A ttem pts to quantify PI-TP by establishing an E L IS A system w ere unsuccessful as a consequence o f the antisera, even after affinity purification, being unable to function as a capture phase when coated onto the plastic o f E L IS A plates. B iotinylated PI-TP w as used to probe the antibody coated plates and binding o f the biotinyl protein was measured using Extravidin-HRPO with O PD substrate. N o
Supernatant
PI-TP
RIPA treated cell pellet
PI-TP
Tim e (s) 0 15 30 45 60 120 300
Fig 4.5 E x am in atio n o f th e re te n tio n o f P I-T P in SL O perm eablised H L 60 cells
In order to investigate the loss o f PI-TP from perm eabilised H L60 cells further, H L60 cells w ere perm eabilised according to the standard protocol, and both the supernatant, and the extracellular m edium probed for the presence o f PI-TP. Earlier attem pts to carry out these
investigations w ere hindered by the viscous nature o f the cell sample w hen solubilised in SDS PAG E sam ple buffer. It w as therefore decided to treat the perm eabilised cell pellet w ith RIPA buffer, in order to strip away proteins bound to cellular m em branes, w hile leaving the nuclear material as insoluble matter. Each lane corresponds to approxim ately
significant capture w as detected. The ELISA strategies considered are schem atically represented in Chapter 7, Fig. 7.1.
4.8 D iscussion:
Studies o f the kinetics with which the phospholipase C P isozym es leak from the permeabilised cell reveal that by comparison with the similarly sized lactate dehydrogenase, a truly cytosolic protein, PLC leakage is considerably retarded, with the process only being completed after at least 45 minutes from th e start o f permeabilisation. This observation is a confirmation o f results made using biochemical m ethods in a variety o f different cell types (200, 201). Even at the end o f this period it appears likely that some at least o f the PLCP present in the cell rem ains associated either w ith the cell membrane itself, or as suggested by the w ork o f C P D ow nes (16), the underlying cytoskeleton. The ability to reconstitute PLC activity w ithout the addition o f exogenous protein, all be it at reduced levels, in extensively permeabilised cells also supports the premise that some o f the activity remains in th e cell and is dependent upon the maintenance o f some degree o f structural integrity - purified m em branes from disrupted cells produce an extremely poor PLC response to GTPyS stim ulation in com parison to SLO permeabilised cells.
Exam ination o f PI-TP and the kinetics with which it leaves the cell reveals a som ew hat m ore rapid process. This effect is illustrated in Fig. 4.4. Fig. 4.4 B illustrates results obtained from earlier w ork conducted by Dr. E.M . Cunningham in this laboratory using biochemical assays to determine the release o f PI-TP, PLC activity and LD H release from permeabilised H L60 cells. These experim ents w ere conducted in parallel with stimulation o f cells with GTPyS to determ ine the rate at which PLC activity decays in the stimulated cell. As can be seen, P l-transfer activity accum ulated in the supernatant quite rapidly, with 80% release having occurred within 10 m inutes o f permeabilisation. This process is closely paralleled by th e loss o f LD H from the cell. It is particularly w orthy o f note that the responsiveness o f PLC in the permeabilised cell to GTPyS decays over a very similar tim e course. These biochemical observations are confirmed by investigating permeabilisation using
immunological methods. In Fig] 4.4 A the kinetics o f PLC leakage from permeabilised cells follows very closely the kinetics dem onstrated by in vitro assays for the individual activities. This supports the model in which PI-T P plays an im portant role in the function o f this signalling pathway in intact cells (2).
A ttem pts to study the extent to which the protein leaked from the cell by W estern blotting supernatant and residual cell pellets from permeabilisation experim ents revealed that to all intents and purposes, PI-TP com pletely leak from the cell within a period o f approximately five minutes.
R ecent w ork has identified the existence o f another isoform o f P I-T P within mammalian cells with very high degree o f conservation, designated as P I-T P P (146). The sequences o f a and P PI-TP can be found in Chapter I, Fig 1.5. Im m unoreagents have been raised against the a isoform and a panspecific antibody has also been raised in the lab o f K. W irtz and have been used to study the subcellular localisation o f PI-T P in Swiss 3T3 fibroblast cells (168, 202-204). In this case, the differential retention o f detectable levels o f the tw o proteins in various membrane com partm ents including the nucleus and Golgi has been dem onstrated even after extensive permeabilisation. The apparent discrepancy between these observations and W estern blot analysis may be explained as a result o f the different methodologies used, or as a function o f th e cells used in th e tw o different studies. It should also be remembered th at the reagents used in these experiments w ere raised against the P I-T P a and that while considerable hom ology exists between the tw o proteins, the antibodies raised against P I-T P a will not be completely cross reactive with the other isoform. Thus it is conceivable that this reagent will yield results skewed in favour o f the a-isoform and may m isrepresent, or even fail to detect the presence o f the p isoform.