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At least 973 records · Page 54Linked to original sources
PubMed1993

The affinity-labelling of cathepsin S with peptidyl diazomethyl ketones. Comparison with the inhibition of cathepsin L and calpain.

Since peptidyl diazomethyl ketones are useful irreversible inhibitors for inactivating cysteinyl proteinases in vitro and in vivo and in order to reveal their role, we set out to obtain selective and effective reagents for cathepsin S. A number of such derivatives with hydrophobic amino acid residues, such as valine, leucine and tryptophane in positions adjacent to the primary specificity site were synthesized and these provided inhibitors rapidly acting at high dilution. For example, 1 nM Z-Leu-Leu-Nle-CHN2 inactivates cathepsin S with k2nd = 4.6 x 10(6) M-1 x s-1 at pH 6.5, 25 degrees C. Similarities to the specificities of cathepsin L and calpain were evident. However, Z-Val-Val-NleCHN2 is over 300 times more effective in inactivating S than L. On the other hand, Z-Phe-Tyr(t-Bu)CHN2 is about 10(4) more effective against L than S. Reagents are thus now available for a clear discrimination between these proteases.

Amino Acid Sequence↗
PubMed1989

Size heterogeneity of affinity labeled estrogen receptors in the MtTF4 tumor whose growth is inhibited by estradiol, in pituitary gland and uterus.

UNLABELLED: Estrogen receptors (ER) of the MtTF4 tumor whose growth is inhibited by estradiol (E2) were analyzed and compared to those of tissues whose growth is stimulated by E2 (uterus and pituitary gland). Cytosol prepared in buffer containing protease inhibitors was incubated with [3H]tamoxifen aziridine ([3H]TAZ) in the presence or absence of non-radioactive competitor. The labeled proteins were precipitated, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in denaturing conditions and detected by fluorography. Two classes of ER were identified. The first class is of high molecular weight (Mr = 65,000-64,000). In normal tissues, it is indeed frequently made up of two subtypes as revealed by the presence of a doublet on autoradiograms. In the MtTF4 tumor these subtypes were only rarely suspected and never they were as marked and distinct as in normal tissues. The second class, of low molecular weight (Mr ! 54,000-52,000), is also frequently made up of two subtypes in the uterus and the proportion of this class is higher in the uterus of mature than of immature rats. The MtTF4 tumor contains this class of ER but, due to the presence of non-specifically labeled proteins in this region, its relative amount cannot be estimated and the doublet was exceptionally revealed. In the pituitary gland, this small receptor has not been found. CONCLUSIONS: (i) On the basis of molecular weight analyses, estrogen receptors are heterogeneous, (ii) the ER pattern depends on the type of tissue and the sexual maturity of rats but all the tissues examined contained at least one type of the "classic" high molecular weight receptor, and (iii) no evident correlation was found between the ER pattern and the positive or negative response to estradiol.

Animals↗
PubMed2004

Affinity labeled glutaryl-7-amino cephalosporanic acid acylase C130 can hydrolyze the inhibitor during crystallization.

7Beta-bromoacetyl amino cephalosporanic acid (BA-7-ACA), an analog of glutaryl-7-amino cephalosporanic acid (GL-7-ACA), can inhibit and specifically alkylate GL-7-ACA acylase (C130) from Pseudomonas sp.130, forming a carbon-carbon bond between BA-7-ACA and the C-2 on indole ring of Trp-beta4 residue of C130. Here we reported that BA-7-ACA labeled C130 (BA-C130) could self-catalyze the hydrolysis of BA-7-ACA during crystallization process. The hydrolysis was confirmed to be a reaction analogous to the one of GL-7-ACA by comparative matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry analysis. BA-C130 was inactive at room temperature, but in the process of crystallization at 18 degrees C it catalyzed the hydrolysis of BA-7-ACA, and thus made the latter become a substrate. Meanwhile, in crystals, 7-ACA was released but the acetic acid still bound with Trp-beta4, and as a result, the enzyme remained to be inactive. These results demonstrated that Trp-beta4 in the alphabetabetaalpha motif was critical and sensitive for the activity of C130 and also suggested that there was a conformational change induced by deacylation during the process of crystallization.

Acetic Acid↗