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PubMed1988

Experience with the use of pronase to eliminate interference factors in the latex agglutination test for cryptococcal antigen.

Cryptococcal antigen titers of 70 cerebrospinal fluid and 57 serum specimens from patients suspected of having cryptococcosis were determined both before and after treatment with pronase. Median titers of cerebrospinal fluid specimens before and after treatment were 128 and 128, respectively; mean geometric titers of these specimens before and after treatment were 102 and 204, respectively. Median titers of the serum specimens before and after treatment were 16 and 512, respectively; mean geometric titers of these specimens before and after treatment were 19 and 631, respectively. The modified latex agglutination test did not detect antigen in any of 50 cerebrospinal fluid and 51 serum specimens from patients not suspected of having cryptococcosis. These results suggest that the pronase modification increases the sensitivity of the latex agglutination test and that the modification be routinely incorporated into it.

Agglutination Tests↗
PubMed1983

Pronase reduces intramembranous particle density in uterine epithelial cells in vivo.

The proteolytic enzyme, pronase, was injected into the uterine lumen of rats. This treatment removed half the intramembranous particles (IMPs) from the apical plasma membrane of the uterine epithelial cells but tight junctions of these cells were unaffected. We conclude that at least some of the IMPs are proteinaceous in nature and suggest that IMPs not affected by pronase may be deeply embedded in the lipid bilayer.

Animals↗
PubMed1985

Effect of an N-succinyl-L-trialanine p-nitroanilide-hydrolyzing protease from pronase on glucose metabolism in mice.

Effect of an N-succinyl-L-trialanine p-nitroanilide-hydrolyzing protease (STA-protease) purified from Pronase on glucose metabolism was investigated by an intravenous injection into fasted mice. The maximum decrease in blood sugar level by STA-protease was observed 2 h after the injection of a dose of 5.0 mg/kg. The hypoglycemic activity was observed with other microbial proteases, such as Pronase E and subtilisin BPN', whereas no activity was found with the modified enzymes which almost wholly lost their proteolytic activities by the treatment with diisopropyl fluorophosphate or guanidine HCl. The increase in blood sugar level by epinephrine and the conversion of [1-14C] pyruvate into blood glucose were distinctly suppressed with STA-protease as well as 5-methoxyindole-2-carboxylic acid (MICA), an inhibitor of gluconeogenesis. A slight increase in glycogen content in diaphragm of mice was observed during 2-4 h after the injection of STA-protease, though it was markedly increased by insulin. In test of glucose tolerance, the increase in blood sugar was distinctly suppressed by insulin but not by STA-protease. Blood lactate level was not subjected to change by STA-protease, in Therefore, STA-protease may affect the in vivo metabolism of glucose in a different way from the in vitro action which has been reported to react with cell surface and consequently to mimic the actions of insulin.

Aluminum Silicates↗
PubMed1975

Separation of gastric mucosal cells of rat with proteolytic enzymes, pronase and trypsin, with special reference to the collection, morphology and viability of the generative cells.

Methods to separate and collect gastric mucosal cells of the rat using proteolytic enzymes were devised. Pronase (1.0%) achieved better results than did trypsin (2.0%) in collecting single isolated cells with higher cell yields and viability. The cells dissociated with trypsin retained glandular structures as in situ. The measurement of radioactivity revealed that the incorporation of 3H-thymidine into generative cells was highest in the cell suspension collected by the second 15 min dissociation. It was concluded that the most effective method to obtain dissociated cells from the generative zone of the mucosa is to collect the cells dissociated with 1.0% pronase continuously for a period from 15 to 45 min after the start of dissociation. On autogradiographic analysis with 3H-thymidine, the ratio of generative cells was 10%, approximately 3 X 10(5) cells, in the specimens.

Animals↗
PubMed1995

Deafferentation-induced expression of GAP-43, NCAM, and NILE in the adult rat dorsal horn following pronase injection of the sciatic nerve.

The expression of growth-associated protein 43 (GAP-43), neural cell adhesion molecule (NCAM), and nerve-growth-factor-inducible large external glycoprotein (NILE) in the adult rat dorsal horn was examined at several survival times after unilateral pronase injection of the sciatic nerve. Pronase injection produces a permanent major loss of sciatic primary afferents in the dorsal horn, and there is a later sprouting of saphenous afferents into the sciatic territory. Small-diameter myelinated and nonmyelinated saphenous afferents sprout within the superficial dorsal horn, and larger, myelinated afferents sprout within the deep dorsal horn. In the present study, GAP-43 and NCAM immunoreactivity increased in the superficial dorsal horn by 10 days after injection. By 20 days, the increase spread into the deep dorsal horn; NCAM returned to normal after 1-2 months, but GAP-43 persisted up to 4 months. NILE immunoreactivity appeared in laminae I and II by 10 days and increased up to 30 days; by 2 months no NILE remained. NILE never spread into the deeper dorsal horn, regardless of survival time. These data suggest a correlation in the expression of both NCAM and NILE with the sprouting of fine-diameter sprouting afferents in laminae I and II, and of NCAM expression with the sprouting of larger-diameter afferents in the deep dorsal horn.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗
PubMed1980

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.

Animals↗
PubMed1974

Isolation and characterization of an esterase-active enzyme from pronase with special reference to activation of Clostridium botulinum type E progenitor toxin.

(1) By sequential chromatography on CM-Sephadex, SP-Sephadex and Sephadex G-100, an esterase-active enzyme was isolated from a commercial preparation of pronase, (2) The purity of the isolated enzyme was proven by disc electrophoresis and sedimentation analysis. (3) The molecular weight of the esterase-active enzyme was approximately 16,000. The sedimentation coefficient was 1.85 S. The N- and C-terminal amino acids were asparagine and valine, respectively. (4) The enzyme was similar to that of bovine trypsin in the substrate specificity. It appeared to be the same as the trypsin-like enzyme isolated from pronase by Wähyby (1968). However, the relative rates of hydrolyses of different substrates by the enzyme differed from those of bovine trypsin. (5) The enzyme activated Clostridium botulinum type E progenitor toxin. Even a higher toxicity was resulted by treating the progenitor toxin with this enzyme than with bovine trypsin. (6) The results support the hypothesis that activation of type E progenitor toxin involves hydrolysis of a lysine ester bond.

Amino Acids↗
PubMed1989

Characterisation of the first monoclonal antibody against the pronase resistant core of the Alzheimer PHF.

A monoclonal antibody, NOAL 6.423 has been produced which unequivocally identifies a protein which is a constituent of the pronase resistant core of the Alzheimer PHF. Although the site recognised by this antibody is contained somewhere within the highly conserved triple repeat region of the tau molecule, no experiments have succeeded so far in demonstrating NOAL 6.423 reactivity in any brain proteins other than those derived from PHFs. A site which can be recognised by NOAL 6.423 cannot be generated in preparations of normal porcine tau by simple proteolytic cleavage with pronase. Likewise reactivity with the PHF peptide recognised by NOAL 6.423 is not altered by alkaline phosphatase. The unusual properties of NOAL 6.423 are consistent with the possibility that its recognition site is not determined solely by the primary structure of the conserved region of the tau molecule.

Alzheimer Disease↗
PubMed1988

Proteolysis of spectrin by trypsin and pronase in the presence of phospholipid suspensions.

The effect of phospholipid suspensions on the proteolysis of isolated spectrin was examined by SDS-polyacrylamide gradient gel electrophoresis. Proteolysis of spectrin in the membranes by trypsin and pronase was also studied. It was found that electrophoretic patterns of spectrin fragments were influenced by the presence of the suspension prepared from phosphatidylethanolamine:phosphatidylserine (60:40) mixture and of phosphatidylcholine. Qualitative changes in the proteolytic patterns obtained after proteolysis of spectrin by pronase in the presence of phosphatidylcholine suspension were observed. The changes in the sensitivity of spectrin towards proteases result probably from changes in the accessibility of some peptide bonds upon the interaction of this extrinsic protein with phospholipids.

Animals↗
PubMed1985

Splenic B cells from CBA/N mice acquire responsiveness to anti-immunoglobulin after a brief treatment with pronase.

Although splenic B cells of CBA/N mice do not synthesize DNA in response to anti-mouse IgM (mu-chain specific), the cells respond readily to Sepharose linked anti-mu. Subsequent to a brief treatment with pronase, CBA/N splenocytes exhibited anti-mu-mediated DNA synthesis at 40 to 100% of the DNA synthetic capacity detected with Sepharose linked anti-mu. Furthermore, spleen cell populations treated with anti-Thy-1.2 and complement or populations purified on anti-immunoglobulin-coated Petri plates (greater than 90% surface immunoglobulin positive) acquired responsiveness to anti-mu after pronase treatment.

Animals↗
PubMed1976

Proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (Pronase). Purification and characterization of the carboxypeptidase.

We described earlier the facilitated purifications of the trypsin and aminopeptidase components present in Pronase (Vosbeck, K. D., Chow, K. -F., and Awad, W. M., Jr. (1973) J. Biol. Chem. 248, 6029-6034). A partially resolved protein mixture left over after one of the steps in that procedure was passed through a Sephadex G-75 column. By this means, a component with carboxypeptidase activity was separated from associated serine endopeptidases. Further purification of this exopeptidase to apparent homogeneity was acheived by refiltration through the same Sephadex column and by CM-cellulose chromatography. A single protein band was observed after acrylamide gel electrophoresis; analysis by sedimentation equilibrium using the meniscus depletion method gave a molecular weight of 30,300. This enzyme demonstrates activity against Nalpha-benzyloxycarbonylglycyl-L-leucine and hippuryl-D,L-phenyllactate; no activity was found against Nalpha-acetyl-L-tyrosine ethyl ester, Nalpha-benzoyl-D,L-arginine-p-nitroanilide, or L-leuckne-p-nitroanilide. The maximum activity lies between pH values of 7 and 8; the enzyme is stable between pH values of 6 and 10. At room temperature 1,10-phenanthroline inactivates the enzyme completely whereas EDTA has no effect. Of the many cations tested, only Co2+, Ni2+, or Zn2+ restores activity to the 1,10-phenanthroline-treated enzyme; Co2+ provided 3 times the native activity. The metal in the native protein was found to be zinc. These findings are similar to those recorded with bovine pancreatic carboxypeptidase A, and suggest the possibility that the present enzyme may ge genetically related to the mammalian protein, as in previously noted examples of homology of three Pronase endopeptidases to pancreatic serine enzymes.

Amino Acids↗
PubMed1983

[Effect of collagenase, hyaluronidase and pronase, alone or combined with cationic markers, on the renal filtration barrier in the rat].

The authors investigate the effect of collagenase, hyaluronidase and pronase, used either alone or associated with cationic tracers such as Alcian Blue, Ruth enium Red and Polyethyleneimine on the Rat glomerular filtration barrier. Collagenase appears to promote the perviousness of the three constitutive layers of the glomerular basement membrane. The effects of hyaluronidase and pronase seem to be specific and permit to suppose that the glomerular basement membrane supports several types of anionic sites. The affinity of cationic tracers seems to fluctuate according to the constitution of these sites.

Alcian Blue↗
PubMed1982

Antibody restores human alternative complement pathway activation by mouse erythrocytes rendered functionally deficient by pretreatment with pronase.

Activation of the human alternative pathway of complement (C) by surfaces requires the initial deposition of C3b by fluid-phase C3 convertase and sustained C3 cleavage by C3 convertases fixed to the surface. Nonactivating particles have previously been characterized by an inability to sustain the function of C3 convertases on their surfaces because these sites were susceptible to the regulatory action of the control proteins. Pronase converts the mouse erythrocyte (E) from an activator to a nonactivator by markedly decreasing the ability of the cell to affix C3b generated by a fluid-phase C3 convertase; this conversion is unrelated to the action of the control proteins on bound C3b. The capacity of antibody and its F(ab')2 or Fab' fragments to restore the alternative pathway-activating function of pronase-treated mouse E indicates that the contribution of antibody is mediated by its combining site without a requirement for bridging or for the Fc portion. The fab'-dependent activation of the alternative pathway of C relates to the deposition of C3b on the particle surface, which is a first and continuing step in alternative pathway activation. The antibody effect is not directed to the action of the regulatory proteins. Thus, particle-dependent activation of the alternative C pathway has been shown for the first time to be abolished and restored by cell surface-directed mechanisms that function independently of the regulatory proteins.

Animals↗
PubMed1979

Sensitivity and background staining of the unlabeled antibody enzyme (PAP) method. new approaches to the investigation of the effects of non-immune serum, primary antiserum concentration and pronase digestion using formalin fixed paraffin embedded material.

The effect of primary antibody concentration, non-immune serum, and pronase digestion on the stainability of immunoglobulin containing cells (plasma cells) and background stainability was investigated using the peroxidase-antiperoxidase (PAP) method and antigens consisting of formalin fixed paraffin embedded maxillary mucosa, colonic mucosa, inflammatory synovia and soft-tissue plasmocytoma. The effect of primary anti-serum concentration and non-immune serum on cell and background stainability was smaller, but pronase digestion clearly improved positive stainability and reduced background stainability.

Animals↗
PubMed1993

Deafferentation-induced changes in neuropeptides of the adult rat dorsal horn following pronase injection of the sciatic nerve.

The effect of deafferentation on the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), somatostatin (SS), and cholecystokinin (CCK) in the lumbar dorsal horn of the adult rat was examined by the indirect immunohistochemical method. Deafferentation was induced by injecting the sciatic nerve of anesthetized rats with proteolytic enzymes (20 mg pronase), which cause selective death of the nerve's ganglion cells and degeneration of their terminal arborization in the spinal cord. The density of immunolabel of each peptide was determined by using a computerized densitometry analysis system in two animal groups, i.e., short-term (10-13 days after injection) and long-term (4-9 months). In both groups, the deafferentation produced a significant ipsilateral depletion of CGRP, SP, CCK, and SS immunoreactivity. This depletion was limited to the area occupied by the sciatic terminals in the dorsal horn. In the long-term group, the loss of CGRP and SP staining was significantly less than that in the short-term animals, thus indicating partial recovery. A similar, but not statistically significant, trend was observed for CCK and SS. The large decrease in CGRP and SP seen in short-term animals reflects the large contribution of the sciatic nerve to the lumbar dorsal horn. The partial recovery of peptides demonstrates the plasticity of the nervous system and may parallel sprouting of primary afferents from other nerves, such as the saphenous nerve, as we have demonstrated in previous studies.

Animals↗
PubMed1999

Unusual phosphatase activity resistant to SDS and pronase treatments in Xenopus ovary.

An unusual phosphatase, which is resistant to treatment by 5% SDS and proteolytic enzymes, was isolated as two types, 1 and 2, from pronase-treated homogenates of Xenopus ovary. The molecular sizes of types 1 and 2 were estimated as about 140 kDa and more than 2 x 10(4) kDa, respectively, by gel filtration, but as 140 and 130 kDa as a catalytic unit, respectively, by electrophoresis, implying that whereas type 1 might be composed of catalytic unit alone, type 2 is a multicomponent complex consisting of a 130-kDa catalytic unit. Both activities were sensitive to nucleases but resistant to tested proteolytic enzymes. These findings suggest that the unusual phosphatase activity is attributable to a polynucleotide.

Animals↗