Similarities between single-chain tissue plasminogen activator and the cell-surface proteinase, guanidinobenzoatase.
AuthorsSteven FS, Griffin MM
JournalBiochem Soc Trans
PubMed ID2125944
Amplified fluorescence sensing of protease activity with conjugated polyelectrolytes.
AuthorsPinto MR, Schanze KS
JournalProc Natl Acad Sci U S A
PubMed ID15136727
'Fluorescent conjugated polyelectrolytes with pendant ionic sulfonate and carboxylate groups are used to sense protease activity. Inclusion of the fluorescent conjugated polyelectrolyte into the assay scheme leads to amplification of the sensory response. The sensing mechanism relies on an electrostatic interaction between the conjugated polyelectrolyte and a peptide substrate that ... More
Sensitive method to identify and characterize proteinases in situ after SDS-PAGE.
AuthorsWilliams J, McGrath WJ, Mangel WF
JournalBiotechniques
PubMed ID11084874
'Cells and body fluids contain numerous, different proteinases; to identify and characterize them are both important and difficult tasks. Especially difficult to identify and characterize are highly specific proteinases. Here, we present an extremely sensitive and quantitative method to characterize proteinases fractionated by SDS-PAGE that cleave specific rhodamine-based fluorogenic substrates. ... More
Observations on the inhibition of serum and cell surface enzymes by eicosapentaenoic acid.
AuthorsSmith KL, Steven FS, Tisdale MJ
JournalJ Enzyme Inhib
PubMed ID1284967
'The relationship between serum and tumour cell surface proteolytic enzymes and the development of muscle breakdown in cancer cachexia has been studied in a murine model of the condition (MAC16). The surface of the MAC16 tumour cells carried a proteolytic enzyme referred to as guanidinobenzoatase (GB). Serum from mice also ... More
Rhodamine-based compounds as fluorogenic substrates for serine proteinases.
AuthorsLeytus SP, Melhado LL, Mangel WF
JournalBiochem J
PubMed ID6342611
'A new fluorogenic substrate for serine proteinases, bis(N-benzyloxycarbonyl-L-argininamido)Rhodamine [(Cbz-Arg-NH)2-Rhodamine], was synthesized, purified and chemically and enzymically characterized. This compound, which employs Rhodamine as a fluorophoric leaving group, is the first in a series of substrates designed to measure the amidase activity of proteinases. Cleavage of one of the amide bonds ... More
The status of trypsin-like enzymes in squamous-cell carcinoma of the head and neck region.
AuthorsSteven FS, Williams LA, Maier H, Arndt J, Weidauer H, Born A
JournalJ Cancer Res Clin Oncol
PubMed ID2312606
'The activity of two proteases associated with tumour cells was studied using frozen sections of squamous-cell carcinoma and fluorescent probes for the enzymes. Four fluorescent probes were used to define the enzymic status of guanidinobenzoatase on the surface of the squamous carcinoma cells. Each of four probes demonstrated the location ... More
Interaction of tetrapyrrolic rings with rhodamine 110 and 123 and with rhodamine 110 derivatives bearing a peptidic side chain.
Rhodamines 110 and 123, and rhodamine 110 linked via peptide bonds to Arg, Cbz-Arg and Cbz-Ile-Pr-Arg interact with free base porphyrins or cytochrome C. Rhodamines 110 and 123 essentially form 1:1 complexes while the other derivatives form 2:1 complexes. The possible biological implications of these results are discussed. ... More
Inhibition of guanidinobenzoatase by a substrate for trypsin-like enzymes.
AuthorsSteven FS, Griffin MM, Mangel WF, Maier H, Altmannsberger M
JournalJ Enzyme Inhib
PubMed ID2467971
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 ... More
Theory and experimental method for determining individual kinetic constants of fast-acting, irreversible proteinase inhibitors.
AuthorsLeytus SP, Toledo DL, Mangel WF
JournalBiochim Biophys Acta
PubMed ID6204689
A theory and experimental method are presented to characterize the kinetics of fast-acting, irreversible proteinase inhibitors. The theory is based upon formal analysis of the case of an irreversible inhibitor competing with a substrate for the active-site of a proteinase. From this theory, an experimental method is described by which ... More
A fluorescent study of ligands for guanidinobenzoatase, a protease associated with tumour cells.
AuthorsSteven F, Griffin MM, Williams LA, Feichter G
JournalAnticancer Res
PubMed ID3218954
We have employed ethanol-fixed wax embedded sections of human breast tumours and smears of rat leukaemia cells to provide test systems with recognisable tumour cells amongst normal cells. We have used 9-aminoacridine to locate cells possesing guanidinobenzoatase, an enzyme which degrades fibronectin and which binds 9-aminoacridine to its active centre. ... More
Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.
AuthorsBoonacker E, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID11724895
Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative ... More
A homogeneous, nonradioactive high-throughput fluorogenic protein kinase assay.
AuthorsKupcho K, Somberg R, Bulleit B, Goueli SA
JournalAnal Biochem
PubMed ID12758259
Protein kinases play an important role in many cellular processes and mediate cellular responses to a variety of extracellular stimuli. They have been identified by many pharmaceuticals as valid targets for drug discovery. Because of the large number of protein kinases, and the large number of compounds to be screened, ... More
Studies on the activity of a protease associated with cells at the advancing edge of human tumour masses in frozen sections.
AuthorsSteven FS, Griffin MM, Maier H, Weidauer H, Mangel WF, Altmannsberger M
JournalBr J Cancer
PubMed ID3166893
A fluorescent probe has been employed to study the status of a tumour associated protease, guanidinobenzoatase, in frozen sections of human tumours obtained from the head and neck regions. The results indicate that in vivo a naturally occurring inhibitor of guanidinobenzoatase effectively controls the activity of this enzyme on the ... More
A homogeneous, nonradioactive high-throughput fluorogenic protein phosphatase assay.
AuthorsKupcho K, Hsiao K, Bulleit B, Goueli SA
JournalJ Biomol Screen
PubMed ID15140384
Protein phosphatases are critical components in cellular regulation; they do not only act as antioncogenes by antagonizing protein kinases, but they also play a positive regulatory role in a variety of cellular processes that require dephosphorylation. Thus, assessing the function of these enzymes necessitates the need for a robust, sensitive ... More
New class of sensitive and selective fluorogenic substrates for serine proteinases. Amino acid and dipeptide derivatives of rhodamine.
AuthorsLeytus SP, Patterson WL, Mangel WF
JournalBiochem J
PubMed ID6228222
A series of dipeptide derivatives of Rhodamine, each containing an arginine residue in the P1 position and one of ten representative benzyloxycarbonyl (Cbz)-blocked amino acids in the P2 position, has been synthesized, purified and characterized as substrates for serine proteinases. These substrates are easily prepared with high yields. Cleavage of ... More