----4)GlcNAc(1----6)[alpha Fuc(1----2)beta Gal(1----3)]GalNAc-ol; alpha Fuc(1----2)beta Gal(1----3)/6)[alpha NeuAc(2----3)beta Gal(1----4)GlcNAc(1----3)/6)]GalNAc-ol; alpha GalNAc(1----3)beta Gal(1----3)[alpha NeuAc(2----3)beta Gal(1----4)GlcNAc(1----6)]GalNAc-ol; beta GlcNAc(1----3)[alpha Fuc(1----2)]beta Gal(1----3)[alpha NeuAc(2----6)]GalNAc-ol; beta Gal(1----3)-[alpha NeuAc(2----6)]GalNAc-ol; alpha NeuAc(2----6)GalNAc-ol; and beta Gal3SO3(1----4) GlcNAc(1----6)[alpha Fuc(1----2)beta Gal(1----3)]GalNAc-ol.

Animals↗
PubMed2006

Immunofluorescence on pronase-digested paraffin sections: a valuable salvage technique for renal biopsies.

Direct immunofluorescence (IF) on frozen tissue is the method of choice for the study of medical renal diseases. When no glomeruli are available, IF can be performed on the formalin-fixed paraffin-embedded tissue allocated for light microscopy after antigen retrieval with proteases. In this study, the results of IF on frozen tissue (IF-F) and on deparaffinized, pronase-treated tissue (IF-P) were compared in 71 renal biopsies representing 12 major renal diseases. Using IF-P, diagnostic findings were obtained in 100% of cases of lupus nephritis, acute post-infectious glomerulonephritis, cryoglobulinemic glomerulonephritis, fibrillary glomerulonephritis, primary amyloidosis, myeloma cast nephropathy, and light-chain Fanconi syndrome (LCFS), 88% of cases of immunoglobulin (Ig)A nephropathy, 80% of cases of light-chain deposition disease, 60% of cases of membranoproliferative glomerulonephritis type 1, 50% of cases of idiopathic membranous glomerulopathy (MGN) and 20% of cases of anti-glomerular basement membrane (GBM) disease. IF-P was less sensitive than IF-F for the detection of C3 in all disease categories and for the detection of IgG in cases of MGN and anti-GBM disease. The diagnostic kappa light-chain staining was demonstrated in 100% of cases of LCFS by IF-P versus 40% by IF-F. We conclude that IF-P is a valuable salvage immunohistochemical technique for renal biopsies lacking adequate cortical sampling for IF-F, and is superior to IF-F for the diagnosis of LCFS.

Antigen-Antibody Complex↗
PubMed1994

Chemical modification of aminopeptidase isolated from Pronase.

Chemical modification of aminopeptidase from pronase has revealed two important histidines in enzyme catalysis. In the absence of metal ions, modification of the readily-modified histidine (pKa 6.9 +/- 0.5) results in a drastic loss of activity, indicating that this residue is indispensible for enzyme activity. In the presence of CaCl2, the modified enzyme still retains approx. 60% of the activity, whereas modification of another histidine (pKa 7.7 +/- 0.2) leads to a dramatic loss of activity. In fact, the enzyme with the first histidine being modified is active only in the presence of metal ions. Moreover, modification of the second histidine is prevented by the presence of Ca(II). These results indicate that the second histidine is serving as a ligand for Ca(II) and the bound Ca(II) is directly involved in enzyme catalysis. The c.d. spectra of the modified and unmodified enzymes in the absence or presence of CaCl2 are all very similar, indicating that no gross conformational changes in protein occur upon modification or by the presence of Ca(II). Modification of both histidines is prevented by the presence of a competitive inhibitor, suggesting that they are located in the active centre. Modification of 11 amino groups, two tyrosines, or four arginines causes no appreciable inactivation of the enzyme, indicating that these residues are not directly involved in enzyme catalysis.

Aminopeptidases↗
PubMed1986

Immunohistochemical localization of CA 125 antigen in formalin-fixed paraffin sections of ovarian tumors with the use of Pronase.

The OC 125 monoclonal antibody was used to localize CA 125 antigen in routine paraffin sections of ovarian tumors with the use of a modified avidin-biotin-peroxidase complex (ABC) technic. Pretreatment of the paraffin sections with Pronase allowed subsequent detection of CA 125 antigen. OC 125 stained 4 (80%) of 5 benign and borderline serous ovarian tumors, 12 (86%) of 14 serous adenocarcinomas, and 3 (23%) of 13 benign and malignant mucinous ovarian tumors. The pattern of distribution of CA 125 antigen was mostly at the intraluminal and peripheral cell surfaces, while intracytoplasmic staining also was seen. Overall, CA 125 antigen detectability rate in paraffin sections was found to be compatible with those reported in frozen sections. The method allows retrospective immunohistochemical examination of a large number of cases with ovarian tumors.

Antibodies, Monoclonal↗
PubMed1988

Purification and characterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from a commercial preparation (Pronase).

A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.

Amino Acids↗
PubMed1976

Studies on the mitogenic activity of trypsin, pronase and neuraminidase on human peripheral blood lymphocytes.

Human peripheral blood lymphocytes were cultured in the presence of protease (trypsin and pronase) and Vibrio choleraneuraminidase. T and B lymphocyte populations were separated and the effect of these enzymes plus phytohaemagglutin or Tuberculin was studied. The results of these experiments show that proteases moderately stimulate spontaneous deoxynucleic acid synthesis of control cells and potentiate the effect of tuberculin on sensitized cells. These enzymes act specifically on B lymphocytes. Neuraminidase also increases spontaneous deoxyribonucleic acid synthesis of control cells and augments significantly the in vitro response to PPD. There is no additive effect of neuraminidase on phytohaemagglutin stimulated cells. Neuraminidase seems to stimulate specifically T lymphocytes. Some possible mechanisms of action of these enzymes are proposed and discussed.

B-Lymphocytes↗