Dataset Information

Design of a transferrin-proteinase inhibitor conjugate to probe for active cysteine proteinases in endosomes.

ABSTRACT: A new technique has been developed to identify active proteinases in endosomes that does not require prior isolation of organelles and extraction of the active enzymes. [125I]Iodotyrosylalanyldiazomethane was reversibly conjugated to transferrin to selectively deliver it to endosomes. The protein was conjugated to the inhibitor via a disulphide bond using N-succinimidyl 3-(2-pyridyldithio)propionate. The inhibitor portion of the conjugate bound irreversibly to active cathepsins B and L, and subsequently the reacted enzymes were separated from the transferrin after SDS/PAGE under reducing conditions. Uptake of the protein-inhibitor conjugate and incorporation of inhibitor into cathepsins was blocked at 4 degreesC, demonstrating that the conjugate enters cells by receptor-mediated endocytosis. Furthermore, endocytosed transferrin-inhibitor conjugate could be recycled back to the extracellular medium and binding to the transferrin receptor could be blocked by native transferrin. Labelling of the enzymes was not blocked by incubating cells at 16 degreesC, consistent with the majority of the reagent being targeted to endosomes. The inhibited enzymes remained conjugated to transferrin, showing that the disulphide bond between the transferrin and inhibitor was not reduced in the endosome. Results from these studies show that endosomes contain both intermediate and late biosynthetic forms of active cathepsin B, which are indistinguishable from those found in mature lysosomes. These results indicate that the active enzymes in endosomes are not early biosynthetic forms in transit to lysosomes but most probably enter the endosome via retrograde traffic from the lysosome.

SUBMITTER: Xing R

PROVIDER: S-EPMC1219918 | biostudies-other | 1998 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. Different probe designs were investigated with respect to sensitivity and selectivity of microarray hybridization. An array with 11 different variants of oligonucleotides for miR-122a and miR-16 was produced. 5 µg of respective total RNA was fluorescently labelled by 3’ ligation. Total RNA was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 5 fmol of each of 816 non redundant miRNAs sequences and miRControl 3 sequences. Local background was subtracted from the signal to obtain the net signal intensity and the mean of the net signal intensities of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was two-fold the mean background value (2bkg dataset).

Dataset Information

Design of a transferrin-proteinase inhibitor conjugate to probe for active cysteine proteinases in endosomes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets