Avtor/Urednik		Plemenitaš, A
Naslov		Semi-intaktne celice: eksperimentalni model za študij metaboličnih procesov
Prevedeni naslov		Semi-intact cells, an experimentalmodel for the studies of metabolic processes
Tip		članek
Vir		Farm Vestn
Vol. in št.		Letnik 47, št. 3
Leto izdaje		1996
Obseg		str. 281-6
Jezik		slo
Abstrakt		Molecular mechanisms of complex metabolic proceses in which intact subcellular organelles are essential have been difficult to investigate with standard cell-free systems. A model of CHO-215 semi-intact cells was aplied for the studies of posttranslational regulatory mechanism(s) of the 3-hydroxy-3-methylglutaryl coenzyme (HMGCoA) reductase activity. HMGCoA reductase is considered to be a rate limiting enzyme in the cholesterol biosynthesis and is a subject of control at multiple levels in animal cells. Our attention is focused on the mechanisms of short-term regulation of HMGCoA reductase activity mediated by cholesterol at the osttranslational level. HMGCoA reductase is a membrane bound enzyme of endoplasmic reticulum and structurally with its seven membrane spanning regions resembles the structure of rhodopsin and beta-adrenergic receptors. The mode of action of these proteins is connected to G-proteins. The question arose whether G-proteins are also involved in regulatory processes of HMGCoA reductase activity. To address these questions we needed a cell-free system that would respond upon perturbation in the similar way as intact cells. Since all attempts with cell-free systems prepared by standard homogenization techniques failed to give statisfactory results, a model of semi-intact cells was applied. In such system by removing the portions of plasm membrane large performations are created that allow the release of soluble cytoplasmic components but leaves the intracelular organelles functionally active and intact. The so celled semi-intact cells have been introduced to study vesicular transport along secretory and endocytic pathways. Semi-intact cells allow direct access of chemicals, different probes and antibodies to the cytoplasmic domain of the cells. In our studies, semi-infact cells prepared from CHO-215 cells were found to respond upon cholesterol addition in the similar way as intact cells. Abstract truncated at 2000 characters.
Izvleček		Delo obravnava možnost uporabe semi-intaktnih celic za študij metaboličnih procesov v celici, konkretno v našem primeru, za uravnavanje aktivnosti 3-hidroksi-metilglutaril A reduktaze (HMGCoA reduktaze) s holesterolom na potranslacijskem nivoju. Pri študiju mehanizmov, s katerimi povečan nivo holesterola v celici posreduje signal za zmanjšanje encimske aktivnosti HMGCoA reduktaze, smo uporabili sistem semi-intaktnih celic CHO-215 (Chinese Hamster Ovary), ki so avksotrofne za holesterol. Pokazalo se je, da semi-intaktne celice, to so celice, ki imajo v plazemski membrani zadosti velike pore, da je omogočen prost pretok vseh topnih citosolnih faktorjev, reagirajo v smislu uravnavanja aktivnosti HMGCoA reduktaze s holesterolom podobno kot intaktne celice. Ugotovili smo tudi, da so za takšen efekt nujno potrebni citosolni faktorji in da so v mehanizem uravnavanja vključeni G-proteini. Semi-intaktne celice omogočajo vnos tako manjših molekul (na različne označene probe), kot tudi vnos protiteles. Sistem smo uspešno testirali z vnosom protiteles, ki reagirajo s HMGCoA reduktazo in s protitelesi, ki reagirajo s fosforiliranimi proteini. Rezultati kažejo, da je tudi v tem smislu sistem semi-intaktnih celic uporaben za študij metaboličnih procesov v celici.
Deskriptorji		CHO CELLS HYDROXYMETHYLGLUTARYL COA REDUCTASES CHOLESTEROL HAMSTERS MODELS, BIOLOGICAL ANTIBODIES