Biomedical subjects
Publications and source records attributed to W N Kuo.
The interactions of a number of basic proteins with stimulatory protein kinase modulator (PKMs) from the mouse brain were measured by absorbance at 360 nm. Such interactions were not altered in the presence of Mg2+ at low concentrations.
Three peak-activity fractions of megamodulin were noted in Sephadex G-100 filtration of its crude preparation from baker's yeast (Saccharomyces cerevisiae). Megamodulin thus prepared and its Mg2+-binding complex were shown not only to augment the activity of rabbit brain megamodulin-dependent protein kinase I (M-PK1) but also to interact with arginine-rich histone.
Megamodulin was partially purified from E. coli by the steps of Bio-Gel P-6DG and Sephadex G-100 filtrations, and DEAE-cellulose ion-exchange. It is a heat-stable, trypsin-labile, and acidic protein which appears to be a large molecule in aggregated 'holo' form. In addition, its dissociation into smaller fragments, cation-binding capacity, interaction with basic proteins, and stimulation of megamodulin-dependent protein kinases (M-PK) from rabbit brain or skeletal muscle, were also demonstrated.
The versatile multication binding protein, otherwise known as the stimulatory protein kinase modulator (PKMs) or megamodulin has been isolated from Escherichia coli and it has been found to stimulate E. coli RNA polymerase holoenzyme. It has been hypothesized that megamodulin may regulate RNA polymerase activity by blocking the pausing mechanism and/or increased chain elongation activity during the process of transcription, and also may play an important role in protein synthesis.
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The formation of complexes between cations and stimulatory protein kinase modulator (PKMs) were performed by preincubation and gel filtration. The stimulatory effects of Fe3+, Fe2+, Mg2+, and Co2+ complexes on cerebellar modulator-dependent protein kinases (M-PK) were similar to those without preformation of complexes by preincubation and gel filtration. Antagonism by Ca2+ on the stimulatory effect of Mg2+ was noted in spite of the ineffectiveness of Ca2+ when present alone.
The levels of modulator-dependent protein kinases and protamine-dependent protein kinase(s) in various tissues of adult mice were compared. Cerebellum contained the highest levels of both modulator-dependent protein kinases and protamine-dependent protein kinase(s), whereas skeletal muscle contained no detectable enzymes. The lung and the ileum were also rich in modulator-dependent protein kinases, while other tissues were poor sources of these enzymes.
The separation of modulator-dependent protein kinase I from modulator-dependent protein kinase II obtained from the lungs of sexually premature male mice was accomplished by Sephadex G-200 gel filtration. After preincubation of a mouse lung cytosol fraction with arginine-rich histone, theophylline, cyclic GMP and crude protein kinase modulator a cyclic GMP-dependent protein kinase activity peak present in a non-preincubated sample completely disappeared and was replaced by a late-eluted modulator-dependent protein kinase II peak. There was a difference in substrate specificity between modulator-dependent protein kinase I and modulator-dependent protein kinase II despite their similar dependence on crude protein kinase modulator or partially purified stimulatory protein kinase modulator for their maximal activities.
The spontaneous conversion of mammalian cyclic GMP-dependent protein kinase (G-PK) into modulator-dependent protein kinase (type II) (M-PKII) in the absence of cGMP or histone was observed in vitro. The findings, together with similarity in substrate protein specificity, suggest that M-PKII is the catalytic subunit of mammalian G-PK.
A new type of enzyme, modulator-dependent protein kinase (type I) (M-PKI), was successfully isolated from the cytosol fraction of mouse testes. It was eluted slightly after the peak of cyclic GMP-dependent protein kinase (G-PK) by Sephadex G-200 gel filtration. Unlike either cyclic AMP-dependent protein Kinase (A-PK) or G-PK, its maximal activity depended exclusively on the presence of crude protein kinase modulators (PKM) or partially purified stimulatory modulator (PKMs).
Decreased activities of both the inhibitory modulator of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase (A-PK) as well as the stimulatory modulator of guanosine 3':5'-monophosphate (cGMP)-dependent protein kinase (G-PK) from the mouse cerebellum were noted due to the administration of excessive doses of ethanol, caffeine, and phenobarbital for up to 28 days. The dose-dependent of the inhibition of A-PK or the stimulation of G-PK was observed as a function of the amount of protein kinase modulators in the cerebellum of mice receiving different doses of ethanol.
Changes in relative levels of cyclic AMP-dependent protein kinase (A-PK) and cyclic GMP-dependent protein kinase (G-PK) in prostates, epidymides and testes from guinea-pigs were examined at 3 different ages. During postnatal development, a decrease in the ratio of the 2 classes of protein kinases was seen in prostates, whereas increases of the ratios of the enzymes were found in epididymides and testes.
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Possible involvement of cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and cyclic nucleotide phosphodiesterases in functions of vascular tissues were investigated in the dog. All of the above activities, localized in the smooth muscle-rich inner layer of the blood vessels, were found to be higher in the arteries than in the veins. The peripheral arteries were disproportionately richer in cyclic GMP-dependent protein kinase (as indicated by high ratios of cyclic GMP-dependent to cyclic AMP-dependent protein kinase) than were the veins, with the exception of the pulmonary artery, an atypical arterial tissue exposed to low blood pressure. Interestingly, the protein kinase ratio for the aorta, an artery with no significant role in blood pressure regulation, was not higher than that for the vena cava. Creation of femoral arteriovenous fistulae in the dogs led to preferential reductions in the cyclic GMP-dependent enzyme activity both in the proximal and distal arteries, whereas it was elevated in the stressed vein distal to the anastomotic site. The cyclic GMP-dependent enzyme was preferentially reduced in the saphenous artery distal to occlusion. Changes in the cyclic GMP-dependent enzyme activity appeared to precede gross atrophy or hypertrophy of the vessels. It is suggested that the vascular cyclic GMP-dependent protein kinase may be closely related to peripheral resistance and its regulation.
The stimulatory and inhibitory activities in the crude preparation of protein kinase modulator from dog heart were separated by Sephadex G-100 gel filtration, and the stimulatory modulator was further purified by DEAE-cellulose chromatography. The isolated stimulatory modulator, as the crude modulator preparation, stimulated the activity of the purified guanosine 3':5'-monophosphate (cGMP)-dependent protein kinases of both mammalian and arthropod origins in the presence of cGMP. The cGMP-dependent protein kinases were not activated by cGMP in the absence of either the isolated stimulatory modulator or the crude modulator. The stimulatory modulator, unlike the crude modulator had no effect on the activity of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase. The stimulatory modulator was a protein since its activity was destroyed by trypsin but was resistant to hydrolysis by DNase, RNase, phospholipase C, and lysozyme. The isolated inhibitory modulator, presumably the same as the protein inhibitor of cAMP-dependent protein kinase reported by Walsh et al. (Wash. D.A., Ashby, C.D., Gonzalez, C., Calkins, D., Fischer. E.H., and Krebs, E.G. (1971) J. Biol. Chem. 246, 1977-1985), depressed the cAMP-stimulated activity of cAMP-dependent protein kinase as did the crude preparation of protein kinase modulator. The isolated inhibitory modulator, unlike the crude preparation, was without effect on cGMP-dependent protein kinase. The present findings provide evidence to support that in mammals there are separate proteins for the stimulatory and the inhibitory activities of protein kinase modulator, in contrast to the modulator from an arthropod tissue (lobster tail muscle, Donnelly et al. (Donnelly, T.E., Jr., Kuo, J.F., Reyes, P.L., Liu, Y.P., and Greengard, P. (1973) J. Biol. Chem. 248, 190-198) which has been shown to possess both activities.
The crude protein kinase modulator preparations obtained from several rat tissues (aorta, brain heart, liver, lung, skeletal muscle, small intestine and testis) were separated into their stimulatory and inhibitory modulator components by Sephadex G-100 gel filtration. The isolated stimulatory modulator augmented the activity of guanosine 3':5'-monophosphate-dependent protein kinase. The isolated inhibitory modulator, on the other hand, depressed the activity of cyclic AMP-dependent protein kinase; it was without effect on the activity of cyclic GMP-dependent protein kinease. The present findings indicate that in the mammal, apparently in contrast to the arthropoda, separate proteins are responsibile for the stimulatory and the inhibitory activities of protein kinase modulator and that the two classes of cyclic nucleotide-dependent protein kinase are regulated in an opposing manner by these two types of modulators.