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  • 1
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 48 (1992), S. 411-423 
    ISSN: 0730-2312
    Keywords: cognate peptide substrate ; preprotein processing ; prepro TGFα ; HeLa cells ; cell surface proteases ; aminopeptidases ; endopeptidases ; product profiling ; thin layer chromatography ; factor regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A radiometric assay has been developed for the detection of proteolytic activity capable of releasing transforming growth factor alpha (TGFα) from its membrane bound precursor. The assay is dependent upon the separation by thin layer chromatography of hydrolytic products of a nonapeptide substrate containing a radioactive iodinated tyrosine residue as a reporting group N-terminal to an octapeptide which is cognate to the N-terminal cleavage sequence of TGFα. We describe the selectivity of the peptidase assay with commercially purified proteases and with cell-associated peptidases, its exquisite sensitivity, and its applicability to defining peptidase activity, which may be responsible for the processing of the membrane-bound prepro TGFα. The activity of two different elastases had different profiles which thus may be of use in characterizing them. The characteristics of the intact and extracted HeLa cell assay with respect to time, cell density, and peptidase concentration are defined, as are conditions needed to remove endogenous, confounding, proteolytic activity from the serum used to support cell culture. Intact HeLa cell cultures exhibit both exo- and endo-peptidase activity at approximately equal levels in both sparse and dense monolayer culture without relationship to cell density, and at a level equal to 1-2% of total cell activity of these enzyme classes.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 54 (1994), S. 320-331 
    ISSN: 0730-2312
    Keywords: apoptosis ; cell surface proteases ; ectopeptidases ; prepro TGFα ; UVC-irradiation ; growth factor regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have previously characterized the stimulation of HeLa cell surface peptidase activity directed toward a nonapeptide substrate in response to low fluences of ultraviolet irradiation [Brown et al. (1993): J Cell Biochem 51:102-115]. To explore the hypothesis that this comprised a global response to cell stress featuring the interruption of DNA synthesis, a variety of agents affecting macromolecular synthesis were applied to HeLa cell cultures. Actinomycin D, 5,6-dichloro-1 β-ribofuranosyl benzimadazole, mitomycin C, ultraviolet light, and cycloheximide at doses which inhibited cell growth, but fell short of increasing the proportion of cells which had lost cell membrane impermeability to trypan blue, resulted in the concentration dependent increase in both amino- and endo-peptidase activities of intact HeLa cell cultures. γ-Irradiation, despite inhibiting an increase in cell number over a 20-h observation period, had no effect on the expressed level of cell surface peptidase activity nor did the accumulation of cells in S or G2 phase by thymidine parasynchronization. Some of these agents were found to increase the proportion of cells in the culture undergoing apoptosis (programmed cell death), and a strong correlation was found between the extent of apoptosis and the degree of elevation in cell surface peptidase activity. Higher concentrations of perturbants in some instances increased the percentage of cells that were nonviable and an associated release of intracellular proteases overwhelmed the linear correlation with apoptotic cells. The present data do not distinguish between a homogeneous elevation of surface peptidase activity in all cells of treated cultures or the heterogeneous increase in only preapoptotic or apoptotic cells. Since sunburn of the skin increases both the occurrence of apoptotic keratinocytes (sunburn cells) in the affected epidermis and the release of membrane bound cell activators such as transforming growth factor α, it is suggested by way of extrapolation of these in vitro results, that the increase in cell surface proteolytic activity plays an integral part in the reparative responses of the epidermal cells in vivo.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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