Takaisin Ajatusvarikolle - Back to the Thought Deposit
- Dinoglyfs - Esihistorialliset elšimet historiankirjoissa - Prehistoric Creatures Documented by the Ancient Man

Footnotes and extra appendixes transferred away from the PhD manuscript for the lack of space




No biochemistry has been carried out with twinfilin in its tissue-extracted, possibly modified, physiological form. This is a caveat in comparison to the AC- and drebrin studies, where the work started from the native proteins. Surprises or enhancement for the recombinant forms were seen e.g. in severing activity of cofilin (Ichetovkin et al 2000).


It remains to be studied, whether twinfilin is expressed in the highly specialized cell types such as neutrophiles or platelets. The differences in the PIP2-binding pattern of yeast profilin and cofilin was a surprise. The differential nature had not been found before and awaits clarification.The main difference between AC and twf still is the ATP-binding detected by Beeler et al (1994) and Rohwer et al (1997), although the affinity and the relevance of the ATP/ADP-binding remain to be confirmed. Affinities with physiological significance to ATP range from picomolar (myosin) to millimolar (glycolytic enzymes) KDs, so a quantitative measure has a value as such.


Kinetic studies in (~7 %) glycerol with viscosity as a parameter to follow collisions and intraprotein substrate shuffling should be sought in order to confirm the conformational change and the role of wt twinfilin in it. The linker and tail regions could be classified as motifs in the databases, if they were studied in more detail. I continued the study of the putative hinge region by NMR to get a definite answer, whether the 9s-1 refers to actin or twinfilin. In biphasic binding, what is calculated from the steady state (equilibrium) kinetics is an apparent KD, which incorporates both of the steps. A real equilibrium constant for the first binding step could also be calculated e.g. from the double reciprocal plot Ė if reliable data only was available. {If 1/kobs was plotted against 1/[twf], then intercept on y-axis = 1/k2; slope = 1/(k1 x k2).}


The shortcoming in determining the rate for the first step apart from the second one in the (at least) two-step interaction of wt twf is mainly due to the experimental system that is limiting. To fit and measure the exponential second order rate constant one would need to use very low twinfilin concentrations (below 100 nM or so) and, consequently, much lower NBD-actin concentrations to simplify data analysis. This would obviously limit the amplitude of the fluorescence signal even in the case of an external photomultiplier and our 500 nm cut-off filter. The major question would be the physiological significance of this elaboration, given the high micromolar concentrations of twinfilin from yeast. The only interest would be in elucidation of the molecular mechanism and the precise role of the N-terminal domain.


To get a fluorescence signal from the NBD-label, the binding has to be direct (proximal) with a maximum distance of some 20 Ň, and many ACs cannot be measured by these means. To avoid the NBD-caveat, FRET could be applied. FRET permits the accurate distance measurements of complexes over distances from ~10 to 100 Ň with an accuracy of ~1 nm, in real time (Deniz et al 2000).


The mammalian cofilin pseudogene is considered nothing but an annoyance. When the human genome draft was released, over 30 actin pseudogenes were found (Pollard 2001). Where are they derived from? Could the intriguing question of the physiological role of introns be answered by phylogenetics at a nucleotide level in the light of the conserved actin? Are introns a method of keeping the genes in their place, away from degenerating shuffling and cut'n paste errors? Mutations accumulate faster on pseudogenes, so pseudogene databanks should be self-evident in order for the evolutionary claims to be verifiable. Copying of deliberate and trivial errors in software is the most convincing evidence for piratism in the court. What if the best way to evaluate the probabilities and barriers of variation, adaptation and speciation in nature, would turn out to be pseudogene statistics?