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W F Mangel

Publications and source records attributed to W F Mangel.

15 recordsLinked to original sources
PubMed1992

Membrane permeable fluorogenic rhodamine substrates for selective determination of cathepsin L.

The dipeptidyl rhodamine diamide substrates (Z-Phe-Arg)2-R110 and (Z-Arg-Arg)2-R110 are 820- and 360-fold more selective for cathepsin L than for cathepsin B allowing a sensitive determination of cathepsin L activity in the presence of high activity of cathepsin B. The results obtained with cell lysates suggest that the cysteine proteinase activity of vital macrophages detected by flow cytometry with these substrates is mainly due to cathepsin L.

Animals↗
PubMed1992

Flow cytometric analysis of protease activities in vital cells.

The analysis of lysosomal proteases in cell lysates is complicated by pH-dependent and oxidative changes of their activity and complex formation with cytosolic inhibitors. Therefore, new flow cytometric methods were developed for the intracellular measurement of protease activities in viable cells. Intracellular cleavage of substrates such as Z-Arg-Arg-4-trifluoromethylcoumarinyl-7-amide to green fluorescent 7-amino-4-trifluoromethylcoumarin (AFC) in viable neutrophils and monocytes was only detected following phagocytosis of Escherichia coli. A measurement of the cysteine or serine proteinase activities in resting human leukocytes was, however, not possible with AFC derivatives as the overlapping blue fluorescence of the substrates reduces sensitivity. Nonfluorescent bis-substituted peptide derivatives of rhodamine 110 (R110), which are intracellularly cleaved to green fluorescent mono-substituted R110 and free R110 proved to be more sensitive substrates. The activity of the lysosomal cysteine proteinases of human monocytes or rat macrophages, i.e. cathepsin B and L, was specifically measured with (Z-Arg-Arg)2-R110, (Z-Phe-Arg)2-R110, or (Z-Ala-Arg-Arg)2-R110. Fluorescence generation was completely inhibited by Z-Phe-Ala-diazomethane or E-64. The serine proteinases of human neutrophils were analyzed with Elastase-substrates such as (Z-Ala-Ala)2-R110 or (Z-Ala-Ala-Ala)2-R110. Specificity was shown by inhibition with diisopropylfluorophosphate.

Amino Acid Sequence↗
PubMed1991

Conformation of one- and two-chain high molecular weight urokinase analyzed by small-angle neutron scattering and vacuum ultraviolet circular dichroism.

The structures of one- and two-chain high molecular weight human urokinase were analyzed by small-angle neutron scattering and vacuum ultraviolet circular dichroism. Both one- and two-chain high molecular weight urokinases exhibited a radius of gyration of 31 A and a maximum dimension of 90 A. Neither parameter was affected by the presence of lysine sufficient to saturate all the lysine-binding sites in human plasminogen. These physical parameters are consistent with the sedimentation coefficient of high molecular weight urokinase and indicate that both proteins are highly asymmetric. Neither protein contained much alpha-helix or parallel beta-sheet. Most of the secondary structure was in the form of antiparallel beta-sheet and beta-turns, very similar to the secondary structure of plasminogen. The macroscopic kinetic constants, Km and kcat, for the hydrolysis of (pyroGlu-Gly-Arg-NH)2-rhodamine by two-chain high molecular weight urokinase and low molecular weight urokinase which lacks the epidermal growth factor and kringle domains were similar. These structural and kinetic data are consistent with the domains in both forms of urokinase being independent structural and functional units.

Circular Dichroism↗
PubMed1991

Conformation of Lys-plasminogen and the kringle 1-3 fragment of plasminogen analyzed by small-angle neutron scattering.

Native human Glu-plasminogen (Glu1-Asn791) was previously shown to have a radius of gyration of 39 A and a shape best described by a prolate ellipsoid [Mangel, W. F., Lin, B., & Ramakrishnan, V. (1990) Science 248, 69-73]. Upon occupation of a weak lysine-binding site, the shape reversibly changes to that best described by a Debye random coil with a radius of gyration of 56 A. Conversion from the closed to the open form is not accompanied by any change in secondary structure, hence the closed conformation is formed by interaction between domains, the five kringles and the protease domain, and this is abolished upon conversion to the open form. Here we analyzed by small-angle neutron scattering the conformations of human Lys-plasminogen (Lys78-Asn791) and the fragment K1-3 that contains the first three kringles of plasminogen (Tyr80-Val338 or Tyr80-Val354). The shape of Lys-plasminogen was best described by a Debye random coil with a radius of gyration of 51 A, and occupation of its lysine-binding sites by 6-aminohexanoic acid did not dramatically alter its conformation. Thus Lys-plasminogen was in the open form, similar to that of Glu-plasminogen with its lysine-binding sites occupied. The fragment K1-3 in the absence or presence of 6-aminohexanoic acid had a shape best described equally either by an elongated prolate ellipsoid or by a Debye random coil, with a radius of gyration of 29 A. Our model for the two forms of plasminogen is that, in the closed form, domain interaction generates a compact, almost globular, structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Structure of alpha 2-macroglobulin from the arthropod Limulus polyphemus.

A structural and functional homologue of vertebrate alpha 2-macroglobulin (alpha 2M) has been identified in the hemolymph and blood cells of the arthropod Limulus polyphemus, one of the oldest living fossil invertebrates (Quigley, J. P., and Armstrong, P. B. (1985) J. Biol. Chem. 260, 12715-12719). The subunit molecular mass is 185 kDa. The native molecular mass, determined by scanning transmission electron microscopy (STEM) under conditions in which the linear relationship between the STEM large angle detector signal and specimen mass thickness allows the determination of the total macromolecular mass, was 354 +/- 35 kDa. Sedimentation equilibrium measurements gave a value of 366 kDa, independent of solute concentration. Sedimentation velocity experiments indicated a homogeneous component with a frictional ratio of 1.41. Thus, the native structure appears to be a dimer, with a somewhat extended conformation. The behavior during gel permeation chromatography was anomalous, yielding an apparent molecular mass approximately half-way between that expected for the dimeric and tetrameric configurations. Transmission electron microscopy of negatively stained preparations revealed a dimeric butterfly-like structure that collapsed following reaction with chymotrypsin.

Animals↗
PubMed1990

Structure of an acyl-enzyme intermediate during catalysis: (guanidinobenzoyl)trypsin.

The crystal and molecular structure of trypsin at a transiently stable intermediate step during catalysis has been determined by X-ray diffraction methods. Bovine trypsin cleaved the substrate p-nitrophenyl p-guanidinobenzoate during crystallization under conditions in which the acyl-enzyme intermediate, (guanidinobenzoyl)trypsin, was stable. Orthorhombic crystals formed in space group P2(1)2(1)2(1), with a = 63.74, b = 63.54, and c = 68.93 A. This is a crystal form of bovine trypsin for which a molecular structure has not been reported. Diffraction data were measured with a FAST (Enraf Nonius) diffractometer. The structure was refined to a crystallographic residual of R = 0.16 for data in the resolution range 7.0-2.0 A. The refined model of (guanidinobenzoyl)trypsin provides insight into the structural basis for its slow rate of deacylation, which in solution at 25 degrees C and pH 7.4 exhibits a t1/2 of 12 h. In addition to the rotation of the Ser-195 hydroxyl away from His-157, C beta of Ser-195 moves 0.7 A toward Asp-189 at the bottom of the active site, with respect to the native structure. This allows formation of energetically favorable H bonds and an ion pair between the carboxylate of Asp-189 and the guanidino group of the substrate. This movement is dictated by the rigidity of the aromatic ring in guanidinobenzoate--model-building indicates that this should not occur when arginine, with its more flexible aliphatic backbone, forms the ester bond with Ser-195. As a consequence, highly ordered water molecules in the active site are no longer close enough to the scissile ester bond to serve as potential nucleophiles for hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Characterization of an extremely large, ligand-induced conformational change in plasminogen.

Native human plasminogen has a radius of gyration of 39 angstroms. Upon occupation of a weak lysine binding site, the radius of gyration increases to 56 angstroms, an extremely large ligand-induced conformational change. There are no intermediate conformational states between the closed and open form. The conformational chang is not accompanied by a change in secondary structure, hence the closed conformation is formed by interaction between domains that is abolished upon conversion to the open form. This reversible change in conformation, in which the shape of the protein changes from that best described by a prolate ellipsoid to a flexible structure best described by a Debye random coil, is physiologically relevant because a weak lysine binding site regulates the activation of plasminogen.

Aminocaproic Acid↗
PubMed1988

Inhibition of guanidinobenzoatase by a substrate for trypsin-like enzymes.

Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. Guanidinobenzoatase has been shown to be an arginine selective protease and is distinct from trypsin, plasmin and thrombin, the latter enzymes can be assayed with bis(carbobenzyloxycarbonyl-L-argininamido)-Rhodamine or BZAR. Guanidinobenzoatase is inhibited by BZAR when the enzyme is assayed in free solution and when the enzyme is cell-bound in frozen sections of tumour containing tissues. It is proposed that BZAR and its analogues may be of value in inhibiting tumour cell invasion in vivo and also that the selectivity of BZAR may be used to direct cytotoxic drugs to tumour cells possessing active guanidinobenzoatase.

Animals↗
PubMed1978

Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2.

Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded.

Capsid↗
PubMed1976

Location of the sequences coding for capsid proteins VP1 and VP2 on polyoma virus DNA.

The 19S and 16S polyoma virus late mRNAs have been separated on sucrose-formamide density gradients and translated in vitro. The 16S RNA codes only for polyoma capsid protein VP1, while the 19S RNA codes in addition for capsid protein VP2. Since the 19S and 16S species have been previously mapped on the viral genome, these results allow us to deduce the location of the sequences coding for VP1 and VP2. Comparison of the chain lengths of the capsid proteins with the size of the viral mRNAs coding for them suggests that VP1 and VP2 are entirely virus-coded. Purified polyoma 19S RNA directs the synthesis of very little VP1 in vitro, although it contains all the sequences required to code for the protein. The initiation site for VP1 synthesis which is located at an internal position on the messenger is probably inactive either because it is inaccessible or because it lacks an adjacent "capped" 5' terminus. Similar inactive internal initiation sites have been reported for other eucarotic viral mRNAs (for example, Semliki forest virus, Brome mosaic virus, and tobacco mosaic virus), suggesting that while eucaryotic mRNAs may have more than one initiation site for protein synthesis, only those sites nearer the 5' terminus of the mRNA are active.

Base Sequence↗
PubMedDate not supplied

Evidence for the functional similarity between tumour cell surface guanidinobenzoatase and tissue type plasminogen activator.

Tumour cells possess a cell surface protease referred to as guanidinobenzoatase (GB). The active centre of GB binds the fluorescent probe 9-amino acridine (9-AA) and this binding enables cells possessing active GB to be located by fluorescent microscopy. GB binding of 9-AA was inhibited by prior treatment of sections of tumour tissue with a specific polyclonal antibody recognising the tumour associated protease tissue plasminogen activator (t-PA). GB binding of 9-AA was also inhibited by prior treatment of sections of tumour tissues with PAI-I, a protein inhibitor of plasminogen activatory. We conclude from these studies and kinetic analyses that GB and t-PA are very similar both in structure and function.

Aminacrine↗