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Type of publication: Baccalaureate Thesis
Type of document:

Year: 2011

Authors: Weeger, Jasmin

Title: Charakterisierung der Aktivität verschiedener Hämproteine des Endoplasmatischen Retikulums in kultivierten Hepatozyten der Ratte.

Other title: Characterization of the activity from different hemeproteins of the endoplasmatic reticulum in cultivated hepatozytes of the rat

Source: Bakkalaureatsarbeit, Vet. Med. Univ. Wien, pp. 50.


Advisor(s):

Duvigneau Catharina

Reviewer(s):
Gille Lars

Vetmed Research Units:
Institute for Medical Biochemistry


Graduation date: 29.08.11


Abstract:
During this work we analyzed activities of the heme enzymes hemoxygenase and the isoenzymes CYP1A2 and CYP2B1 of the endoplasmatic reticulum in cultivated hepatozytes of the rat (BRL3a). Therefore we determined if it is easier to detect the activity in-vitro or in-situ. Aim of the study was to analyze if the product accumulates in the culture supernatant, as it would be an easy way for the determination of the enzyme activities. For measuring the activity in-situ the supernatant was collected after different incubation times and the pellets were processed for other methods. In parallel the activities were determined in cell homogenates (in-vitro) in comparison to liver. Additionally different substrate concentrations and time points were tested in the all assay formats. To that aim, liver cells of the rat (BRL3a) were cultivated and treated with different substrates: hemin for hemeoxygenase, resorufin ethyl ether (REE) for CYP1A2 and resorufin benzyl ether (RBE) for CYP2B1. DMSO was used for vehicle control and the homogenized liver as positive control. The enzyme activity of the BRL3a cells was compared to the activity of liver cells. The products, which were formed by the cells, were extracted into organic solvents and quantified by different means. For measurement of bilirubin, the reaction product of the HO activity, the organic solvents were subjected to photometric analyses, whereas resorufin, the reaction product of the CYP activity, was measured by HPLC. We found, that only stimulation with hemin resulted in a sufficient accumulation of product in the supernatant of BRL3a in function to the time. The supernatant of cells, which were incubated with REE or RBE, did not show any reasonable accumulation of the product resorufin, possibly because it was not stable in medium. In the cell homogenates we could find an in-vitro activity of CYP1A2 which however was much lower than that detected in liver homogenate. Supernatant and cell pellets of cultivated BRL3a cells of the rat can be used for determination of the HO activity in-situ. Due to the relative weak activity it is easier to determine the HO activity in this format, than using an in-vitro assay. For detection of the CYP activity the supernatant could not be used, however the cell homogenates show a weak activity, lower than that found in liverhomogenats with same protein concentration. BRL3a cells are thus suitable for measurement of the HO activity in-situ, but less suitable for the detection of the CYP activity. The substrates REE and RBE seem to be not steady in culture medium, because a product formation can also be found in the absence of cells. Therefore for these assays an accumulation of product can be expected only after a short incubation time and using high substrate concentrations.


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