Fluorescence-Quenched Solid Phase Combinatorial Libraries in the Characterization of Cysteine Protease Substrate Specificity†

1999 ◽  
Vol 1 (6) ◽  
pp. 509-523 ◽  
Author(s):  
Phaedria M. St. Hilaire ◽  
Marianne Willert ◽  
Maria Aparecida Juliano ◽  
Luiz Juliano ◽  
Morten Meldal
Keyword(s):  
Substrate Specificity ◽  
Cysteine Protease ◽  
Solid Phase ◽  
Combinatorial Libraries ◽  
Protease Substrate

Characterization of the West Nile virus protease substrate specificity and inhibitors

2007 ◽  
Vol 39 (3) ◽  
pp. 606-614 ◽  
Author(s):  
Niklaus H. Mueller ◽  
Changsuek Yon ◽  
Vannakambadi K. Ganesh ◽  
R. Padmanabhan
Keyword(s):  
West Nile Virus ◽  
Substrate Specificity ◽  
The West ◽  
West Nile ◽  
Protease Substrate

Characterization of HIV-1 Protease Substrate Specificity by Use of Synthetic Peptides

1989 ◽  
pp. 155-164
Author(s):  
K. Moelling ◽  
M. T. Knoop ◽  
S. Billich ◽  
I. Bláha ◽  
L. Pavlíčková ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Synthetic Peptides ◽  
Protease Substrate ◽  
Hiv 1

scholarly journals Characterization of the substrate specificity of the major cysteine protease (cruzipain) from Trypanosoma cruzi using a portion-mixing combinatorial library and fluorogenic peptides

1997 ◽  
Vol 323 (2) ◽  
pp. 427-433 ◽  
Author(s):  
Elaine DEL NERY ◽  
Maria A. JULIANO ◽  
Morten MELDAL ◽  
Ib SVENDSEN ◽  
Julio SCHARFSTEIN ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Substrate Specificity ◽  
Solid Phase ◽  
Cathepsin L ◽  
Amino Acid Residues ◽  
General Preference ◽  
Human Cathepsin ◽  
Hydrolysis Of ◽  
Positively Charged

The substrate specificity of the major cysteinyl proteinase of the parasitic protozoan Trypanosoma cruzi (cruzipain) was investigated, by combinatorial replacement of amino acid residues at positions P5–P´5, using a fluorescent quenched solid-phase library assay. Positively charged residues appear to be a general preference in the P5–P3 and the P´5–P´3 positions, while a hydrophobic residue was always required at the P2 position. A broad range of amino acids could be accepted at the P´1 position. A clear difference in terms of specificity between cruzipain and human cathepsin L was observed for the accommodation of Pro at the P2 position. The P1 specificity was investigated by a more detailed enzyme kinetic analysis using peptidyl-MCA (where MCA is methylcoumarin amide) and Abz-peptidyl-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] as substrates, and the results were compared with those obtained using human cathepsin L. Cruzipain showed a clear preference for benzyl-Cys or Arg at the P1 position. Human cathepsin L presented similar behaviour to that of cruzipain for the hydrolysis of the ε-NH2-Cap-Leu-Xaa-MCA (where Cap is ε-aminocaproyl) and Abz-Lys-Leu-Xaa-Phe-Ser-Lys-Gln-EDDnp series, whereas the mammalian enzyme was able to tolerate large P1 residues, such as phenylalanine, better than cruzipain in the latter series.

Application of solid-phase immune electron microscopy to study physical and stability characteristics of enterically transmitted non-a, non-b hepatitis virus

1988 ◽  
Vol 46 ◽  
pp. 80-81
Author(s):  
Charles D. Humphrey ◽  
E. H. Cook ◽  
Karen A. McCaustland ◽  
Daniel W. Bradley
Keyword(s):  
Electron Microscopy ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Solid Phase ◽  
Etiologic Agent ◽  
World Health ◽  
Health Concern ◽  
Morphological Identification ◽  
Immune Electron Microscopy

Enterically transmitted non-A, non-B hepatitis (ET-NANBH) is a type of hepatitis which is increasingly becoming a significant world health concern. As with hepatitis A virus (HAV), spread is by the fecal-oral mode of transmission. Until recently, the etiologic agent had not been isolated and identified. We have succeeded in the isolation and preliminary characterization of this virus and demonstrating that this agent can cause hepatic disease and seroconversion in experimental primates. Our characterization of this virus was facilitated by immune (IEM) and solid phase immune electron microscopic (SPIEM) methodologies.Many immune electron microscopy methodologies have been used for morphological identification and characterization of viruses. We have previously reported a highly effective solid phase immune electron microscopy procedure which facilitated identification of hepatitis A virus (HAV) in crude cell culture extracts. More recently we have reported utilization of the method for identification of an etiologic agent responsible for (ET-NANBH).

TEM characterization of Au/Polysilicon interactions

1990 ◽  
Vol 48 (4) ◽  
pp. 650-651
Author(s):  
N. David Theodore ◽  
Leslie H. Allen ◽  
C. Barry Carter ◽  
James W. Mayer
Keyword(s):  
Solid Phase ◽  
Single Crystal Silicon ◽  
Chemical Vapor ◽  
Electrical Contacts ◽  
Silicon Substrates ◽  
Crystal Silicon ◽  
Transmission Electron ◽  
Polysilicon Films ◽  
The Stability

Metal/polysilicon investigations contribute to an understanding of issues relevant to the stability of electrical contacts in semiconductor devices. These investigations also contribute to an understanding of Si lateral solid-phase epitactic growth. Metals such as Au, Al and Ag form eutectics with Si. reactions in these metal/polysilicon systems lead to the formation of large-grain silicon. Of these systems, the Al/polysilicon system has been most extensively studied. In this study, the behavior upon thermal annealing of Au/polysilicon bilayers is investigated using cross-section transmission electron microscopy (XTEM). The unique feature of this system is that silicon grain-growth occurs at particularly low temperatures ∽300°C).Gold/polysilicon bilayers were fabricated on thermally oxidized single-crystal silicon substrates. Lowpressure chemical vapor deposition (LPCVD) at 620°C was used to obtain 100 to 400 nm polysilicon films. The surface of the polysilicon was cleaned with a buffered hydrofluoric acid solution. Gold was then thermally evaporated onto the samples.

Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

1998 ◽  
Vol 79 (01) ◽  
pp. 104-109 ◽  
Author(s):  
Osamu Takamiya
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Human Factor ◽  
Solid Phase ◽  
Three Dimensional ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Dimensional Structure ◽  
Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

Identification of a Coronavirus Hemagglutinin-Esterase with a Substrate Specificity Different from Those of Influenza C Virus and Bovine Coronavirus

1999 ◽  
Vol 73 (5) ◽  
pp. 3737-3743 ◽  
Author(s):  
Alfred Klausegger ◽  
Birgit Strobl ◽  
Gerhard Regl ◽  
Alexandra Kaser ◽  
Willem Luytjes ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Solid Phase ◽  
Hepatitis Virus ◽  
Bovine Brain ◽  
Brain Extract ◽  
Binding Assays ◽  
Bovine Coronavirus ◽  
Close Relationship ◽  
Influenza C Virus ◽  
Cloned Gene

ABSTRACT We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substratesp-nitrophenyl acetate, α-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse α1macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.

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