Muscle pyruvate kinase

Davies, C.E., and Kaplan, J.G. (1972) Use of diimidoester cross-linking reagent to examine the subunit structure of rabbit muscle pyruvate kinase. Can. J. Biochem. 50, 416-422. 

K+. Pyruvate kinase is inhibited by 3-5 mM ATP, as well as by acetyl-S-CoA and long-chain fatty acids. Rabbit muscle pyruvate kinase is typically used in ATP regeneration. 

Affinity Labeling of Catalytic ATP Sites. Residues involved in ATP binding are potentially revealed by the use of affinity labels that are based on ATP s structure. Perhaps the most systematically studied of these compounds is 5 -fluorosulfonylbenzoyladenosine (5 -FSBA) (Figure 3a), which has been reported to label at least six kinases (32-A1). In the case of rabbit muscle pyruvate kinase such work has Indicated the presence of a tyrosine residue within the metal nucleotide binding site and an essential cysteine residue located at or near the free metal binding site (32). A similar reagent, 5 -FSBGuanosine, revealed the presence of two cysteine residues at the catalytic site of this same enzyme, both distinct residues from those modified by 5 -FSBA (33,34). With yeast pyruvate kinase both tyrosine and cysteine residues were modified by 5 -FSBA at the catalytic site ( ), and with porcine cAMP-dependent protein kinase a lysine residue was labeled at the active site (36). 

Stuart DI, Levine M, Muirhead H, Stammers DK. Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 AjMol Biol 1979 134 109-142. 

Muscle pyruvate kinase (PK) responds hyperbolically to its substrate, PEP, but the liver form of the enzyme responds sigmoidally. Fructose-1,6-bisphos-phate is an allosteric activator of liver pyruvate kinase, but it apparently has no effect on the muscle enzyme. 

In protein cross-linking studies, DMP has been used to examine the subunit structure of muscle pyruvate kinase (Davies and Kaplan, 1972), for the cross-linking of lactose synthetase (Brew et al., 1975), and to conjugate a fluorescent derivative of a-lactalbumin to glactosyltransferase (O Keefe et al., 1980). The reagent also has... 

I n this way we have shown that phosphoryl transfer catalysed by Bacillus stearothermophilus and rabbit skeletal muscle phosphofructokinase (6), and rabbit skeletal muscle pyruvate kinase occurs with inversion of configuration at phosphorus (7). The simplest interpretation of these stereochemical results is that phosphoryl transfer occurs by an in-line mechanism in the enzyme substrate ternary complexes. Stereochemical analysis is thus proving to be of considerable importance for delineating the mechanism adopted by phosphokinases. ... 

Creatine kinase was purified from rabbit muscle by the method of Kuby et al, (4). Rabbit muscle pyruvate kinase was purchased from Boehringer. Porcine muscle adenylate kinase was purchased from Sigma, and was further purified by gel filtration on Sephadex G-50. The enzymes were homogeneous as judged by their specific activities and by their migration as single components in sodium dodecyl sulfate gel electrophoresis. Proton NMR spectra at 250 MHz of 0.5-2.0 mM enzyme sites in 0 solution were obtained with a Bruker WM 250 MHz pulse FT spectrometer at 25°. At least 256 transients were accumulated over 8192 data points using 16 bit A/D conversion. Relaxation rates and histidine pK values were determined by standard NMR methods (5, 6),... 

The enzyme that catalyzes the conversion of PEP to pyruvate is pyruvate kinase. Liver pyruvate kinase is stimulated allosterically by fructose-1,6-diphosphate, AMP, ADP, and glyceraldehyde-3-phosphate. It is inhibited by alanine, ATP, NADH, and, more importantly, by cAMP- and Ca2 calmodulin-controlled phosphorylation. High blood glucagon levels thus inhibit the activities of both PFK II and pyruvate kinase in the liver through phosphorylation. Transcription of pyruvate kinase is also decreased by glucagon and increased by insulin. Muscle pyruvate kinase is not subject to cAMP or Ca2+ regulation. The pyruvate kinase reaction is practically irreversible. 

D. If the phosphodiesterase that degrades cAMP were inhibited, cAMP levels would rise. Protein kinase A would become more active in the liver and muscle, pyruvate kinase would become less active, and glycogen synthetase activity would be decreased. 

Tor mammalian liver and whelk muscle pyruvate kinases, data for both the dephosphorylated (Dephos) and phosphorylated (Phos) enzyme forms are given, representing for the whelk enzymes pyruvate kinase in aerobic versus anoxic tissue, respectively. 

Chan, L. M Hickmon, T Collilns, C. J., Davidson, Y. Y. (1986). The interaction of rabbit muscle pyruvate kinase with F-actin, Fed. Proc. 45, 1657. 

Storey, K. B. Hochachka, P. W. (1975). Squid muscle pyruvate kinase control properties in a tissue with an active a-GP cycle. Comp. Biochem. Physiol. 52B, 187-191. 

From 31P-NMR studies at one frequency of the ternary complex of muscle pyruvate kinase, Mn, and phosphoglycolate, a competitive analog of the substrate phosphoenolpyruvate, a Mn to P distance of 3.5 0.3 A was obtained suggesting a distorted inner sphere complex ((15), Fig. 4)). 

Isani, G. Cattani, O. Carpene, E. Cortesi, P. Kinetic properties of liver and muscle pyruvate kinase of a marine teleost, sea bass (Dicentrarchus labrax L.).. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 107, 617-624 (1994)... 

Roberts, B. Anderson, P.J. The purification and kinetic characterization of eel white muscle pyruvate kinase. Comp. Biochem. Physiol. B Comp. Biochem., 80, 51-56 (1985)... 

Guderley, H. Hochachka, P.W. Catalytic and regulatory properties of muscle pyruvate kinase from Cancer magister. J. Exp. Zook, 212, 461-469 (1980)... 

Lonberg, N. Gilbert, W. (1983). Primary structure of chicken muscle pyruvate kinase mRNA. Proc. Nall. Acad. Sci. USA, 80, 3661-5. 

The use of thallium-205 as a probe for potassium has been suggested, - and Kayne and Reuben have used the broadening between the signals of the two paramagnetic ions thallium(i) and manganese(n) to estimate the distance between the monovalent and bivalent activators in rabbit muscle pyruvate kinase. Several cases of the inhibition of Ca + transport processes by lanthanides have been reported, and Williams and co-workers have proposed the use of the lanthanides as probes for calcium. 

Rabbit muscle pyruvate kinase was not inactivated when treated at 0° for 5 hr with A -p-fluorobenzoyl-ATP (100 fiM), iV -p-fluorobenzoyl-ADP (260 IJ.M), or A -o-fluorobenzoyl-ADP (3 mM). Yeast hexokinase was apparently not inactivated by (I) or (II) although searching tests could not be made because of the excellent substrate properties of (I) and (II) in the presence of glucose and because of the marked instability of this enzyme in the absence of glucose. 

Initial reaction velocities were measured at 340 nm. For all experiments (except where noted) the final volume of 1.00 ml of 0.1 M Tris chloride (pH 7.6) contained 13 /ug of rabbit muscle lactic dehydrogenase, 0.05 jug of rabbit muscle pyruvate kinase, 0.1 M KCl, 0.025 M MgS04, 1.5 mM phosphoenolpyruvate (sodium salt), 0.25 mAf ADP (sodium salt), and 0.25 mM NADH. The order of addition of the components was the same as described above for AMP aminohydrolase. 

Fig. 11. 3 P-NMR spectra of equilibrium mixtures of (A) rabbit muscle pyruvate kinase (Nageswara Rao et al 1979) and (B) yeast 3-P-glycerate kinase (Nageswara Rao et al 1978b) at catalytic and stoichiometric enzyme concentrations. Spectra for pyruvate kinase were obtained at 40.3 MHz, 15 C, and pH 8.0 for 3-P-glycerate kinase catalytic equilibrium mixture was recorded at 24 MHz and stoichiometric equilibrium mixtures at 109.3 MHz, 1°C, and pH 7.0. The appearance a small P-enolpyruvate signal in the catalytic equilibrium mixture of pyruvate kinase occurs in the presence of a 15-fold excess of pyruvate over ATP. Shifts upheld are negative.

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