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A D Befus

Publications and source records attributed to A D Befus.

At least 19 recordsLinked to original sources
PubMed1998

Mast cells express novel CD8 molecules that selectively modulate mediator secretion.

CD8, a marker largely restricted to subsets of T lymphocytes and NK cells, was detected on freshly isolated rat peritoneal mast cells (PMC). Using flow cytometry, Percoll-enriched rat PMC (> or = 98% purity) were positive for the hinge region of CD8alpha (67.5 +/- 9.5%; Ab OX8) and CD8beta (27.8 +/- 2.3%; Ab 341). CD8+ PMC consisted of two populations, CD8alpha+ (22.5%) and CD8alpha+ beta+ (15.9%). Interestingly, G28, an Ab that identifies the IgV-like region of CD8alpha on T lymphocytes, did not bind PMC, suggesting that PMC CD8alpha is distinct from that on T lymphocytes. Moreover, a similar pattern of Ab positivity for CD8 was observed on a rat mast cell line, RBL 2H3. The presence of CD8alpha immunoreactivity on rat PMC was further confirmed by confocal microscopy. In situ reverse-transcription PCR and reverse-transcription PCR analysis demonstrated that PMC contained mRNA transcripts encoding CD8alpha. In functional studies of CD8 on PMC, both TNF-alpha and nitric oxide production were induced by OX8 (CD8alpha) and 341 Ab (CD8beta) in a dose-dependent manner. However, neither OX8 nor 341 induced histamine secretion from PMC. Ag-induced secretion of TNF-alpha, nitric oxide, and histamine was not affected by OX8 or 341 Abs, suggesting that there are distinct signaling mechanisms mediated by CD8 and Fc epsilonRI. These results indicate that rat PMC express functional CD8 molecules that may be distinct from those of T lymphocytes. The difference suggests there is a ligand other than MHC class I for mast cell CD8.

Animals↗
PubMed1998

Mechanisms of macrophage stimulation through CD8: macrophage CD8alpha and CD8beta induce nitric oxide production and associated killing of the parasite Leishmania major.

Prior studies demonstrated that rat macrophages express CD8, which differs from T lymphocyte CD8 within the ligand binding domain. We investigated whether stimulation of macrophage CD8 could induce mediator release and regulate host defense. Cross-linking either CD8alpha (OX8, 5 microg/ml) or CD8beta (341, 10 microg/ml) stimulated nitric oxide (NO) production, which correlated with an up-regulation of inducible NO synthase protein. Cell signaling inhibitors were used to elucidate the pathways of CD8alpha and CD8beta stimulation. Genistein (broad spectrum protein tyrosine kinase inhibitor, 10 microg/ml), PP1 (src family kinase inhibitor, 5 microg/ml), polymyxin B (protein kinase C (PKC) inhibitor, 100 microg/ml), and Ro 31-8220 (PKC inhibitor, 1 microM) significantly inhibited anti-CD8alpha- and anti-CD8beta-stimulated NO production and inducible NO synthase up-regulation, suggesting that tyrosine kinase(s) (src family) and PKC are involved in CD8 signaling. In addition, cross-linking CD8alpha stimulated NO-dependent macrophage killing of the parasite Leishmania major. For the first time, this work demonstrates that the beta-chain of macrophage CD8, in addition to the alpha-chain, can regulate mediator release. These results further illustrate the importance of this molecule and support our previous data demonstrating differences between macrophage and T lymphocyte CD8. Additional studies on the signaling mechanisms and possible ligand(s) for macrophage CD8 will lead to a greater understanding of inflammation and host defense.

Animals↗
PubMed1998

Role of intestinal mast cells in modulating gastrointestinal pathophysiology.

OBJECTIVE: This article reviews the current understanding of the pathophysiologic role of intestinal mast cells. DATA SOURCE: Up to date English language publications on mast cell characteristics, heterogeneity and functions were used. Recent articles were used to develop and extend novel concepts about the role of intestinal mast cells. STUDY SELECTION: Reference sources were selected because of their pertinence to the pathophysiological effects of mast cells in intestinal hypersensitivity. Recent publications on the following topics were emphasized: mast cell proteases in intestinal anaphylaxis; effects of nitric oxide in gastrointestinal pathophysiology; involvement of cytokines derived from mast cells in tissue damage and repair. RESULTS: Mast cells are clearly implicated in the pathology of intestinal disease. Growing evidence suggests physiological roles for mast cells in the protection of tissues from inflammatory damage, and in intestinal maturation. Mast cells can release cytokines, such as tumour necrosis factor-alpha and interleukin-10, which were originally thought to contribute to inflammatory damage, but which may also have anti-inflammatory properties. Interestingly, mast cell function can be regulated by nitric oxide, and mast cells themselves are sources of this important mediator. Nitric oxide has protective as well as detrimental effects in the intestine. CONCLUSIONS: Intestinal mast cells have physiologic regulatory effects in addition to their pathologic effects. However, relatively little is known about the mechanisms of these regulatory effects. Mast cells are likely in an ongoing fluctuating balance between physiological functions and pathological effects in normal individuals. Poorly known factors can create an imbalance and lead to pathologic reactions.

Anaphylaxis↗
PubMed1998

Rat mucosal mast cells: the cultured bone marrow-derived mast cell is biochemically and functionally analogous to its counterpart in vivo.

Mast cells (MC) are biochemically and functionally heterogeneous and the mixture of MC phenotypes varies according to anatomical location. Intestinal mucosal MC (IMMC) have been used to study the mucosal MC subset in the rat, but they are difficult to isolate in sufficient numbers and with consistent purity and viability. Bone marrow-derived MC (BMMC), with an apparent mucosal MC phenotype, can be cultured in large numbers and with high purity from normal rat bone marrow using supernatants from mesenteric lymph node cells of rats infected with the nematode, Nippostrongylus brasiliensis. We have compared serine proteinase content, tumour necrosis factor-alpha (TNF-alpha) storage and secretion, and TNF-alpha-dependent cytotoxicity of IMMC and BMMC to assess the appropriateness of BMMC as in vitro models of mucosal MC. Two-dimensional gel electrophoretic analysis revealed that the overall protein constituents of BMMC and IMMC were highly homologous. Immunoblotting confirmed that both MC types expressed the MMC-associated enzyme, rat mast cell proteinase-2 (RMCP-2), but not RMCP-1, mast cell proteinase-5 (MCP-5) or carboxypeptidase A (CPA), which characterize the connective tissue MC in the rat and which were detected in a representative of this subset, namely, the periotoneal MC (PMC). BMMC demonstrated levels of TNF-alpha-dependent cytotoxicity that were equivalent to those of IMMC. Like IMMC, BMMC contained little stored TNF-alpha, in comparison with PMC, but both MC types generated substantial amounts of TNF-alpha 6 hr following IgE-mediated activation. Pretreatment of PMC with recombinant rat interferon-gamma (IFN-gamma) for 20 hr inhibited anti-immunoglobulin E (anti-IgE)-mediated release of the granule-associated enzyme, beta-hexosaminidase, whereas identically treated BMMC were unresponsive to this cytokine. Similar results have previously been reported for IMMC. Rat BMMC, unlike their more immature and less phenotypically committed counterparts in the mouse, appear therefore to be more appropriate models for studies on the mucosal MC.

Animals↗
PubMed1998

Effects of toxin A from Clostridium difficile on mast cell activation and survival.

Toxins A and B from Clostridium difficile are the main cause of antibiotic-associated diarrhea and pseudomembranous colitis. They cause fluid accumulation, necrosis, and a strong inflammatory response when inoculated in intestinal loops. Since mast cells are a rich source of inflammatory mediators, abundant in the gut, and known to be involved in C. difficile-induced enteritis, we studied the in vitro effect of toxin A on isolated mast cells. Normal rats sensitized by infection with Nippostrongilus brasiliensis were used to isolate peritoneal mast cells (PMC). PMC from naive rats were stimulated with calcium ionophore A23187 as a model of antigen-independent activation, and PMC from sensitized rats were stimulated with N. brasiliensis antigens to study immunoglobulin E-dependent mast cell activation. After 4 h, toxin A did not induce release of nitric oxide or histamine in naive PMC. However, 10 ng of toxin per ml caused a significant release of tumor necrosis factor alpha (TNF-alpha). In contrast, 1 microg of toxin per ml inhibited antigen or A23187-induced histamine release by PMC. Toxin A at 1 microg/ml for 4 h caused disruption of actin which aggregated in the cytoplasm and around the nucleus. After 24 h, chromatin condensation, cytoplasmic blebbing, and apoptotic-like vesicles were observed; DNA fragmentation was documented also. These results suggest that mast cells may participate in the initial inflammatory response to C. difficile infection by releasing TNF-alpha upon interaction with toxin A. However, longer exposure to toxin A affects the release of inflammatory mediators, perhaps because of the alteration of the cytoskeleton and induction of apoptosis. The impaired functions and survival of mast cells by C. difficile toxin A could hamper the capacity of these cells to counteract the infection, thus prolonging the pathogenic effects of C. difficile toxins.

Animals↗
PubMed1997

Differential regulation of mast cell function by IL-10 and stem cell factor.

In the development of rodent mast cells (MC), IL-10 significantly enhances the growth factor activity of stem cell factor (SCF). The differential effects of IL-10 and SCF on function of rat peritoneal MC (PMC) are investigated in this study. IL-10 inhibits both constitutive and Ag-induced nitric oxide production by PMC in a dose-dependent manner, whereas SCF does not affect nitric oxide production by PMC. Short term (20-min) incubation with IL-10 does not affect Ag-induced histamine secretion, whereas long term (24-h) incubation with IL-10 significantly potentiates Ag-induced histamine secretion, an effect similar to those reported for IL-3 and IL-4 in mouse MC. By contrast, SCF significantly potentiates Ag-induced histamine secretion in both short term (20-min) and long term (24-h) experiments. Both constitutive and Ag-induced TNF-alpha production by PMC are dose-dependently inhibited by IL-10, whereas they are not affected by SCF at all doses tested (2-500 ng/ml) and incubation times observed (3-24 h). Interestingly, rat PMC constitutively express IL-10 mRNA and proteins, as tested by reverse transcription-PCR and immunocytochemistry. In addition, it was found by flow cytometry that 23.5% PMC express surface IL-10. Moreover, treatment of PMC with anti-IL-10 Ab for 6 h significantly potentiates constitutive and Ag-induced TNF-alpha production by rat PMC. Thus, IL-10 and SCF exert different regulatory effects on MC secretory function. IL-10 produced by MC has the potential to regulate MC function in an autocrine manner, an effect that may be highly relevant to responses involving MC activation.

Animals↗
PubMed1997

Determination of cell surface charge by photometric titration.

A colloid titration method has been frequently used to determine the number of charged residues at the cell surface. Here we present a new version of this technique, based on photometric measurements of a metachromatic shift in the maximum absorption of toluidine blue as it binds to the cell surface. The major improvements are: (1) simplified methodology and (2) increased precision of equivalence point determination. The data are analyzed using Gran's theory, which allows measurements to be taken at regular intervals instead of being concentrated around the equivalence titration point. We used this method to characterize the cell surface charge of three populations of rat mast cells: (1) peritoneal mast cells (PMC), (2) bone marrow-derived mast cells (BMMC) and (3) a rat cultured mast cell line (RCMC). Our results indicate that PMC have (4.23 +/- 0.59) x 10(8), while BMMC (8.58 +/- 0.26) x 10(7) negatively charged residues per cell. The results for RCMC were similar to those for BMMC. Taking into account the size differences between PMC and BMMC, the average charge density of PMC was also significantly higher than that of BMMC. The differences in cell surface charge were analyzed in the light of different sensitivities of mast cells to polycationic secretagogues.

Animals↗
PubMed1997

Optimization of the isolation and effective use of mRNA from rat mast cells.

To define the molecular regulation of mast cell phenotype and function optimized procedures must be available to study mRNA from mast cells freshly isolated from tissues. However, rat peritoneal mast cells (PMC) contain large amounts of the proteoglycan heparin, and unfortunately, this molecule which is a potent inhibitor of reverse transcriptase (RT) and Taq polymerase and thus RT-PCR, copurifies with RNA. Here we describe an optimized protocol for extracting and amplifying RNA from rat PMC. Mast cells were isolated from rat peritoneum and a method modified from that of Chomczynski and Sacchi (1987) was used to extract the RNA. Following the removal of heparin by heparinase digestion, first strand cDNA synthesis was primed with oligo-dT and the resulting cDNA was quantified by rapid paper chromatography. The use of a detection system for the reverse transcription reaction ensured that the production of cDNA had occurred and allowed subsequent PCR testing to be optimal. cDNA thus produced can be used to detect relatively specific (histidine decarboxylase) and non-specific (beta-actin) mast cell products. Our PCR studies have shown a 300-fold increase in sensitivity over RNA processed by other methods.

Animals↗
PubMed1997

A novel CD8 molecule expressed by alveolar and peritoneal macrophages stimulates nitric oxide production.

Macrophages play an essential role in host defense, and we have identified a novel CD8 molecule, on alveolar and peritoneal macrophages, that may be involved in regulating this function. Flow cytometric analysis of bronchoalveolar lavage from normal rats identified a large number of CD8-positive cells that could not be accounted for by T lymphocytes. Within the scatter profile region in which the majority of cells were alveolar macrophages (OX41; 89 +/- 1%), 63 +/- 5% of the cells stained positively for CD8alpha (OX8) and 52 +/- 3% for CD8beta (341). Double-staining of lavage cells confirmed the presence of CD8 on alveolar macrophages. Interestingly, flow cytometry showed differences between CD8 on alveolar macrophages and on T lymphocytes within the ligand binding domain for MHC class I. Reverse transcription-PCR analysis on FACS-enriched alveolar macrophages showed the presence of CD8alpha mRNA, determining that macrophages synthesize CD8. Further studies identified both the alpha (49 +/- 8%)- and beta (37 +/- 4%)-chains of CD8 on peritoneal lavage cells (86 +/- 3% macrophages (OX42, CD11b)). As with alveolar macrophages, there were differences within the ligand-binding domain of CD8 on peritoneal macrophages compared with T lymphocytes. Functional studies determined that anti-CD8alpha (OX8) stimulated a dose-dependent release of nitric oxide, indicating that CD8 can directly regulate macrophage function. Thus, macrophages express an unusual CD8 molecule that differs within its ligand-binding domain, compared with T lymphocytes, and these findings suggest hitherto unknown ligand(s) for CD8. These findings will lead to a greater understanding of macrophage function and regulation.

Animals↗
PubMed1997

Anti-inflammatory effect of beta 2-agonists: inhibition of TNF-alpha release from human mast cells.

Beta 2-agonists inhibit the release of preformed mediators such as histamine and newly synthesized mediators such as prostaglandin D2 from mast cells. However, although mast cells have been identified as an important source of several cytokines including tumor necrosis factor-alpha (TNF-alpha), there is no information about their regulation by beta 2-agonists. Thus given the importance of TNF-alpha in inflammation and the widespread use of beta 2-agonists, we investigated the effect of long-acting (salmeterol) and short-acting (salbutamol) beta 2-agonists on the secretion of TNF-alpha from human skin mast cells. Treatment of mast cells with salmeterol or salbutamol (100 nmol/L) inhibited the IgE-dependent release of TNF-alpha (82% and 74%, respectively). Moreover, 2-hour treatment with salmeterol, isoproterenol, or salbutamol inhibited mast cell cytotoxicity against a TNF-alpha-sensitive cell line, WEHI-164, with an IC50 of 71, 50, and 29 nmol/L, respectively. Specificity for beta-adrenergic receptors was shown with propranolol. The inhibitory effect of beta 2-agonists was observed after only 20 minutes of treatment but was lost by 24 hours after removal of salbutamol and isoproterenol (7% and 11% inhibition remaining, respectively). In contrast, the inhibition of TNF-alpha release was increased 1 hour after removal of salmeterol and remained significant 24 hours later. Furthermore, beta 2-agonists did not show tachyphylaxis for the inhibition of TNF-alpha release. Thus selective beta2-agonists demonstrate anti-inflammatory activity by inhibiting the release of TNF-alpha from mast cells stimulated through their IgE receptor or by a tumor target cell. This inhibitory effect of beta-agonists may be important in their mode of action in the treatment of allergic diseases.

Adrenergic beta-Agonists↗
PubMed1997

A novel submandibular gland peptide protects against endotoxic and anaphylactic shock.

Submandibular glands release peptides and proteins that, through exocrine and endocrine actions, facilitate tissue repair in the oral cavity, gastrointestinal tract, and more distal sites such as liver. It has been shown that salivary gland factors also modulate inflammatory responses, because we found that removal of the submandibular glands increases the hypotensive responses to endotoxin. From this observation we proposed that these glands contain a factor that regulates cardiovascular response to shock. With the use of classical peptide isolation procedures, a heptapeptide (TDIFEGG) called submandibular gland peptide T was identified in rat submandibular glands. A synthetic form of this peptide reduced endotoxic shock in sialadenectomized rats by 50% at doses as low as 1 microgram/kg and prevented allergen-induced hypotension by 90% in rats with intact salivary glands at a dose of 100 micrograms/kg. This novel peptide is probably generated from a prohormone, submandibular gland rat 1 protein, a product of the VCSA1 gene. These data indicate that submandibular glands participate in the regulation of systemic homeostasis.

Amino Acid Sequence↗
PubMed1997

TGF-beta1 inhibits the release of histamine and tumor necrosis factor-alpha from mast cells through an autocrine pathway.

Transforming growth factor beta1 (TGF-beta1) is a member of a gene superfamily involved in the regulation of cell growth and differentiation, tissue repair, fibrosis, and inflammatory responses. Given the role of the mast cell (MC) in inflammation and fibrosis, the effect of TGF-beta1 on MC mediator release was studied. In vitro treatment of rat peritoneal MC (PMC) with TGF-beta1 (10(-10) M) for 20 h followed by washes inhibited (23%) antigen stimulated histamine release. Similar pretreatment of PMC with TGF-beta1 (10(-10) M) inhibited (27%) tumor necrosis factor-alpha (TNF-alpha) dependent cytotoxicity and reduced (31%) mRNA levels of TNF-alpha, but did not inhibit nitric oxide (NO) release. By contrast, the presence of TGF-beta1 throughout the cytotoxic assay, but without pretreatment of PMC did not modulate TNF-alpha release. At least 2 h pretreatment with TGF-beta1 was required to inhibit MC TNF-alpha-dependent cytotoxicity. This inhibitory effect of TGF-beta1 was abrogated by antibody to TGF-beta1. Interestingly, the treatment of PMC with anti-TGF-beta1 antibody alone significantly increased the release of histamine and TNF-alpha. Furthermore, freshly isolated rat PMC (10(7)) contained 35 +/- 7 pg latent TGF-beta1 and 51 +/- 9 pg was spontaneously released within 30 min of culture. However, stimulation of PMC with antigen inhibited the spontaneous release of TGF-beta1 by 43%. The duration of pretreatment with TGF-beta1 required to inhibit MC TNF-alpha release was similar to that required for downregulation of MC TNF-alpha-dependent cytotoxicity by IFN-gamma. TGF-beta1 and IFN-gamma had an additive inhibition on TNF-alpha release by PMC. This inhibitory effect was abrogated and TNF-alpha-dependent cytotoxicity was enhanced by the addition of anti-TGF-beta1 antibody, but not by anti-IFN-gamma. These results suggest MC mediator release is regulated by TGF-beta1 in an autocrine manner.

Animals↗
PubMed1996

Inhibitory effects of sulfasalazine and its metabolites on histamine release and TNF-alpha production by mast cells.

Sulfasalazine is an effective treatment in some inflammatory diseases that exhibit mast cell (MC) hyperplasia. However, its effect on MC has been incompletely studied. We have established that sulfasalazine inhibits the release of histamine and TNF-alpha from MC. Sulfasalazine and its metabolites, 5-aminosalicylic acid (5-ASA) and to a lesser extent sulfapyridine, inhibited Ag-stimulated histamine release from rat peritoneal MC in a concentration-dependent manner with a 50% inhibitory concentration of 6 x 10(6)M, 8 x 10(-6)M, and 3 x 10(-4)M, respectively. Similar results were observed with sulfapyridine and 5-ASA on Ag-stimulated histamine release of another population of MC, namely rat intestinal mucosal MC, but sulfasalazine was markedly less potent than its metabolites. Interestingly, sulfasalazine and sulfapyridine, but not 5-ASA, inhibited Ag-stimulated TNF-alpha released by MC. Similar results were observed with MC-mediated cytotoxic activity in which sulfasalazine and sulfapyridine, but nor 5-ASA, inhibited MC TNF-alpha-dependent cytotoxicity in a concentration-dependent manner. The addition of sulfasalazine to MC, up to 12 h after the cytotoxic assay (16 h) had started, significantly inhibited cytotoxic activity, suggesting that sulfasalazine inhibited the cytotoxic mediator, TNF-alpha. Indeed, affinity studies demonstrated that sulfasalazine binds TNF-alpha. Furthermore, the inhibition of MC cytotoxicity by sulfasalazine appeared to require new protein synthesis. Pretreatment of MC with sulfasalazine also inhibited the release of TNF-alpha and reduced the levels of TNF-alpha mRNA. Thus, sulfasalazine inhibits MC-mediated, TNF-alpha-dependent cytotoxicity by multiple mechanisms: competitive inhibition of soluble TNF-alpha, reduction of levels of TNF-alpha mRNA, and inhibition of TNF-alpha release.

Animals↗
PubMed1996

Stem cell factor potentiates histamine secretion by multiple mechanisms, but does not affect tumour necrosis factor-alpha release from rat mast cells.

The effect of stem cell factor (SCF) on histamine and tumour necrosis factor-alpha (TNF-alpha) release from rat peritoneal mast cells (PMC) was determined and the intracellular pathways involved in the potentiation of histamine secretion were investigated. The effects of SCF (2-100 ng/ml) were examined following both short-term (0 and 20 min) and long-term (up to 24hr) preincubations with SCF. Pretreatment of PMC with SCF for 0 min (concurrent) or 20 min did not induce histamine secretion directly, but significantly increased antigen (Ag)-induced histamine secretion. SCF potentiated Ag-induced intracellular Ca2+ increase and calcium ionophore A23187-induced histamine secretion. Pertussis toxin (PT) inhibited SCF-induced potentiation of IgE-dependent histamine secretion, indicating that PT-sensitive G-proteins are involved in the immediate effects of SCF. In long-term incubation experiments, SCF pretreatment for 18-24 hr significantly enhanced Ag-induced histamine secretion, but did not affect Ag-induced intracellular Ca2+ levels. The effects of long-term incubation with SCF, but not the short-term effects, were blocked by cycloheximide. Interestingly, spontaneous and Ag-induced TNF-alpha release from rat PMC were not affected by pretreatment with SCF (2-500 ng/ml) for 1 to 24 hr. Thus, through immediate and delayed mechanisms, SCF potentiates histamine release from PMC, but has not effect on TNF-alpha release. The regulation of MC by SCF may be important in allergic and other inflammatory diseases.

Animals↗
PubMed1996

Regulation of mRNA levels of TNF-alpha and the alpha chain of the high-affinity receptor for IgE in mast cells by IFN-gamma and alpha/beta.

Rat mast cell lines (hybrid rat mast cells, HRMC, and rat cultured mast cells, RCMC) and mast cells from the rat body cavity were used to test the hypothesis that IFN-alpha/beta and IFN-gamma inhibit tumor necrosis factor alpha (TNF-alpha)-mediated cytotoxicity by depressing the steady-state levels of mRNA for TNF-alpha. In vitro treatment of mast cells with IFN-gamma and IFN-alpha/beta depressed mRNA levels. By contrast, IFN pretreatment of mast cell lines induced an increase in levels of mRNA for the IFN-inducible gene, 2'5'-oligoadenylate synthetase and also for high-affinity IgE-receptor-alpha. The IFN-mediated regulation of mast cells may be an important mechanism in the control of inflammatory pathways characterized by Th1- and Th2-type responses.

Animals↗
PubMed1995

Proteinases of rat mast cells. Peritoneal but not intestinal mucosal mast cells express mast cell proteinase 5 and carboxypeptidase A.

Six basic proteins of 26 to 38 kDa with isoelectric points (pI) > or = 8.5 were abundant in proteins separated by two-dimensional SDS-PAGE from adult rat peritoneal mast cells (MC). One was identified previously as rat mast cell proteinase (RMCP) 1, a chymase of 26 to 28 kDa, pI > 9.0. Microsequence analyses showed that two polypeptides of about 29 and 30 kDa had NH2 terminal amino acid sequences homologous to mouse MC proteinase 5 (MCP-5), whereas the amino terminals of the 33, 35, and 36 kDa proteins were homologous to MC carboxypeptidase A (MC-CPA). Rabbit Abs produced against synthetic peptides of the identified NH2 terminal sequences were used in immunoblot studies. At least three proteins reacted with Abs to MC-CPA, whereas Abs to MCP-5 detected three adjacent polypeptides, rather than just the two identified by using microsequence analysis. Removal of oligosaccharide side chains using peptide:N-glycosidase F reduced the heterogeneity of each set of three polypeptides (MCP-5 and MC-CPA) to a band of each protein of a lower M(r). The serine proteinase inhibitor [3H]diisopropylfluorophosphate ([3H]DFP) bound to a proteinase of 30 to 35 kDa, which is probably MC tryptase (pI < or = 6.0). Immunoblot analysis of proteins from intestinal mucosal mast cells showed RMCP-2, but not RMCP-1, MCP-5, or MC-CPA. This is the first report of MCP-5 in the rat and of clearly distinguishable glycosylated forms of MC CPA. These proteinases appear to be restricted in their distribution to selected MC populations, but little is known about their functions.

Amidohydrolases↗