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1

Influence of organic solvents on papain kinetics

100%
Szabelski M., Stachowiak K., Wiczk W.
Acta Biochimica Polonica
|
2001
|
tom 48
|
nr 4
Papain activity was studied in water-organic solvent mixtures using the fluorogenic substrate Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. The increase of organic solvent (MeOH, EtOH, iPrOH, TFE, MeCN, (MeO)2Et and DMF) concentration in the mixture caused a substantial decrease the initial rate of papain-catalyzed hydrolysis. More­over, the number of papain active sites decreased with the increase of DMF and MeOH concentration.
2

Aldehyde dehydrogenase isoenzymes in tumours - assay with possible prognostic value for oxazaphosphorine chemotherapy

72%
Wroczynski P., Laskowska A., Wierzchowski J., Szubert A., Polanski J., Slowiaczek M.
Acta Biochimica Polonica
|
1998
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tom 45
|
nr 1
A novel fluorimetric assay, allowing independent measurement of the activities of two principal cytosolic forms of human aldehyde dehydrogenase, ALDH-1 and ALDH-3 (known as a tumour-associated ALDH) was applied to estimate the activities of these isoenzymes in human liver and thyroid tumours. The assay is based on two artificial substrates, 6-methoxy-2-naphthaldehyde (MONAL-62) and 7-methoxy-1-naphthaldehyde (MONAL-71), exhibiting excellent substrate properties toward various forms of human ALDH (see Wierzchowski et al., 1997, Anal. Biochem. 245, 69-78). We have found significant differences in ALDH activities between malignant and non-malignant tissue fragments, particularly in cancerous livers. Out of 16 tumours examined, only 4 exhibited ALDH-1 activities comparable to that found in the tumour-free tissue (0.5-2.5 U/g), while in the remaining 12 this activity was at least 10-fold lower. The ALDH-3 activity was detectable in about 40% of both tumour and tumour-free liver samples (maximum value 1.5 U/g). Comparison of 13 pathological thyroid fragments revealed ALDH activities in the range of 0.02 to 0.35 U/g, with two malignant samples showing activities of 0.27 and 0.18 U/g. Both substrate specificity and kinetic behaviour of the thyroid ALDH (Km values for the fluorogenic naphthaldehydes as well as propanal inhibition profile) were similar to those of the purified ALDH-1. In 5 thyroid samples traces of ALDH-3 activity was detected, using MONAL-62 and NADP+ as substrates (maximum value 0.04 U/g). Possible prognostic value of the foregoing measurements for cyclophosphamide chemotherapy is discussed.
3

Influence on Me2SO and incubation time on papain activity studied using fluorogenic substrates

72%
Szabelski M., Stachowiak K., Wiczk W.
Acta Biochimica Polonica
|
2001
|
tom 48
|
nr 4
Papain activity in a buffer containing Me2SO was studied using fluorogenic sub­strates. It was found that the number of active sites of papain decreases with increas­ing Me2SO concentration whereas the incubation time, in a buffer containing 3% Me2SO does not affect the number of active sites. However, an increase of papain in­cubation time in the buffer with 3% Me2SO decreased the initial rate of hydrolysis of Z-Phe-Arg-Amc as well as Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. Moreover, an in­crease of Me2SO concentration in working buffer decreased the initial rate of papain-catalysed hydrolysis of both substrates. A rapid decrease of the initial rate (by up to 30%) was observed between 1 and 2% Me2SO. Application of the Michae- lis-Menten equation revealed that at the higher Me2SO concentrations the apparent values of kcat/Km decreased as a result of Km increase and kcat decrease. However, Me2SO changed the substrate binding process more effectively (Km) than the rate of catalysis kcat.
4
Content available remote

Metabolic mapping by enzyme histochemistry in living animals, tissues and cells

58%
Van Noorden C. J. F.
Journal of Physiology and Pharmacology. Supplement
|
2009
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tom 60
|
nr 4
5
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Optimization of measurement enzyme activity using fluorogenic substrates in water

58%
Skorczewski P., Mudryk Z., Kulinski B.
Baltic Coastal Zone. Journal of Ecology and Protection of the Coastline
|
1999
|
tom 03
6

Fluorogenic peptide substrates for carboxydipeptidase activity of cathepsin B

58%
Stachowiak K., Tokmina M., Karpinska A., Sosnowska R., Wiczk W.
Acta Biochimica Polonica
|
2004
|
tom 51
|
nr 1
Cathepsin B is a lysosomal cysteine protease exhibiting mainly dipeptidyl carboxypeptidase activity, which decreases dramatically above pH 5.5, when the en­zyme starts acting as an endopeptidase. Since the common cathepsin B assays are performed at pH 6 and do not distinguish between these activities, we synthesized a series of peptide substrates specifically designed for the carboxydipeptidase activity of cathepsin B. The amino-acid sequences of the P5-P1 part of these substrates were based on the binding fragments of cystatin C and cystatin SA, the natural reversible inhibitors of papain-like cysteine protease. The sequences of the P'1-P'2 dipeptide fragments of the substrates were chosen on the basis of the specificity of the S'1-S'2 sites of the cathepsin B catalytic cleft. The rates of hydrolysis by cathepsin B and papain, the archetypal cysteine protease, were monitored by a continuous fluores­cence assay based on internal resonance energy transfer from an Edans to a Dabcyl group. The fluorescence energy donor and acceptor were attached to the C- and the N-terminal amino-acid residues, respectively. The kinetics of hydrolysis followed the Michaelis-Menten model. Out of all the examined peptides Dabcyl-R-L-V-G-FE(Edans) turned out to be a very good substrate for both papain and cathepsin B at both pH 6 and pH 5. The replacement of Glu by Asp turned this peptide into an exclusive substrate for cathepsin B not hydrolyzed by papain. The substitution of Phe by Nal in the original substrate caused an increase of the specificity constant for cathepsin B at pH 5, and a significant decrease at pH 6. The results of kinetic studies also suggest that Arg in position P4 is not important for the exopeptidase activity of cathepsin B, and that introducing Glu in place of Val in position P2 causes an increase of the substrate preference towards this activity.
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