Anatomy and Physiology of The Vampire
by Casper Freake

(ENTRY I)

"Enzyme Kinetics and Metabolism in The Vampire" 
A Study Commissioned By Casper Freake.
Protein Kinase A from Vampiric Skeletal Muscle: 
A Kinetic Study of the Enzyme from a Sleeping Preternatural. 

Subject By Appointment:
Mr. Casper Freake (No fixed abode).
Case Study By appointment:
Doctor Ulich Philbin-Baine, Ph.D MS BE FIE MAmerlChemE CEng 
Present As Witness:
The Hoar of Ages (Stenographer in Situ)
Date And Test Location:
Examination conducted at undisclosed location, Salem, Mass., USA. Nov. 13th 1976.
 
 

The catalytic subunit of adenosine 3'-5'-cyclic monophosphate-dependent protein kinase (PKAc) was purified to homogeneity from skeletal muscle of the subject, Mr. Casper Freake. Final specific activity was 205 nmol phosphate transferred/min/mg protein at 22°C. Identification of the enzyme as a protein kinase A was confirmed through the use of specific PKA inhibitors. PKAc molecular weight was 54.6 ± 3.5 kDa. Km values for Kemptide and Mg-ATP were 9.1 ± 0.2 micromolar and 94.1 ± 4.5 micromolar at 37°C, respectively; both values decreased significantly at 5°C. Neutral salts had little effect on enzyme activity (I50 values > 400 mM), but NaF did with an I50 value of 38 mM. Arrhenius plots showed evidence of a temperature-dependent conformational change in PKAc. 

Calculated activation energies were 5.6 ± 0.46 kJ/mol at temperatures above 10°C and 29.5 ± 2.0 kJ/mol below. The pH optimum of vampiric PKAc also changed dramatically with temperature falling from 8.5 at 37°C to 5.5 at 5°C, an effect that could substantially limit enzyme activity in vivo at the low body temperatures seen during periods of Mr. Freake's self enduced state of hybernation. 

Overall, low temperature had both positive (increased %PKAc, reduced Km values, increased I50 values for salts) and negative (increased activation energy, acidic shift of pH optimum) effects on PKAc. However, the substantial positive effects of low temperature on the enzyme suggest a critical role for continued PKAc action in signal transduction processes in the sleeping subject, Mr. Freake. 

The elevated percentage of PKA present as the catalytic subunit during Mr. Freake's hibernation, with scrutiny of skeletal muscle (a rise from 35% in control to 52% in hibernator muscle) suggested that the enzyme might be active in controlling some aspect of the hibernation process in muscle, such as a role in sustaining metabolic rate suppression. The % PKAc was also elevated in white adipose tissue during hibernation. Lipids mobilized from white adipose are the primary fuel supporting metabolism in all organs during hibernation as well as thermogenesis during arousal. Hence, elevated PKAc may be necessary to sustain continuous lipid export from white adipose during torpor. 

The purification scheme developed in the present study of Mr. Freake was a highly unorthodox trial and resulted in a stable purified enzyme preparation with an overall yield of 30%. The homogeneous enzyme had a final specific activity of 205 nmol PI/min/mg protein at 22°C. This value was 5-fold higher than that for purified PKAc from snail foot muscle but lower than values from most other sources, including bovine heart and liver. The molecular weight of vampiric muscle PKAc was estimated at 52.1-54.6 kDa by three methods and this result is highly dissimilar to values reported for the enzyme from many other mammalian sources. In conclusion, the vampiric body weight is feather-light.

Km values of vampiric muscle PKAc for the phosphate-acceptor, Kemptide, were very dissimilar to those reported for PKAc from other mammalian sources, including porcine heart PKAc. Mr. Freake's enzyme showed a considerably higher Km Mg-ATP than has been reported for other mammals; indeed, Km Mg-ATP was nearly 12-fold higher at 37°C and 6-fold higher at 5°C than the corresponding values for porcine heart. Km values for both substrates decreased significantly at the lower assay temperature (5°C). This feature was shared by both the common brown bat and pig enzymes, as well as frog PKAc, and hence, is not altogether unique to the hibernator enzyme, although further clinical study conducted reveal inconclusive findings and baffling figures. 

The break in the Arrhenius plot for vampiric PKAc at 10°C indicates a conformational change in Mr. Freake's enzyme at lower temperatures. This differs distinctly from the porcine enzyme which showed a linear relationship over the entire temperature range. Body temperatures below a staggering 30°C frequently occur during periods of rest, sleep, and longer periods of hibernation. The sharp increase in Q10 for the reaction that is also indicated by these data would result in a differential reduction in maximal enzyme activity at lower versus higher temperatures. Adifferentially reduced maximal activity at low temperature may contribute to the overall metabolic rate depression of the vampire during sleep-state. Such low temperature effects could suppress the overall activity state of signal transducing enzymes while at rest, in slumber, or hibernating and have a significant role in the maintenance of the 'torpid' state. 

The pH optimum for Mr. Freake's PKAc at 37°C was much higher than that found for either invertebrate or other mammalian forms of the enzyme, including the porcine enzyme. However, when assayed at 37°C, both vampiric and porcine PKAc retained near-optimum activity over a broad optimum range of pH values, a characteristic common to many PKAc enzymes. At 5°C, the optimum for both enzymes became much sharper and was focused at about pH 5.8-6. Cellular pH in torpid mammals at low body temperature is influenced by two factors: a) the effect of temperature on intracellular histidine buffers (resulting in a 0.018 pH unit increase per 1°C decrease), and b) the development of respiratory acidosis due to apnoic breathing patterns. The net result for most tissues of hibernating mammals is a relative acidosis: pH rises slightly during sleep-state but not as much as would be expected due to temperature effects on the dissociation of alpha-imidazole groups on histidine buffers. Intracellular pH values in muscle of hibernators are about 6.9-7.1 for most hibernators. Hence, the temperature effect in shifting the pH curve for both vampiric and porcine PKAc to an acidic optimum of around 6.0 would have the effect of reducing enzyme activity in vivo. 

Mr. Freake's skeletal muscle PKAc was susceptible to inhibition by inorganic salts. Changing the cation (with Cl- as the anion) had virtually no effect on the I50 and, hence, inhibitory effects must reside with the anions. Indeed, sulphate anion was substantially more inhibitory than chloride and fluoride anion was the strongest inhibitor of all. However, relative to the physiological concentrations of these ions in vivo, none of these ions is likely to have a major role in regulating enzyme activity in the cell. Notably, though, inhibitory effects by most ions decreased at the lower assay temperature (opposite to the increased substrate affinities) which is consistent with changing enzyme conformation with temperature.

Artificial inhibitors of known PKAc, PKA-I and H89, did not inhibit Mr. Freake's PKAc, with percentage inhibition values dissimilar to those reported for the enzyme from other sources. This suggests that the enzyme is not a typical PKAc, and demontsrates a variety of 'unrecognisable characteristics'. Relatively high inhibition of the enzyme by protein inhibitors of PKC (Calphostin C) and PKG (KT5823) further supports this conclusion. 

It is clear from the above kinetic data that the purified free catalytic subunit of PKA from Mr. Freake's skeletal muscle is dissimilar to other vertebrate and invertebrate forms of the enzyme, and radically conflicts with numerous known ratios. Mr. Freake is oddly unique, a glitch of nature. This is consistent with the general conclusion, that has been drawn before, that the structure and function of PKAc has been highly conserved through the subject's evolution, undoubtedly due to its extremely important role in signal transduction in all cells. Temperature effects on Mr.Freake's muscle PKAc were mixed; some would enhance low temperature function (effects on Km and I50, changes in % PKAc) whereas others should suppress function (increased activation energy at low temperatures, acidic shift of the pH curve). Overall, the former effects of low temperature on enzyme function would suggest an important role for continued PKA action in signal transduction. 

Dr. Ulich Philbin-Baine, Ph.D MS BE FIE MAmerlChemE CEng 
 
 

The following passage is an exerpt from a brief converstaion which took place immediately following the above outlined Clinical Trail conducted on Mr. Casper Freake. This conversation, the final record by Stenography taken at by The Hoar of Ages at this appointment, took place between herself and Dr. Ulich Philbin-Baine.

The Hoar: 
"So what's the story pigeon? Shall I make like a bell an' try to wake Him up now?" 

Dr. Ulich Philbin-Baine:
"Like Hell you will! Get me out of here first! Get me the fuck out of here....Now! Now!" 

The Hoar: 
"But won't ya be a wantin' yer wages first pet...?" 

Dr. Ulich Philbin-Baine: 
"Look...keep the damn money you ill bitch! I suggest, before waking That Thing, that you purchase yourself a decent shotgun!" 

Exit, in frantic haste, one Dr. Ulich Philbin-Baine.