Search

The chromatin fraction was prepared from yeast Saccharomyces cerevisiae free from cytoplasmic contamination except for a trace of mitochondria. When the yeast chromatin was incubated with histones as a substrate it showed three peaks of proteolytic activity as approximately pH 4, pH 7 and pH 11. These activities were separated from each other by differential extractions from chromatin and successive gel filtration through Sephadex G-100. Proteases were partially characterized by affinity labeling with [3H]diisopropylfluorophosphate (iPr2P-F) and by various protease inhibitors. The neutral and the alkaline proteases were serine proteases with a molecular mass of 35 kDa and 25 kDa respectively. The acidic protease showed a molecular size larger than 100 kDa on the gel filtration, and was probably an aspartyl protease because it was most strongly inhibited by pepstatin. A iPr2P-F-binding protein with a molecular mass of 66 kDa, found in chromatin, was likely to be converted to the alkaline protease of 25 kDa when chromatin was incubated at pH 10 or in 6 M urea/0.1 M phosphoric acid at the extraction. The distribution of proteolytic activities and iPr2P-F-binding proteins were compared among chromatins from different strains and from cells in different growth phases and it was found that these three proteases were present in all of them but with different proportions. Considering that rat liver chromatin contains equivalents to these proteases [Tsurugi, K. and Ogata, K. (1982) J. Biochem. (Tokyo) 92, 1369-1381], the results suggested that they play some important roles in the function of eukaryotic chromatin.

Secretory granules of the rat basophilic leukemia (RBL-1) cell, a chemically generated tumor cell line maintained in tissue culture, were shown to stain with alcian blue but not with safranin counterstain and to have sparse, small, electron-dense granules. A Mr 25,000 protein was the major [3H]diisopropyl fluorophosphate-binding protein in extracts of RBL-1 cells. Double-immunodiffusion analysis of extracts revealed immunoreactivity for rat mast cell protease (RMCP)-II, a Mr 25,000 neutral protease present in the secretory granules of rat mucosal mast cells and cultured rat bone marrow-derived mast cells, but no immunoreactivity for RMCP-I, the predominant neutral protease of rat connective tissue mast cells. By radial immunodiffusion, there was 66.8 ng of RMCP-II per 10(6) cells. Whereas rat connective tissue mast cells stain with alcian blue and safranin and contain heparin proteoglycan, rat mucosal and rat bone marrow-derived mast cells stain with alcian blue only and contain a non-heparin proteoglycan and lesser amounts of histamine. Proliferation of rat mucosal mast cells in vivo and rat bone marrow-derived mast cells in vitro requires T-cell factors, whereas no comparable requirement has been observed for connective tissue mast cells. The transformed RBL-1 tumor cells, whose growth is independent of factors other than those present in standard tissue culture medium, has previously been shown to contain predominantly chondroitin sulfate di-B proteoglycans and low amounts of histamine. The similar histology and secretory granule biochemistry of the rat mucosal mast cells, rat culture-derived mast cell, and RBL-1 cell suggest that they comprise a single mast cell subclass distinct from the rat connective tissue mast cell.

Atrial natriuretic factor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, but circulates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. It has previously been shown that proANF is secreted intact from cultured atrial cardiocytes and can be cleaved by a serum protease to smaller, 3-kDa peptides believed to be the circulating forms. This report describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of 35S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Cleavage in serum was not dependent on the presence of platelets or other cellular elements. Complete inhibition of proANF cleavage was obtained with the protease inhibitors benzamidine, leupeptin, phenylmethanesulfonyl fluoride, and diisopropyl fluorophosphate (DFP) but not with aprotinin, soybean trypsin inhibitor, pepstatin, or hirudin. Unlike the vitamin K dependent plasma proteins, the proANF-cleaving protease did not adsorb to barium sulfate. With the sequential application of ion-exchange, hydroxylapatite, lectin affinity, and gel filtration chromatography, a 5000-6000-fold purification of the enzyme from rat serum was achieved. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A.(ABSTRACT TRUNCATED AT 250 WORDS)

The experiments described in this paper were designed to isolate [3H]di-isopropyl phosphorofluoridate-binding proteins by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis for the purpose of characterizing and identifying potential initiation sites for organophosphorus-compound-induced delayed neurotoxicity. The major Paraoxon-insensitive Mipafox-sensitive binding protein (Mr 160 000) was found to be identical with one previously identified as neurotoxic esterase, an enzyme that has been proposed to be the target site for organophosphorus-compound-induced delayed neurotoxicity. However, two other binding proteins with suitable binding characteristics were also found in smaller amounts, one of which has not been detected previously. Di-isopropyl phosphorofluoridate was found to phosphorylate all three of these proteins at rates similar to the rate at which neurotoxic esterase is inhibited by di-isopropyl phosphorofluoridate. Varying the concentration of di-isopropyl phosphorofluoridate or the time of incubation produced similar increases in binding to each of the labelled proteins. This suggests that the reaction rates of di-isopropyl phosphorofluoridate with proteins may be described by first-order kinetics, and the concentration of the Michael is complex formed during binding is minimal for all the phosphorylated proteins. The recovery of the binding activity in the 160 000-Mr band was found to be similar to the recovery of neurotoxic esterase activity, lending further support to the contention that this band is identical with neurotoxic esterase.

The influenza C virus (INF-C) hemagglutinin recognizes 9-O-acetyl-N-acetylneuraminic acid. The same protein contains the receptor-destroying enzyme (RDE), which is a 9-O-acetyl-esterase. The RDE was inactivated by the serine esterase inhibitor di-isopropyl fluorophosphate (DFP). [3H]DFP-labeling localized the active site to the heavy chain of the glycoprotein. DFP did not alter the hemagglutination or fusion properties of the protein, but markedly decreased infectivity of the virus, demonstrating that the RDE is important for primary infection. Finally, DFP-treated INF-C bound specifically and irreversibly to cells expressing 9-O-acetylated sialic acids. This provides a probe for a molecule that was hitherto very difficult to study.

Cultured thioglycollate-stimulated peritoneal macrophages synthesize, accumulate, and continuously release high levels of plasminogen activators for at least 4 days whereas cultures of unstimulated macrophages do not; the higher specific catalytic activity of released vs. cell-associated enzyme suggests that the plasminogen activators are actively secreted. The major macrophage plasminogen activator is a serine protease of mol wt 48,000, and thus resembles the comparable enzyme released by virally transformed fibroblasts. Macrophages release a second plasminogen activator of mol wt 28,000 that is also a serine enzyme. The secretion products released by stimulated and unstimulated macrophages have been compared by SDS-polyacrylamide gel electrophoresis after chemical labeling with (3)H-DFP or biosynthetic labeling with (14)C-amino acids. These procedures show that some proteins are formed in both cultures, whereas others are uniquely secreted by each type of macrophage. The serine enzymes released by the two kinds of macrophages differ in specificity and electrophoretic mobility.

In addition to its ability to hydrolyze acetylcholine, purified eel acetylcholinesterase possesses a trypsin-like endopeptidase activity. The tryptic activity is associated with a serine residue at a site that is distinct from the esteratic site. To label both the esteratic and tryptic sites, the enzyme was incubated with the serine hydrolase inhibitor [3H]diisopropyl fluorophosphate. This compound labelled the protein in a biphasic manner, with both slow and rapid labelling kinetics. The time course of the rapid phase was similar to the time course of inactivation of the esteratic activity. The time course of the slow phase was similar to the time course of inactivation of the tryptic activity. Labelling of the nonesteratic site was inhibited by the trypsin inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone. The total number of sites labelled by [3H]diisopropyl fluorophosphate on eel acetylcholinesterase was 2.6 mol/280,000 g protein, whereas the number of tryptic sites was less (0.52 mol/280,000 g). The results suggest that a subpopulation of acetylcholinesterase molecules may possess tryptic activity. Extensive chromatography of the purified enzyme by ion-exchange and gel filtration failed to separate the labelled tryptic component from acetylcholinesterase. On sodium dodecyl sulfate-polyacrylamide gels, the labelled tryptic component comigrated with a polypeptide of 50,000 molecular weight, which is a major proteolytic digestion product derived from the intact acetylcholinesterase monomer. Because of its localization in many noncholinergic peptide-containing cells, acetylcholinesterase could act as a neuropeptide processing enzyme in these cells.

The acute mortality caused by two irreversible inhibitors of cholinesterases [diisopropylfluorophosphate (DFP) and diethoxyphosphorylthiocholine, 217 MI-phospholine iodide] has been investigated on chick embryos at different stages of development. The results demonstrate that the above compounds do not show any acute lethal action when administered before the 9th day of incubation; on the other hand, the administration is regularly followed by death after the 9th day of incubation. The doses are comparable to those causing death in hatched chicks. It has also been observed that no appreciable difference exists in DFP uptake from the yolk before and after the 9th day of incubation and that drug-induced cholinesterase inhibition is of the same order of magnitude at any developmental stage; the compound pyridine-2-aldoxime methanesulfonate (2-PAM) was a good antidote against DFP acute lethality. It seems likely that between the 8th and the 9th day of incubation the target system of organophosphorus inhibitors, that is, the cholinesterase enzymatic system, reaches a new point of maturation.

Neurotoxic esterase activity was measured in homogenates of human placenta and hen brain, spinal cord, liver, kidney and spleen. The activity in liver comprised less than 20% of the Paraoxon-resistant esterases, but in the other tissues neurotoxic esterase accounted for over 50%. The same tissues were labelled with [3H]di-isopropyl phosphorofluoridate, and any isopropyl group transferred on to protein during 'aging' of the labelled enzymes (alkali-volatilizable tritium) was measured. No Paraoxon-sensitive labelled sites were found to age in this way in any tissue. In brain, the Paraoxon-resistant alkali-volatilizable-tritium-labelled sites correlated with the number of neurotoxic esterase labelled sites, indicating that 'aging' and isopropyl group transfer were 100% efficient. The site receiving the transferred isopropyl group was characterized by analysing the distribution of radiolabelled proteins on gel-filtration chromatography in the presence of SDS. In particulate preparations from each tissue, the protein-bound alkali-volatilizable tritium (transferred isopropyl group) was attached to a polypeptide of Mr 178 000. This same polypeptide also bore the isopropyl-phosphoryl group of neurotoxic esterase, indicating that aging of neurotoxic esterase is an intramolecular group transfer. The apparent turnover number for the enzyme (average 1.6 X 10(5) min-1) was approximately the same in each hen tissue, confirming that closely similar enzymes were present in brain, spinal cord, liver and spleen. The apparent turnover for the human enzyme was 1.8-fold higher than that for the hen enzyme. The concentration of the neurotoxic esterase phosphorylated subunit in brain, spinal cord, spleen, placenta and liver was 14.6, 3.8, 7.4, 3.3 and 3.8 pmol/g of tissue. The evidence indicated that neurotoxic esterase is present in each tissue except kidney, and that isopropyl group transfer on 'aging' occurs on this enzyme only. This process is an intramolecular transfer of the group within the same polypeptide.

One protein in canine seminal plasma accounts for over 90% of the total protein and is present at the high concentration of approximately 10 mg/ml. We demonstrate that this predominant protein is a proteolytic enzyme. The enzyme has been purified and migrates as a single symmetrical peak of apparent molecular mass of 29,000 daltons on a column of Sephadex G-75 and as a single band of approximately 30,000 daltons when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, the enzyme dissociates into subunits of 15,000 and 12,000-14,000 daltons. The 15,000-dalton subunit contains the enzyme active site as determined by labeling with [3H]diisopropyl fluorophosphate. The enzyme hydrolyzed the synthetic ester substrates N alpha-benzoyl-L-arginine ethyl ester and N alpha-tosyl-L-arginine methyl ester with maximum specific activities at 25 degrees C of 105 mumol/min/mg and 33 mumol/min/mg, and Km values of 7.4 and 9.1 mM, respectively. The enzyme exhibited a pH optimum of 8.0. The metal ions, Cu2+, Zn2+, Cd2+, and Co2+ were reversible inhibitors and diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride irreversible inhibitors of enzymatic activity. By immunofluorescence, the enzyme can be detected on the tail and postacrosomal regions of washed ejaculated canine sperm, but it is absent from epididymal sperm.

The functional activity and active site of plasmin in full-term newborns have been studied and compared to those in adults in order to investigate the nature of the abnormality found in newborn plasminogen described in a previous paper. The functional activity of newborn plasminogen measured on chromogenic substrate was approximately 18% that of adult plasminogen when streptokinase was used as an activator and 12% when urokinase was used. Proteolysis of newborn plasminogen by urokinase yielding a two-chain plasmin form occurred normally, but the incorporation of diisopropylphosphorofluoridate into the light chain of newborn plasmin was approximately 23% of that observed in the light chain of adult plasmin. These observations suggest that the abnormality of full-term newborn plasminogen is located in the active site of the molecule.

Human embryonic lung fibroblast (MRC5) cells secreted small amounts of plasminogen activators into normal, serum-free harvest medium. Stimulation with calcium led to markedly enhanced levels of activator. The major species of plasminogen activator in the harvest medium of the stimulated cultures resembled u-PA when analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by fibrin zymography. This activator was partially-purified using controlled-pore glass and Blue Sepharose CL6B. Characterisation by fibrin zymography in the presence of specific antibody, by 3H-DFP labelling and by fibrin-binding ability indicated that the activator was indistinguishable from high molecular weight u-PA. The possible physiological role of the production of relatively large amounts of u-PA by a lung cell capable of producing both u-PA and t-PA is discussed.

An intracellular clotting factor, factor B, which is closely associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus), was purified and characterized. The purified preparation gave a single band (Mr = 64,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while three bands (Mr = 64,000, 40,000, and 25,000) were detected on SDS-PAGE after reduction. This preparation was converted by limulus clotting factor C to an activated form, factor B, with Mr = 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000) bridged by disulfide linkage(s). The factor B, which was produced separately by treating the partially purified factor B with factor C, was also purified. It gave a single band on unreduced SDS-PAGE and two bands on reduced SDS-PAGE. The purified factor B had Mr of 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000). These results indicated that the purified factor B zymogen is a mixture of single-chain and two-chain forms, both of which have the same molecular weight of 64,000, and that these two forms are converted to factor B by factor C. The diisopropyl phosphorofluoridate-sensitive site of factor B was found in the heavy chain. The reconstitution studies using purified factor C, factor B, proclotting enzyme and coagulogen in the presence of lipopolysaccharide indicated that factor B is an essential component to complete sequential activation of the limulus clotting system, and that it specifically activates proclotting enzyme to the active clotting enzyme.

Activated Factor B, incorporated into the cobra venom factor (CVF)-dependent C3/C5 convertase, was inactivated by diisopropylfluorophosphate (DFP). Inactivation was time- and dose-dependent and was enhanced by the presence of substrate. Treatment of the zymogen of Factor B with DFP effected significant inactivation. Incorporation of [3H]diisopropylphosphate into the zymogen and into the activated form of Factor B was demonstrated after [3H]DFP treatment and subsequent electrophoresis of the proteins on polyacrylamide gels containing sodium dodecyl sulfate. Inactivation of activated C2 incorporated into the classical C5 convertase was observed on DFP treatment of the enzyme. DFP also reduced the activity of the C2 zymogen. The description of their serine proteinase nature further emphasizes the close structural and functional relationship of C2 and Factor B.

Mouse bone marrow-derived mast cells, differentiated in vitro with concanavalin A splenocyte-conditioned medium and sensitized with monoclonal IgE, release neutral serine proteases after activation with specific antigen. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of the supernatants from immunologically activated mast cells revealed the presence of four prominent proteins of 27,000, 29,000, 30,000 and 31,000 m.w. When the supernatants and sonicated residual cells from antigen-challenged or nonactivated IgE-sensitized mast cells were incubated with [3H]diisopropylfluorophosphate ([3H]DFP) and the proteins were subjected to SDS-PAGE followed by autoradiography, proteins of 27,000 to 31,000 m.w. were labeled with [3H]DFP. The antigen-dependent release of labeled proteins was accompanied by a corresponding depletion of similarly sized [3H]DFP-labeled proteins from these cell pellets relative to unactivated cells. The SDS gels were also stained with Coomassie Blue and were sectioned to separate the individual proteins for measurement of their incorporated radioactivity; the net percent antigen-dependent release of all four [3H]DFP-labeled proteins ranged from 64 to 68% and was comparable to that of the secretory granule markers, beta-hexosaminidase and histamine. That the [3H]DFP-labeled proteins were derived from the secretory granules of the cells was supported by studies in which mast cells were cultured for 4 days in the presence of 1 mM sodium butyrate. This treatment produced a differential increase in their cellular content of histamine (10-fold), [3H]DFP binding proteins (two- to fourfold), and beta-hexosaminidase (minimally), while the net percent antigen-dependent release of each of these constituents was unchanged. After sensitization and antigen activation, the net percent release of histamine, beta-hexosaminidase, and the four [3H]DFP-labeled proteins was 51, 59, and 53 to 61%, respectively, for sodium butyrate-treated cells, and 53, 60, and 64 to 68%, respectively, for cells not exposed to sodium butyrate. Human plasma fibronectin was used as a substrate to demonstrate that the exocytosed proteins possessed proteolytic activity. As assessed by optical density scanning of stained SDS-PAGE gels of the substrate, the proteases present in the supernatants of antigen-activated cells, but not of sensitized unchallenged cells, rapidly degraded native fibronectin at pH 7.0. This degradation was prevented by pretreatment of the exocytosed proteins from immunologically activated cells for 90 min at 37 degrees C with 2 mM DFP.(ABSTRACT TRUNCATED AT 400 WORDS)

A method has been developed for measuring neutrophil cellularity in normal human bone marrow, in which the neutrophil-erythroid ratio was determined from marrow sections and marrow normoblasts were estimated by the erythron iron turnover. Neutrophil maturational categories, defined by morphologic criteria, were supported by autoradiographs of marrow flashed-labeled with 3H-thymidine. Correction for multiple counting error was empirically derived by counting serial sections through cells of each maturational category. The normal neutrophil-erythroid ratio in 13 normal human subjects was 1.5 +/- 0.07. The mean number of normoblasts in the same subjects was estimated to be 5.07 +/- 0.84 X 10(9) cells/kg. Total marrow neutrophils (X 10(9) cells/kg) were 7.70 +/- 1.20, the postmitotic pool (metamyelocytes, bands, and segmented forms) was 5.59 +/- 0.90 and the mitotic pool (promyelocytes + myelocytes) was 2.11 +/- 0.36. Marrow neutrophil ("total") production has been determined from the number of neutrophils comprising the postmitotic marrow pool divided by their transit time Transit time was derived from the appearance in circulating neutrophils of injected 3H-thymidine. The postmitotic pool comprised 5.59 +/- 0.90 X 10(9) neutrophils/kg, and the transit time was 6.60 +/- 0.03 days. From these data marrow neutrophil production was calculated to be 0.85 X 10(9) cells/kg per day. Effective production, measured as the turnover of circulating neutrophils labeled with 3H-thymidine, was 0.87 +/- 0.13 X 10(9) cells/kg per day. This value correlated well with the calculation of marrow neutrophil production. A larger turnover of 1.62 +/- 0.46 X 10(9) cells/kg per day was obtained when diisopropylfluorophosphate-32P was used to label circulating neutrophils. Studies using isologous cells doubly labeled with 3H-thymidine and diisopropylfluorophosphate-32P demonstrated a lower recovery and shorter t1/2 of the 32P label.

Acute administration of the organophosphate diisopropyl-fluorophosphate (DFP) produces behavioral and physiological symptoms indicative of excessive cholinergic stimulation. This behavioral toxicity was found to be incompatible with the occurrence of sleep, despite the fact that chronic administration of DFP has been shown to increase the rapid eye movement stage of sleep. DFP was found to decrease all stages of sleep and to increase wakefulness in a dose-dependent manner. Atropine sulfate, at doses of 3.0 mg/kg, was ineffective in blocking the DFP effects upon sleep.

1. The bivalve Rangia cuneata can enzymatically detoxify the organophosphorus acetylcholinesterase inhibitors DFP and soman. 2. Digestive gland homogenates contained Mazur-type DFPases based on response to Mn2+ ions, and relative rates of DFP: soman hydrolysis. Squid-type DFPase contributed little to the total organophosphate acid (OPA) anhydrase activity of these preparations. 3. The natural substrate(s) and physiological role(s) of OPA anhydrase in R. cuneata has yet to be determined; however, DFPase specific activity was pronounced in the digestive gland, the primary organ involved in bioconcentration and biotransformation of xenobiotics, and in the gills, which are in continuous contact with water-borne chemicals.

Virally transformed cells show an increased production of proteolytic enzymes. These might be involved in transformation-dependent alterations of cell surface glycoproteins. The possibility arises that some of these proteases might be membrane-bound. To investigate this possibility, we have undertaken a comparative study of the reactivity of intact normal and transformed cells with the tritium labelled protease inhibitor diisopropylfluorophosphate, in parallel with fibrinolytic assays. Using these two approaches in concert, it was possible to identify and localize in the transformed cells several proteases which were present in the particulate cell fraction and were probably membrane bound. In particular, a diisopropylfluorophosphate-reactive polypeptide of 62,000 was increased 5--8-fold on transformation. It comigrated with a fibrinolytic activity. Other particle-bound activities were also detected. While diisopropylfluorophosphate-labelling can be useful for detecting proteases inside cells, it does not appear to be specific for surface proteases.

Recently, we and others reported on the expression of a serine proteinase in long-term cultured murine T lymphocyte cell lines. In an attempt to explore the distribution and possible regulation of this enzyme in T lymphocyte subsets, we performed the presented detailed study. We found that the proteinase is not expressed by thymocytes and resting T cells but can be induced by lectin or antigen in combination with lymphokine sources in vitro in macrophage-depleted unselected T cells as well as in both T cell subsets (Lyt-2+,L3T4- and Lyt-2-,L3T4+) separated by flow cytofluorometry. Furthermore, it appears that cell-associated proteinase activity is increasing with prolonged culture period of sensitized T lymphocytes and that it is higher in antigen-activated as compared to lectin-activated T cells. When tested for substrate specificity the T cell-associated proteinase was shown to preferentially cleave model peptide substrates carrying L-arginine at position P1 in combination with nonpolar amino acids at position P2 and P3. As concluded from its sensitivity to proteinase inhibitors the enzyme can be classified as a serine proteinase and by molecular sieving at high ionic strength it was shown to have a mol. mass of approximately 50-60 kDa. Analysis of in vivo activated T cells revealed that this particular proteinase was expressed in flow cytofluorometry sorted lymphocytic choriomeningitis virus-specific Lyt-2+,L3T4- cytolytic T lymphocytes but not in Lyt-2-,L3T4+ T cells presensitized with either Listeria monocytogenes or I-A alloantigens. The data demonstrate that the two T cell subsets (Lyt-2+,L3T4-; Lyt-2-,L3T4+) have distinct in vitro induction requirements for the expression of proteinase and that after activation of T cells in vivo the enzyme is preferentially associated with Lyt-2+,L3T4- effector cells.

A new technique is described for measuring antibodies to an enzyme which is not available in pure form. The secretions of the rat parasitic nematode, Nippostrongylus brasiliensis, were treated with tritiated diisopropylfluorophosphate. Only one component, an acetylcholinesterase, was radiolabelled. Antibodies to this enzyme in rat antisera were estimated by the Farr technique using the labelled enzyme as antigen. The acetylcholinesterase secreted by Necator americanus, the human hookworm, was similarly specifically labelled.

We have compared the plasminogen activating capacity of one- and two-chain urokinase-type plasminogen activator (u-PA). In a 125I-plasminogen conversion assay in the presence of high amounts of a plasmin inhibitor, one-chain u-PA pretreated with diisopropyl fluorophosphate had no detectable activity, the detection limit corresponding to the activity of a 400-fold lower amount of two-chain u-PA. In coupled assays in which generated plasmin was measured with a synthetic substrate, activity was clearly observed with the one-chain preparation, but the initial rate of plasminogen activation was lower than that of a 250-fold smaller concentration of two-chain u-PA. The coupled assays for one-chain u-PA are self-activating because plasmin catalyzes conversion of one- to two-chain u-PA, and it is not possible to decide whether the low activity of one-chain u-PA observed with this type of assay is intrinsic or due to contaminations. On the basis of these findings and a discussion of previous studies, it is concluded that one-chain u-PA has a variety of properties similar to the one-chain proenzyme forms of other serine proteases and that it should, therefore, be considered as a genuine proenzyme form of u-PA.

The present study shows that tissue kallikrein is present in rat pancreas as a proenzyme that can be converted by autolysis to a 38 000 Da active enzyme. The activation of pancreatic prokallikrein was examined by direct radioimmunoassay, enzymatic assays, active-site labeling with immunoprecipitation, and Western blot analyses. A monoclonal antibody (V1C3), which binds only active kallikrein, was used in a direct radioimmunoassay to monitor the appearance of the active enzyme. During a 22-h autolysis of pancreatic extract, a time-dependent increase in active kallikrein concentration paralleled the increase of kallikrein activities measured by both TosArgOMe esterase and kininogenase assays. The activation process was further analyzed by labeling the pancreatic extract with [14C]diisopropylphosphorofluoridate [( 14C]DFP) followed by immunoprecipitation with sheep anti-kallikrein antiserum. Pancreatic prokallikrein was not labeled by [14C]DFP; however, upon autolysis, a 38 000 Da active kallikrein can be labeled with [14C]DFP and increase in quantity with time. Western blot analysis, using a monoclonal antibody (V4D11) which recognizes both latent and active tissue kallikreins, identified a 39 000 Da pancreatic prokallikrein prior to autolysis and a 38 000 Da active kallikrein after 7 h of autolysis. The results indicate that the pancreatic prokallikrein exists as a 39 000 Da protein which may be converted to a 38 000 Da active kallikrein, indistinguishable from purified urinary, brain, spleen or submandibular gland kallikrein.

Purified acetylcholinesterase from Drosophila melanogaster is composed of a 55 kDa and a 16 kDa noncovalently associated subunit. Cleavage of disulfide bonds reveals that two 55 kDa polypeptides are linked together in native dimeric AChE. Western blots with two antibodies directed against the N- and C-termini of the predicted AChE primary sequence show that the 55 and 16 kDa polypeptides originate from proteolysis of the same precursor encoded by the Ace locus.

Mean red cell life-span in normal subjects and in patients with various hematological disorders was examined with 51Cr and DF32P. The results with 51Cr were corrected for 51Cr elution using correction factors. The results by the two methods agreed fairly well with each other. Elution rate in various hematological disorders was 2.3% per day or less except for the patients with extracorpuscular hemolytic agents such as autoimmune hemolytic anemia or congestive splenomegaly. It is concluded that estimates of mean red cell life-span by corrected 51Cr method are more useful and sufficient than uncorrected 51Cr or DF32P method in general hematological disorders.

An enzyme that hydrolyzes soman (1,2,2-trimethylpropyl methylphosphonofluoridate) and two other phosphonofluoridates, but does not hydrolyze DFP (diisopropylphosphorofluoridate), has been partially purified from a rod-shaped spore-forming gram-positive OT (obligate thermophilic) bacterium. The enzyme shows a marked Mn2+ stimulation, and in this and its substrate preference does not resemble the organophosphorus acid anhydrolase (sometimes termed DFPase) found in squid. Like the squid enzyme, it is not inhibited by mipafox (N,N'-diisopropylphosphordiamidofluoridate), is not inactivated by ammonium sulfate, and does hydrolyze the acetylcholinesterase-inhibitory pair of diastereoisomers of soman as well as the relatively noninhibitory pair, thus detoxifying soman. In these three properties the OT enzyme does not resemble the ubiquitous organophosphorus acid anhydrolase often purified from mammalian and bacterial sources by cold ethanol fractionation. Thus this phosphono-specific OT enzyme may have a natural substrate and a physiological role distinct from other organophosphorus acid anhydrolases.

Lecithin:cholesterol acyltransferase (LCAT) was purified from hog plasma and subsequently reacted with [3H]-Diisopropyl fluorophosphate (DFP). The labeled enzyme was digested with pepsin and the peptides separated by high performance liquid chromatography (HPLC). Two radioactive peptides were isolated, subjected to automated amino acid sequencing and yielded the following data: A) Ile-Ser-Leu-Gly-Ala-Pro-Trp-Gly-Gly-Ser, and B) Tyr-Ile-Phe-Asp-x-Gly-Phe-Pro-Tyr-x-Asp-Pro-Val. Both of these sequences represent very highly conserved regions of the enzyme when compared to the sequence of human LCAT. Peptide (A) is considered to represent the catalytic center of LCAT based on comparisons with data reported in the literature.

Recovery of acetylcholinesterase (AChE) at endplates of mouse sternomastoid muscle was studied after inactivation with di-isopropylfluorophosphate in vivo. A short incubation with alpha-bungarotoxin was used to prevent muscle necrosis which usually occurs after esterase inactivation. Under these conditions there was no delay in AChE recovery, unlike what we had previously seen in necrotic muscle. However, even in non-necrotic muscle, the overall recovery of AChE at the postjunctional membrane was very slow, with a half-life of about 20 days. The fact that, at adult neuromuscular junctions, both esterases and receptors turn over about 10 times slower than they do in embryonic muscle suggests a similar regulation of these two molecules by the nerve.

Neuropathy target esterase (NTE) is a membrane-bound carboxylesterase activity that has been proposed as the target site for initiation of organophosphate-induced delayed neuropathy. This activity is identified by its resistance to treatment with Paraoxon and sensitivity to co-incubation with Paraoxon and Mipafox. Sucrose-density-gradient centrifugation of membrane-associated proteins isolated from chick-embryo brains identified three proteins, Mr 161,000, 116,500 and 103,000, that were labelled with [3H]di-isopropyl phosphorofluoridate in an NTE-like manner and that co-migrated with NTE. The 161,000-Mr and 116,500-Mr proteins were identified in both adult and embryo brain. One or both of these proteins may therefore contribute to the activity defined as NTE. In addition, a 61,000-Mr protein was identified that does not comigrate with NTE, but that was labelled with [3H]di-isopropyl phosphorofluoridate in a Paraoxon-resistant and Mipafox-sensitive manner. The effect of Mipafox on labelling, however, was reversibly blocked by co-incubation with Paraoxon. This protein, therefore, is not NTE, but has the necessary inhibitor-sensitivity to be the target site for organophosphate-induced delayed neuropathy.