Pepsin reacts stoicheiometrically with the active-site-directed irreversible inhibitor N-diazoacetyl-l-phenylalanine methyl ester, with concomitant loss of all proteolytic and peptidolytic activity. The reagent esterifies a unique aspartic acid residue in pepsin, which is in the sequence:Ile-Val-Asp-Thr-Gly-Thr-Ser

The total sequence of 365 amino acid residues in bovine prochymosin is presented. Alignment with the amino acid sequence of porcine pepsinogen shows that 204 amino acid residues are common to the two zymogens. Further comparison and alignment with the amino acid sequence of penicillopepsin shows that 66 residues are located at identical positions in all three proteases. The three enzymes belong to a large group of proteases with two aspartate residues in the active center. This group forms a family derived from one common ancestor.Chymosin (EC 3.4.23.4) is the major proteolytic enzyme in the stomach of the preruminant calf. Like pepsin, chymosin belongs to a group of acidic proteases in which two aspartate residues participate in the catalytic mechanism (1, 2). In analogy with the term serine proteases, the term ispartate proteases has been suggested (3).Chymosin is secreted as an inactive precursor, prochymosin, consisting of a single peptide chain with 365 amino acid residues. The zymogen is irreversibly converted into active enzyme by limited proteolysis during which a total of 42 amino acid residues are released from the amino-terminal part of the peptide chain.Prochymosin contains six half-cystine residues forming three disulfide bridges, all located in the enzyme part of the molecule. Investigations on the primary structure began with determination of the amino acid sequences around the disulfide bridges (4). In subsequent papers (5, 6) we reported the sequence of the activation segment and the sequence of the first 61 residues of the active enzyme.This communication presents the complete amino acid sequence of prochymosin B. It is shown that this zymogen is highly homologous to porcine pepsinogen A (7-10). To facilitate the comparison, we have chosen to number the amino acid residues from the NH2-terminus of prochymosin and then continue by counting gaps where such occur in prochymosin relative to pepsinogen. The comparison is further extended to penicillopepsin, the only other aspartate protease of which the sequence is almost completely known (11). METHODSProchymosin and chymosin used in these investigations were prepared in our laboratory according to the methods described previously (12).The sequence was obtained after a series of degradation experiments carried out in parallel. In most cases the first steps were enzymatic cleavage with trypsin or chemical cleavage with cyanogen bromide. To improve solubility of the digest and to reduce the number of fragments, we used maleylated or citraconylated preparations for the digestions with trypsin. The digestions were carried out at 120 (pH 8, for 15-30 order to avoid unspecific, chymotrypsin-like cleavages (13). After such treatment the large fragments were purified by gel filtration on Sephadex G-100 in 0.05 M NH4HCO3, pH 8, with 8 M urea. After cleavage of chymosin with cyanogen bromide the fragments were purified by gel filtration on Sephadex G-100 in 25% acetic acid. The best results were obtained if cleavage was performed on enzyme with inta...

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Stimulation of Ehrlich ascites tumor cells with leukotriene D4 (LTD4) within the concentration range 1-100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca2+ concentration, [Ca2+]i. The Ca2+ peak time, i.e., the time between addition of LTD4 and the highest measured [Ca2+]i value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration of LTD4 (100 nm), the highest measured increase in [Ca2+]i in Ehrlich cells suspended in Ca2+-containing medium is 260 +/- 14 nm and the EC50 value for LTD4-induced Ca2+ mobilization is estimated at 10 nM. Neither the peptido-leukotrienes LTC4 and LTE4 nor LTB4 are able to mimic or block the LTD4-induced Ca2+ mobilization, hence the receptor is specific for LTD4. Removal of Ca2+ from the experimental buffer significantly reduces the size of the LTD4-induced increase in [Ca2+]i. Furthermore, depletion of the intracellular Ins(1,4,5)P3-sensitive Ca2+ stores by addition of the ER-Ca2+-ATPase inhibitor thapsigargin also reduces the size of the LTD4-induced increase in [Ca2+]i in Ehrlich cells suspended in Ca2+-containing medium, and completely abolishes the LTD4-induced increase in [Ca2+]i in Ehrlich cells suspended in Ca2+-free medium containing EGTA. Thus, the LTD4-induced increase in [Ca2+]i in Ehrlich cells involves an influx of Ca2+ from the extracellular compartment as well as a release of Ca2+ from intracellular Ins(1,4,5)P3-sensitive stores. The Ca2+ peak times for the LTD4-induced Ca2+ influx and for the LTD4-induced Ca2+ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35 min in six out of eight experiments, respectively. Stimulation with LTD4 also induces a transient increase in Ins(1,4, 5)P3 generation in the Ehrlich cells, and the Ins(1,4,5)P3 peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4, 5)P3 content seems to increase before the LTD4-induced Ca2+ release from the intracellular stores but after the LTD4-induced Ca2+ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD4-induced increase in Ins(1,4,5)P3 as well as the LTD4-induced increase in [Ca2+]i, indicating that a U73122-sensitivity phospholipase C is involved in the LTD4-induced Ca2+ mobilization in Ehrlich cells. The LTD4-induced Ca2+ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD4-activated Ca2+ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion, LTD4 acts in the Ehrlich cells via a specific receptor for LTD4, which upon stimulation initiates an influx of Ca2+, through yet unidentified Ca2+ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P3 formation and finally release of Ca2+ from the intracellular Ins(1,4,5)P3-sensitive stores.