Regulation of tryptase from human lung mast cells by heparin. Stabilization of the active tetramer

Tryptase was shown to be stabilized as an enzymatically active tetramer by association with heparin and dissociated to inactive monomers in the absence of heparin at 37 degrees C in physiologic buffer and in plasma. There was a 50% loss of tryptase activity at 37 degrees C by 6-8 min in both physiologic buffer and plasma. When heparin glycosaminoglycan was present, tryptase retained nearly full activity for 2 h in buffer and in plasma. Tryptase activity also decayed under standard assay conditions in the presence of synthetic ester and peptide substrates unless bound to heparin. That tryptase is bound to heparin at the pH and physiologic NaCl concentrations employed was shown by chromatography of tryptase on heparin-agarose, gel filtration, and velocity sedimentation. Elution of tryptase from heparin-agarose occurred at 0.8 M NaCl. Maximal stabilization of tryptase by heparin occurred at a weight ratio to tryptase that was equal to or greater than unity. Kcat/Km ratios for tryptase-heparin at 0.15 M NaCl and 37 degrees C were 0.9 X 10(6) s-1 M-1 for tosyl-L-Gly-Pro-Lys-p-nitroanilide and 1.7 X 10(6) s-1 M-1 for p-tosyl-L-arginine methyl ester and are among the highest reported for tryptic enzymes. The mechanism of heparin-dependent stabilization of tryptase was not due to indirect ion binding properties of heparin and was analyzed by Superose 12 high performance liquid chromatography. Active enzyme eluted with an apparent Mr of 132,000 +/- 10,000 (n = 3, +/- S.D.), whereas tryptase inactivated by incubation without heparin eluted with an apparent Mr of 34,000. The tetrameric structure of diisopropyl fluorophosphate-inhibited tryptase was also preserved after incubation with heparin at 37 degrees C but was reduced to monomeric subunits after incubation without heparin. That no appreciable degradation of tryptase occurs under conditions that cause dissociation of subunits was directly shown by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Two different subunits of 34,000 and 33,000 Mr (after reduction) present in the intact enzyme (calculated to be 134,000 Mr) were also detected unchanged after inactivation of tryptase by dissociation of its subunits. Thus, the selective localization and association of heparin and tryptase in the human mast cell secretory granule most likely plays a major role in the regulation of tryptase after secretion.     

Structural studies on a family of cAMP-binding proteins in the nervous system of Aplysia

Five major cAMP-binding proteins that differ in size and charge have been identified in neurons of Aplysia californica by photoaffinity labeling with [32P]8-N3cAMP. These proteins, which we believe are regulatory subunits of cAMP-dependent protein kinase, all differ from the major cAMP-binding protein of buccal muscle. We have compared the structures of these proteins by peptide mapping after chemical and proteolytic cleavage. These analyses indicate that the five binding proteins from nervous tissue and the major muscle protein are closely related to each other. For example, the three neuronal proteins that are most alike and the cAMP-binding protein from muscle have a similar, if not identical, Mr 20,000 domain that contains the 8-N3cAMP-binding site; beyond this domain they diverge. All six proteins appear to belong to a family in which homologous regions have been conserved to maintain common functions. We suggest that the regions of the molecules that differ mediate special functions such as ticketing to particular compartments of the cell. Evidence for regional assortment of the cAMP-dependent protein kinases according to structural type was afforded by subcellular fractionation of Aplysia nervous tissue; photoaffinity labeling of cytoplasm, cytoskeleton, and membrane fractions demonstrated a differential distribution of the five neuronal cAMP-binding proteins. Selective phosphorylation of specific substrates could be a consequence of the compartmentation of diverse cAMP-dependent kinases.     

The effect of tryptase from human mast cells on human prekallikrein

Tryptase, the dominant protease in human mast cells, was examined for its effect on human prekallikrein. Tryptase in the presence and absence of heparin failed to activate prekallikrein as shown in a spectrophotometric assay for kallikrein employing benzoy 1-pro-phe-arg-p-nitroanilide. Treated prekallikrein was converted to active kallikrein by bovine trypsin. Prekallikrein cleavage products were analyzed by electrophoresis in polyacrylamide gels under denaturing conditions (+/- reduction). Tryptase caused no apparent cleavage under conditions where trypsin caused complete cleavage. Thus, tryptase, which has previously been shown to lack kallikrein and kininase activities, neither activates nor destroys prekallikrein.     

Release of atriopeptin in the rat by vasoconstrictors or water immersion correlates with changes in right atrial pressure

Vasopressin, its 1-deamino analog (dAVP), angiotensin II, and phenylephrine, administered intravenously, increased plasma atriopeptin immunoreactivity in chloral hydrate-anesthetized rats. A continuous one hour infusion of either dAVP or phenylephrine caused a sustained elevation in: a) systemic blood pressure; b) right atrial pressure; c) left ventricular end diastolic pressure; and d) plasma atriopeptin immunoreactivity. While continuous infusion of angiotensin II also produced a sustained elevation in left ventricular end diastolic pressure, the changes in right atrial pressure and plasma atriopeptin were only transient. These data suggest that plasma atriopeptin most closely correlates with right atrial pressure. Consistent with this hypothesis, we found that the release of atriopeptin directly correlated with changes in right atrial pressure in anesthetized, water-immersed rats.     

Effect of hyaluronidase treatment of intact cells on hyaluronate synthetase activity

Hyaluronidase treatment of mouse oligodendroglioma cells in monolayer culture resulted in a 4-5-fold stimulation of hyaluronate synthetase, assayed in washed membrane preparations [Philipson, L., & Schwartz, N. B. (1984) J. Biol. Chem. 259, 5017-5023]. We now report studies on the mechanism of the hyaluronidase-induced increase in the specific activity of the membrane-bound synthetase complex. The stimulation was dependent on the concentration of hyaluronidase but not on the particular bond cleaved or the nature of the product generated. Analysis of chain growth during cell-free synthesis by the disaccharide ratio method suggested that substantial internal labeling of hyaluronate chains had occurred. With both treated and untreated membranes, greater than 90% of incorporated (and recovered) radioactivity appeared in unsaturated disaccharides. Further analysis showed that hyaluronidase treatment increased both the rate of elongation and the rate of release of elongated chains from the enzyme complex. Hyaluronidase treatment also caused a change in the apparent steady-state kinetic patterns of double-reciprocal plots from intersecting lines for membranes from control cells to a family of parallel lines. Both the overall stimulation of synthesis and the change in apparent kinetic pattern were reversed by brief incubation of washed cells in the absence of hyaluronidase. These results have led to the development of an explicit kinetic model for hyaluronate synthesis which suggests an explanation for the switch in apparent kinetic patterns based on changing concentrations of a postulated key intermediate.     

Reduction potential of iron in transferrin

The reduction potential of Fe3+ in transferrin was measured spectrophotometrically by equilibration with methyl viologen in the presence of sodium dithionite. For an ionic strength near 0.1 M at 25 degrees C and pH 7.3 under 0.048 atm. CO2, half of the iron is reduced at a potential near -0.40 V (vs. standard hydrogen electrode). At least one disulfide bond of the protein is partially reduced at a potential of -0.44 V, as evidenced by reaction with [14C]iodoacetate.     

Cervical intraepithelial neoplasia and associated condylomatous lesions. A preliminary report on 4,764 women from Northern Israel

During the period spanning the years 1973 to 1981, 4,764 women visited the Gynecology Out-Patient Clinics and Colposcopy Unit of the Nahariyya Hospital to be examined colposcopically and cytologically (and histologically whenever indicated) for precancerous and cancerous lesions of the cervix. Of these women, 2,614 (55%) were referred because of symptoms of cervical pathology and 2,150 (45%) for other (prophylactic) reasons. The subdivision of all women according to their demographic backgrounds afforded a comparison of the findings in Israeli-born Jewesses with those of foreign-born Jewesses and non-Jewish females living in the same geographic area of the Western Galilee district of Israel. Despite the low prevalence of cervical cancer in Jewesses throughout the world, the preliminary report of our pilot study demonstrated that the percentage rates of all degrees of dysplasia/cervical intraepithelial neoplasia (CIN I, II and III) of the uterine cervix of Israeli-born Jewesses was 5.4% in patients with cervical pathology and 3.24% in noncervical-pathology patients. These rates were the highest recorded for any of the demographic groups: 2.06% and 0.33%, respectively, in Moslem women; 1.23% in Christian women with cervical pathology; 2.38% and 1.78%, respectively, in European/American-born Jewesses; and 1.63% and 0.48%, respectively, in Asian/African-born Jewesses. The highest proportion of CIN lesions occurred in the 15- to 30-year-old age groups. Of 100 CIN lesions found in all patients, 45 were cytohistologically associated with the cells of condylomatous lesions. Of 36 patients in whom cervical squamous-cell carcinoma lesions were detected, 18 (50%) were staged (FIGO) as carcinoma in situ (stage 0); the remainder were in stages IA, IB, IIA and IIB, with none in stages III or IV.     

Comparison of dipeptidyl carboxypeptidase and endopeptidase activities in the three enkephalin-hydrolysing metallopeptidases: "angiotensin-converting enzyme", thermolysin and "enkephalinase"

Angiotensin-converting enzyme (ACE), thermolysin and "enkephalinase", three metallopeptidases cleaving the Gly3-Phe4 amide bond of enkephalins, were compared regarding substrate specificity and effects of butanedione, an arginyl-directed reagent. The hydrolysis of enkephalins and analogues was more affected by the nature of P1 and P2 residues in the case of thermolysin than in those of ACE or "enkephalinase"; amidation of the C-terminal carboxylate decreased drastically the hydrolysis by ACE but only marginally by thermolysin and the effect was intermediate for "enkephalinase". With adequate model substrates, the ratio of dipeptidylcarboxypeptidase to tripeptidylcaroxypeptidase (endopeptidase) activities were of 25 for ACE, 3 for "enkephalinase" and only 0.3 for thermolysin. Finally a butanedione treatment increased thermolysin activity, but abolished ACE activity; it reduced "enkephalinase" activity by 80% when measured with a free C-terminal carboxylate enkephalin analogue but only slightly with the corresponding amidated derivative. A critical role of an Arg residue in ACE and, to a lesser extent, in "enkephalinase" (but not in thermolysin) is suggested to be responsible for the preferential dipeptidylcarboxypeptidase activity of these two enzymes.     

Effect of lysosomotropic amines on the secretory pathway and on the recycling of the asialoglycoprotein receptor in human hepatoma cells

We studied the intracellular transport of secretory and membrane proteins in the human hepatoma cell line HepG-2 infected with vesicular stomatitis virus. Cells were pulse-labeled in the presence of [35S]methionine and chased in the presence of the lysosomotropic agent primaquine. At a concentration of 0.3 mM primaquine effectively inhibited the secretion of albumin and, to a lesser extent, that of orosomucoid and transferrin. The drug also prevented the budding of virus particles at the cell surface. The intracellular transport to the Golgi complex of the membrane protein VSV-G was not affected by primaquine as it acquires resistance to endo-beta-N-acetylglucosaminidase H at the same rate as in control cells. Addition of primaquine at various times after the initiation of the chase period indicates that the effect of primaquine occurs just before secretion. In confirmation of the biochemical data, immunocytochemical localization of albumin in cells treated with NH4Cl demonstrated that albumin accumulated in vesicles at the trans side of the Golgi complex. The effect of primaquine on secretion was also compared with its effect on receptor recycling. The dose-response characteristics of the effect of primaquine on receptor recycling are identical to those of the effects on protein secretion and virus budding. These results indicate that both processes involve the same transport mechanism, and/or that they occur via at least one identical intracellular compartment.     

Lupus prone (SWR x NZB)F1 mice produce potentially nephritogenic autoantibodies inherited from the normal SWR parent

The incidence of nephritis in autoimmune NZB mice is low, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. To define the contribution of the normal SWR strain to the development of nephritis, we analyzed 65 monoclonal anti-DNA autoantibodies derived from SNF1 mice and compared them with those obtained from the NZB parent. The majority of the SNF1-derived anti-DNA antibodies were IgG and cationic in charge. By contrast, 77% of the NZB-derived antibodies were IgM. Moreover, all three NZB-derived IgG anti-DNA antibodies were anionic. The cationic property of the SNF1-derived IgG autoantibodies was not restricted to any particular antigenic specificity pattern or IgG subclass, nor was there a preference for the allotype of either parent. However, we identified a set of highly cationic (pI at 8.2 to 8.8 pH) IgG2b anti-DNA antibodies from SNF1 hybrids that had the SWR allotype. Isoelectric focusing of intact antibodies and isolated heavy and light chains showed that the highly cationic charge of these antibodies was determined by the variable regions of their heavy chains. Because IgG anti-DNA antibodies with cationic charge are especially pathogenic, those antibodies bearing the allotype of the normal SWR parent may account for the high incidence of severe nephritis in the F1 hybrids. The results indicate that pathogenic autoantibodies, which are encoded by genes of the nonautoimmune SWR parent, are expressed in the SNF1 mice due to some cellular and genetic regulatory influence of the NZB parent.