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ID naloge: 27 Letnik: 1998 Predmet: patofiziologija
Vezava acetilholinesteraze v motoricnih plocicah zdravih, denerviranih in paraliziranih mišic podgane
Avtor: Blanka Mahne
Mentor: doc. dr. Neva Crne-Finderle
Izhodišce. Prevladuje prepricanje, da so asimetricne oblike AChE prek "kolagenskega repa" vezane na proteoglikane v bazalni lamini živcno-mišicnega stika z ionskimi vezmi. Novejše raziskave na mišicah prepelic pa kažejo, da je v njihovih motoricnih plošcicah vecina AChE vezana bolj trdno, verjetno kovalentno.
Namen. Preverjali smo naslednji hipotezi:
1. V hitrih mišicah podgane je vecji del asimetricnih oblik AChE v motoricnih plošcicah vezan ionsko, manjši pa kovalentno.
2. Razmerje med ionsko in kovalentno vezavo AChE v motoricnih plošcicah mišic podgane se v razlicnih patofizioloških razmerah spreminja.
Metode. Vezavo asimetricnih oblik AChE v motoricnih plošcicah smo proucevali z metodo zaporednega odtapljanja. Poskuse smo delali na normalni podganji mišici extensor digitorum longus (EDL), pa tudi na denervirani, reinervirani in z botulinom paralizirani mišici. Iz plošcicnega podrocja mišic EDL smo v mediju z detergentom najprej odtopili vse globularne oblike AChE. Iz sedimenta vzorca smo nato odtopili AChE v mediju z visoko ionsko mocjo (1 M LiCl). Preostalo plošcicno AChE smo na koncu odtopili z blago proteolizo s papainom (0,2 mg/ml). Aktivnost odtopljene AChE smo dolocali s spektrofotometricno metodo po Ellmanu.
Rezultati. Po predhodnem odtapljanju v mediju z nizko ionsko mocjo in detergentom je v vzorcu iz plošcicnega podrocja normalne mišice EDL 95% celokupne aktivnosti pripadalo plošcicni AChE. Po naknadni inkubaciji v mediju z visoko ionsko mocjo se je iz takih vzorcev odtopilo 67 ± 6% plošcicne AChE; ostalo je še okrog 33% AChE, ki se je odtopila šele po inkubaciji v papainu. Le pri 7 dni denervirani mišici smo lahko dokazali statisticno znacilno (p<0,03), vendar razmeroma majhno zmanjšanje deleža AChE, odtopljenega v mediju z visoko ionsko mocjo (58 ± 7%). Pri vzorcih iz reinerviranih ali paraliziranih mišic EDL pa se ta delež ni statisticno znacilno razlikoval od deleža tako odtopljene AChE iz normalne mišice.
Zakljucki. Rezultati so potrdili dvojno naravo vezave asimetricnih oblik AChE v motoricnih plošcicah hitre mišice podgane in v skladu z našo 1. hipotezo pokazali, da je vecji del AChE v teh motoricnih plošcicah vezan z ionskimi vezmi, manjši pa verjetno kovalentno. Delež ionsko vezane AChE se je rahlo zmanjšal le v denervirani mišici, sicer pa se razmerje med obema vrstama vezave AChE v paralizirani in reinervirani mišici ne spremeni znacilno. Med deležema ionsko in kovalentno vezane AChE v motoricnih plošcicah verjetno obstaja neko ravnovesje, ki se vzpostavlja relativno hitro in je v razlicnih patofizioloških razmerah zelo stabilno.
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[Abstract / English version]
Binding of acetylcholinesterase molecules in neuromuscular junctions of normal, denervated and paralysed rat muscles
Author: Blanka Mahne
Mentor: doc. dr. Neva Crne-Finderle
Background. The asymmetric molecular forms of AChE are probably attached to the basal lamina proteoglycans of the neuromuscular junction (nmj) by ionic interactions involving their "collagenic tail". Nevertheless, recent research on quail muscles showed that most of the AChE in the nmj is more firmly, probably covalently bound.
Objective. We will examine the following two hypotheses:
1. The major part of asymmetric AChE molecules in the motor endplates of fast rat muscles is bound with ionic interactions and the rest is bound covalently.
2. The ratio between ionic and covalently bound AChE in the rat nmj is changes in different patophysiological conditions.
Methods. A sequential solubilization method was used for studying AChE binding in nmj. Experiments were performed on normal rat extensor digitorum longus (EDL) muscles, as well as on denervated, reinnervated and botulinum toxin paralysed muscles. First, all globular AChE forms were solubilized from the nmj region by a detergent solution. Junctional AChE was further solubilized from the pellets in a high ionic strength medium (1 M LiCl). Finally, a mild proteolytic treatment with papain (0,2 mg/ml) was applied to solubilize the residual junctional AChE. AChE activity was measured with the Ellman's spectrophotometric method.
Results. 95% of the AChE activity left over after a detergent treatment in the junctional samples belonged to the junctional AChE. 67 ± 6% of junctional AChE was thereafter solubilized by incubation in a high ionic strength medium; the residual 33% of junctional AChE was extracted only after incubation with papain. In denervated muscles only we could find a small but statistically significant (p<0,03) decrease in the share of AChE which could be solubilized by the high ionic strength medium (58 ± 7%). This share didn't change statistically significantly in reinnervated or paralysed muscle samples in regard to the control muscles.
Conclusions. Our results confirmed the existence of two kinds of binding of the asymmetric AChE forms in the nmj of fast rat muscles. In accordance with our 1st hypothesis, the major part of AChE in these nmj is bound with ionic interactions, and a minor part is bound covalently. The share of AChE bound with ionic forces decreased slightly only in the denervated muscle, during paralysis or after reinnervation the ratio between the two kinds of binding didn't change significantly. A kind of balance probably exists between the ionic and covalently bound AChE in nmj which is established fairly rapidly and is relatively stable in different patophysiological conditions.
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