A fluorometric assay for HIV-protease activity using high-performance liquid chromatography

Tamburini, Paul P.; Dreyer, Robert N.; Hansen, Jutta; Letsinger, Jack; Elting, Jim; Gore-Willse, Ann; Dally, Robert; Hanko, Rudolf; Osterman, David G.; Kamarck, Michael E.; Yoo-Warren, Heeja

doi:10.1016/0003-2697(90)90095-q

Cited by 39 publications

(15 citation statements)

References 15 publications

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“…Rabbit antiserum to human cathepsin D was from Dako Corp. IgG was purified from rabbit sera using Avid AL [23] and coupled to CnBr-activated Sepharose 4B [24]. The methods and instrumentation for peptide synthesis [25], amino acid analysis [26] and mass spectrocopy [26] were as cited previously. The protein content of crude extracts was determined by the Bradford assay [27].…”

Section: Methodsmentioning

confidence: 99%

“…methanol, captopril was absent and the buffer pH was varied from pH 3-8. HPLC instrumentation and general chromatographic conditions were as described [26] except C,, separations were achieved isocratically over 8 min in 100 mM sodium acetate, pH 6.5, containing 27% (by vol.) acetonitrile, and calibrated using authentic standards.…”

Section: Hydrolysis Of N-dansyl-app-(591-60l)-amidementioning

confidence: 99%

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Processing of the Pre‐β‐amyloid Protein by Cathepsin d is Enhanced by a Familial Alzheimer's Disease Mutation

Dreyer

Bausch

Fracasso

et al. 1994

European Journal of Biochemistry

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A major pre-P-amyloid proteinsps (APP,,,) processing activity from Alzheimer's disease brain extracts was identified and found to be indistinguishable from the activity of cathepsin D.APP,, processing activity cleaved APP,,, into a series of fragments that reacted on immunoblots to a monoclonal antibody (C286.8a) against p-amyloid-( 1 -7)-peptide and cleaved N-dansyl-APP-(591-601)-amide at the Glu-Val and Met-Asp bonds. Fragments of 5.5 kDa and 10-12 kDa were formed from the cleavage of APP,,, by cathepsin D at the Glu593-Val594 bond, and had the same Nterminus as a minor form of P-amyloid released by cells. Abbreviations. APP,,,, pre-P-amyloid protein,,, isoform; [N5", L5'6]APP,,,, APP,,, with lysine and methionine at positions 595 and 596 replaced by asparagine and leucine, respectively; dansyl, 5-(dimethylamino)-napthalene-1-sulfonyl; N-dansyl-APP-(591-601)-amide, N-dansyl-ISEVKMDAEFR-amide; C286.8a, IgG,, mAb recognizing P-amyloid residues at positions 1-7; PhMeSO,F, phenylmethylsulfonyl fluoride; E-64, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane; P-2, post-I5000 g pellet.Enzymes. Cathepsin D (EC 3.4.23.5); type I1 site-specific deoxyribonucleases (EC 3.1.21.4).-~ processing enzyme, the activity of which is influenced by a mutation associated with the Swedish pedigree of familial Alzheimer's disease. MATERIALS AND METHODSHuman cathepsin D from Calbiochem was pure by SDS/ PAGE, 93% accurate by amino acid analysis, and contained only cathepsin D derived N-termini, the majority of which were from equimolar amounts of the mature light (GPI-PEVLKNY-), and heavy (GGVKVERQVF-) chains. Pepstatin A and heparin agarose were from Sigma. Mono-Q columns were from Pharmacia. Rabbit antiserum to human cathepsin D was from Dako Corp. IgG was purified from rabbit sera using Avid AL [23] and coupled to CnBr-activated Sepharose 4B [24]. The methods and instrumentation for peptide synthesis [25], amino acid analysis [26] and mass spectrocopy [26] were as cited previously. The protein content of crude extracts was determined by the Bradford assay [27].

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Section: Methodsmentioning

confidence: 99%

Section: Hydrolysis Of N-dansyl-app-(591-60l)-amidementioning

confidence: 99%

Processing of the Pre‐β‐amyloid Protein by Cathepsin d is Enhanced by a Familial Alzheimer's Disease Mutation

Dreyer

Bausch

Fracasso

et al. 1994

European Journal of Biochemistry

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“…NH 2 -terminal sequence analysis was performed essentially as described (13), but with minor modification (6). Amino acid analysis was performed as described previously (14). Reducing SDS-polyacrylamide gel electrophoresis was performed with 10 -20% Tricine-buffered polyacrylamide gels (Novex, San Diego, CA) according to the manufacturer's instructions, and protein was visualized with Coomassie Brilliant Blue R-250 using standard protocols.…”

Section: Measurement Of the Activated Partial Thromboplastin Time (Apmentioning

confidence: 99%

Characterization of Placental Bikunin, a Novel Human Serine Protease Inhibitor

Delaria¹,

Muller²,

Marlor³

et al. 1997

Journal of Biological Chemistry

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102

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We reported previously the cloning of a novel human serine protease inhibitor containing two Kunitz-like domains, designated as placental bikunin, and the subsequent purification of a natural counterpart from human placental tissue (Marlor, C. W., Delaria, K. A., Davis, G., Muller, D. K., Greve, J. M., and Tamburini, P. P. (1997) J. Biol. Chem. 272, 12202-12208). In this report, the 170 residue extracellular domain of placental bikunin (placental bikunin (1-170) ) was expressed in baculovirus-infected Sf9 cells using its putative signal peptide. The resulting 21.3-kDa protein accumulated in the medium with the signal peptide removed and could be highly purified by sequential kallikrein-Sepharose and C 18 reverse-phase chromatography. To provide insights as to the potential in vivo functions of this protein, we performed an extensive investigation of the inhibitory properties of recombinant placental bikunin (1-170) and both of its synthetically prepared Kunitz domains. All three proteins inhibited a number of serine proteases involved in the intrinsic pathway of blood coagulation and fibrinolysis. Placental bikunin (1-170) formed inhibitor-protease complexes with a 1:2 stoichiometry and strongly inhibited human plasmin (K i ‫؍‬ 0.1 nM), human tissue kallikrein (K i ‫؍‬ 0.1 nM), human plasma kallikrein (K i ‫؍‬ 0.3 nM) and human factor XIa (K i ‫؍‬ 6 nM). Conversely, this protein was a weaker inhibitor of factor VIIa-tissue factor (K i ‫؍‬ 1.6 M), factor IXa (K i ‫؍‬ 206 nM), factor Xa (K i ‫؍‬ 364 nM), and factor XIIa (K i ‫؍‬ 430 nM). This specificity profile was to a large extent mimicked, albeit with reduced potency, by the individual Kunitz domains. As predicted from this in vitro specificity profile, recombinant placental bikunin (1-170) prolonged the clotting time in an activated partial thromboplastin time assay.Blood clotting, resulting either from the extrinsic pathway following tissue injury or the intrinsic pathway following contact activation, involves tightly regulated proteolytic cascades (1). The intrinsic pathway is initiated by activation of factor XII either through proteolysis or contact with negatively charged surfaces. Activated factor XIIa, in turn, converts plasma prekallikrein to kallikrein, which can then activate additional factor XII. Factor XIIa activates factor XI, which, in turn, activates factor IX. Activated factor IX forms a complex with factor VIIIa, phospholipid, and calcium, which converts factor X to factor Xa. Factor X is also activated by the factor VIIatissue factor complex operating within the extrinsic pathway. Thrombin generation by factor Xa in complex with factor Va leads ultimately to the formation of the fibrin clot. Thrombus formation is also regulated by the fibrinolytic system whereby plasmin, formed from plasminogen by the action of kallikrein, tissue plasminogen activator (tPA), 1 or urokinase, breaks down both fibrinogen and fibrin (2).Protease inhibitors play critical roles in the regulation of the coagulation and fibrinolytic systems. Tissue factor pathway i...

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“…It is regarded as inconvenient for routine work since column runs are time consuming (typically about 30 min/run). However, reduction of run times to as short as 3 min has been achieved (Tamburini et al, 1990), which makes the method, in combination with autosampler and automatic integration software, applicable for standard assays as well.…”

Section: Discontinuous Assaysmentioning

confidence: 99%