RESULTADOS DEL PROYECTO

L arge volumes o f H L60 cells w ere grow n up for this purpose according to the m ethod already described in Chapter 2. Typically 500x10^ cells w ere cultured, pelleted and washed in 20mM HEPES buffer containing 137mM NaCl, 3m M KCl and lOOjiM EG TA. The cultured cells w ere then treated w ith 2m M D FP on ice for 5 minutes. The cells w ere then washed and resuspended in a hom ogenisation buffer com prising 250m M sucrose, O.IM EGTA, O.IM ED TA, in 20m M PIPES at pH 6.8. Cells w ere then disrupted using a M SE probe sonicator. A post nuclear supernatant w as prepared by centrifugation at 750xg for five minutes. The supernatant w as then further clarified by ultracentrifugation in a Beckm an Ti60 ro to r at 50x10^ rpm fo r 1 hour at 4“C. At the end o f this, the clarified cytosol w as dialysed overnight against several changes o f 3mM KCl, 20mM PIPES pH 6.8, concentrated to approxim ately 5ml in an Amicon concentrator, before being filtered through a 0.45 |iM filter and chrom atographed using 4, 1ml H i-Trap H eparin sepharose columns from Pharmacia.

Initial attem pts at the chromatographic resolution o f the phospholipases C present yielded w hat initially appeared to be a single peak o f PLC activity on the basis

or P IP2 in individual assays it was concluded that there w ere at least tw o distinct activities present, yielding a broader peak o f PIP2 hydrolysing activity, superim posed upon a narrow er, slightly shifted peak o f PI hydrolysing activity. P IP2 is the main

in

vivo

substrate for the activity o f PLCs (145). While under

in vitro

conditions, PLC s can be forced to utilise PI by elevating Ca^^ levels, under the conditions em ployed in the PLC assays under consideration, only PLCsy are able to efficiently hydrolyse PI (144). It w as therefore decided to adjust the FPLC protocol, by running a m ore shallow gradient in order to resolve the peaks o f activity further.

A representative PLC activity profile are illustrated in Fig. 3.1 A. The material w as loaded onto four 1ml H eparin-Sepharose columns, at a rate o f 1ml per minute. A fter loading, the columns w ere run at 2ml per minute with a salt gradient from 0 to IM being applied, 5 minutes after loading and reaching maximal salt concentration at 25 minutes. 2ml fractions w ere collected.

The fractions w ere assayed for the presence o f PLC according to the protocol described in C hapter 2. B oth PI and PIP2 w ere used as substrate in separate assays, in order to help differentiate between the various PLCs present on the basis o f substrate specificity. The results o f the PLC assay are shown in Figure 3 .1 A. As can be seen, utilising P IP2 as substrate, the fractions display three distinct peaks o f activity which are designated as peaks 1 to 3. Using PI as substrate, a single prom inent peak o f activity is observed, corresponding exactly to peak 2 in the P IP2 assay. This suggests the presence o f a PLCy isozyme (145) as observed by S. C ockcroft et al.

3.2.1 Reconstitution of PLC activity in cytosol depleted HL60 cells

A ctive fractions, as determined by PLC assay w ere further examined for their ability to reconstitute GTPyS-stimulated PLC activity in SLO permeabilised H L60 cells. It had previously been observed that the cytolytic bacterial toxin SLO w as able to produce a decay in the responsiveness o f PLCP isozymes to GTPyS and receptor stimulation in HL60 cells. This decay is a consequence o f the loss o f cytosolic com ponents from the cell through toxin-generated pores in the plasma membrane. The ability o f purified com ponents to reconstitute PLC activity could be assessed by means o f adding them back to ^H-inositol labelled, cytosol depleted cells in the

presence o f GTPyS. The resultant level o f activity o f the PLC isozymes present could then be m easured in term s o f the release o f tritiated inositol polyphosphates. The dephosphorylation o f the released inositol phosphates is prevented by the presence o f lithium in the reaction buffer.

The results o f this assay are illustrated in Fig. 3 .2. As can be observed, those peaks from the PLC assay designated as peaks 1 and 2 dem onstrated an ability to stim ulate the hydrolysis o f inositol lipids in the presence o f GTPyS. This assay w as conducted according to the protocol described in C hapter 2. At th e tim e o f perform ing these experiments, it was believed that the reconstituting activity observed w as a consequence o f the presence o f G -protein coupled PLCP isozymes (145).

3.2.2 Western Blotting of resolved PLC activities;

The peaks o f activity identified on the basis o f PLC assay and reconstitution w ere run out on 8% polyacrylamide gels as described in C hapter 2 and transferred onto Im m obilon P, PVD F membrane. The blots w ere then probed w ith antibodies against PLC s p i and 2. The results are shown in Fig. 3.1 B. It w as immediately apparent from the results that a strongly immunoreactive protein w as being recognised by the anti PLC P2 antibodies at a molecular weight o f approxim ately lOOkDa. It w as initially believed that this represented a proteolytic fragm ent o f the full length PLC P2 enzyme.

A fter the initial chrom atography run, further preparations incorporated a cocktail o f protease inhibitors in addition to pre-treatm ent o f the cells w ith D FP, a serine protease inhibitor. N one o f these treatm ents w as able to abrogate the release o f the lOOkDa fragment. A similar observation had been reported by P. G ierschick (Personal communication). From these observations it w as concluded th at P L C p2 w as labile and subject to proteolysis in the course o f preparing th e sample.

A calpain cleavage site was known to be present in the sequence o f PLC P 1 (54) and it w as proposed that PLC P2 may also be cleaved by this activity. T o this end, further runs incorporated a set o f calpain inhibitors. These how ever w ere

A .

In document Nuevas metodologías en el aprendizaje de una lengua extranjera en el primer ciclo de Primaria (página 44-53)