Elisabet Humble

Non-dependence on native structure of pig liver pyruvate kinase when used as a substrate for cyclic 3′,5′-AMP-stimulated protein kinase

Abstract Alkali-inactivated pig liver pyruvate kinase, type L, and a cyanogen bromide fragment from the same enzyme were shown to be phosphorylated by ( 32 P)ATP and cyclic 3′,5′-AMP-stimulated protein kinase. In both cases the rate of phosphorylation was higher than with the native enzyme. Pyruvate kinases types A and M were not phosphorylated under the same conditions. From the 32 P-labelled cyanogen bromide fragment ( 32 P)phosphorylserine was isolated. The electrophoretic pattern of ( 32 P)phosphopeptides obtained on partial acid hydrolysis of the fragment indicated that the phosphorylated site of the fragment was identical with that of the native pyruvate kinase.

Amino acid sequence at the phosphorylated site of rat liver phenylalanine hydroxylase and phosphorylation of a corresponding synthetic peptide

Abstract Purified rat liver phenylalanine hydroxylase was phosphorylated by incubation with ( 32 P)ATP and the catalytic subunit of pig muscle cyclic AMP-stimulated protein kinase. After digestion of the phosphorylated phenylalanine hydroxylase with pepsin, one major ( 32 P)phosphopeptide with the amino acid sequence Ser-Arg-Lys-Leu-( 32 P)SerP-Asx-Phe-Gly-Glx-Glx was isolated. Two non-phosphorylated octapeptides, Ser-Arg-Lys-Leu-Ser-Asp-Phe-Gly and Ser-Arg-Lys-Leu-Ser-Asn-Phe-Gly, were synthesized and characterized as substrates of the protein kinase.

Phosphorylation of human fibrinogen in vitro with calcium-activated, phospholipid-dependent protein kinase and [32P]ATP

Human fibrinogen is a phosphoprotein with part of the phosphorus bound to Iibrinopeptide A [ 1,2]. However, most of the phosphorous remains bound to the fibrin after splitting off the tibrinopeptides 121. Phosphorylserine occurs at 2 or more locations in the a-chain 131, one of which is SerMl [4]. It was recently found that human fibrinogen is a substrate of cyclic AMP-stimulated protein kinase in vitro with a maximal incorporation of z 6 mol phosphate/mol fibrinogen, preferentially into the a-chain [5]. One of the phosphate-accepting sites is Ser44t of the a-chain [5]. 2.1. Materials [32P]ATP was purchased from New England Nuclear (Boston MA). Human fibrinogen was obtained from Kabi AB (Stockholm). Mixed histone (type II-AS), protamine (salmon sperm), phosvitin, 1,2-diolein and phosphatidylserine were products of Sigma. Sephadex was purchased from Pharmacia Fine Chemicals (Uppsala). The DEAE-cellulose and the phosphorylcellulose used were Whatman DE-52 and P11, respectively.

Kinetic properties of pig pyruvate kinases type A from kidney and type M from muscle.

Abstract Kinetic properties of homogeneous preparations of pig kidney and pig muscle pyruvate kinases (EC 2.7.1.40) were studied. Both isozymes showed a hyperbolic relationship to ADP with an apparent Km of 0.3 m m . K+ and Mg2+ were necessary for the activity of both isozymes, and their dependences on these cations were similar. The muscle isozyme expressed Michaelis-Menten type of kinetics with respect to phosphoenolpyruvate, and the apparent Km was the same (0.03 m m ) from pH 5.5 to pH 8.0. In contrast, the dependence on phosphoenolpyruvate changed with pH for the kidney isozyme. It showed similar properties to the muscle isozyme at pH 5.5–7.0 (apparent Km of 0.08 m m ), while two apparent Km values for this substrate were present at pH 7.5–8.0, one low (0.1 m m ) and one high (0.3–0.6 m m ). At pH 7.5, fructose 1,6-bisphosphate converted the kidney isozyme to a kinetical form where only the lower apparent Km for phosphoenolpyruvate was detected. On the other hand, in the presence of alanine or phenylalanine the kidney pyruvate kinase showed only the higher Km for this substrate. At low phosphoenolpyruvate levels both isozymes were inhibited by phenylalanine, and half-maximal inhibition was found at 0.3 and 2.2 m m for the kidney and muscle isozymes, respectively. At a 5 m m concentration of the substrate only the kidney isozyme was inhibited, the apparent Ki being the same. Alanine inhibited the kidney isozyme (apparent Ki at 0.3 m m , irrespective of substrate concentration). No effect was seen on the muscle isozyme. Fructose 1,6-bisphosphate was an activator of the kidney isozyme at phosphoenolpyruvate concentrations below 1.0 m m It also counteracted the inhibition by alanine or phenylalanine of this isozyme. ATP inhibited both isozymes, and this inhibition was not counteracted by fructose 1,6-bisphosphate. The kidney isozyme showed both a high and a low apparent Km for phosphoenolpyruvate in the presence of ATP. The influence of the effectors on the activity of both isozymes varied markedly with pH, except for the action of ATP. At low substrate concentrations, however, the inhibitor action of ATP on the muscle enzyme was diminished around pH 7.5, in contrast to higher or lower pH values. Alanine or phenylalanine were more effective as inhibitors at higher pH values, and fructose 1,6-bisphosphate stimulated the kidney isozyme only at pH levels above pH 6.5. The influence of activators and inhibitors on the regulation of the kidney and muscle pyruvate kinases is discussed.

Phosphorylation of human fibrinogen in vitro with cyclic 3′,5′-AMP-stimulated protein kinase and (32P)ATP

Abstract Human fibrinogen was shown to be a substrate of the catalytic subunit of pig muscle cyclic 3′,5′-AMP-stimulated protein kinase in vitro . Maximally at least 6 mol of (32P)phosphate per mol of fibrinogen was bound, preferentially to the α-chain.

Amino acid sequence at the phosphorylated site of rat liver fructose-1,6-diphosphatase and phosphorylation of a corresponding synthetic peptide

Rat liver fructose-1,6-diphosphatase was phosphorylated with (32P)ATP and the catalytic subunit of cyclic AMP-dependent protein kinase from pig muscle. After digestion with pepsin, α-chymotrypsin and subtilisin a peptide with the amino-terminal sequence Ser-Arg-Tyr-(32P)SerP-Leu-Pro-Leu-Pro was isolated. A synthetic unphosphorylated heptapeptide with the same amino acid sequence, ending with leucine, was phosphorylated with an apparent Km of 400 μM, while the apparent Km value for fructose-1,6-diphosphatase was 30 μM (subunit concentration). The Vmax value was 20 times higher for the peptide than for the enzyme.

Detection and identification of substrates for protein kinases: Use of proteins and synthetic peptides

Publisher Summary This chapter focuses on the detection and identification of substrates for protein kinases active on serine and threonine residues, with emphasis on the substrates and kinases present in mammalian tissues. The crude tissue extract may consist in isolated cell supernatant or the extracts of particulate cell fractions. When the extracts are incubated with [γ-32P]ATP and Mg 2+ , considerable incorporation of [32P]phosphate into proteins occurs, owing to the presence of protein kinases and endogenous substrates. The method of the interruption of phosphorylation greatly influences the amount and type of [32P]phosphoproteins obtained. When further studies on native phosphoprotein are to be made, the reaction may be interrupted by the removal of free Mg 2+ with ethylenediaminetetraacetic acid (EDTA). Phosphoprotein phosphatases may be inhibited by sodium fluoride or orthophosphate. The 32P-labeled phosphoproteins are separated from most of the low molecular weight, 32P-labeled compounds by rapid gel filtration. The considerable amount of these compounds may be adsorbed to the protein of crude extracts after the gel filtration. To obtain reliable estimates of the true protein phosphorylation, the adsorbed material must be removed by denaturation procedures.

Phosphorylation in vitro of fibrinogen from three mammalian species with four different protein kinases

Human, dog, and rabbit fibrinogen served as substrates for calcium-activated, phospholipid-dependent protein kinase, cAMP-dependent protein kinase, casein kinase TS, and casein kinase S. The chains of phosphorylated fibrinogen were separated by polyacrylamide gel electrophoresis and the phosphorylation patterns, obtained on autoradiography of the gels, were found to be characteristic for each of the four protein kinases. Dog, and even more so rabbit, fibrinogen was phosphorylated more rapidly than human fibrinogen by calcium-activated, phospholipid-dependent protein kinase and by casein kinase TS. Dog fibrinogen was not a good substrate for cAMP-dependent protein kinase. The rate of phosphorylation with casein kinase S did not differ very much between the fibrinogens of the three species. In most cases the A alpha-chain was most rapidly phosphorylated. However, in dog fibrinogen incubated with casein kinase TS the B beta-chain was most rapidly phosphorylated. A substantial part of this phosphate seemed to be incorporated as phosphorylthreonine into fibrinopeptide B. In human fibrinogen incubated with the casein kinase TS preparation the gamma-chain as well as the A alpha-chain appeared to be phosphorylated.

Amino acid sequence around the phosphorylated sites of porcine and rat liver pyruvate kinase, type L

Chymotrypsin removed the phosphorylated site of porcine liver pyruvate kinase without inactivating the enzyme. The amino acid sequence of the phosphopeptide obtained was analyzed. By analysis of CNBr fragments containing 33 amino acid residues, further information was obtained on the amino acid sequence around the phosphorylated sites of porcine and rat liver pyruvate kinase. It was found to be (formula see text) for the porcine isozyme and was very similar for the rat isozyme, although the order of the five most C-terminal amino acid residues (Leu, Pro, Ala2, Homoserine) in this fragment was not resolved and Leu was exchanged for Val in position 12 and Arg for Gln in position 26. The chymotryptic porcine isozyme phosphopeptide, composed of 18 amino acid residues, was entirely contained in the corresponding CNBr fragment (residues 7-24).

Phosphorylation of human fibrinogen in vitro with protein kinase C: Characterization of the phosphorylated sites☆

Phosphorylation of human fibrinogen in vitro by incubation with [gamma-32P]ATP and protein kinase C purified from pig spleen, led to incorporation of [32P]phosphate at serine residues located in the A alpha-chain. In order to identify the residues that were phosphorylated, the A alpha-chain of fibrinogen was isolated and subjected to consecutive cleavage by cyanogen bromide, trypsin, and chymotrypsin. The resulting radioactive phosphopeptides were purified by gel chromatography and high-performance liquid chromatography using a reversed-phase column. Subsequent amino acid analysis and manual Edman degradation of the purified phosphopeptides revealed that Ser557, Ser558, Ser559, and Ser599 were phosphorylated. These serine residues are located in the carboxy-terminal part of the A alpha-chain. This region also contains lysine residues participating in the cross-linking of fibrin and, possibly, a site involved in the binding of fibrinogen to receptors on platelets. In addition, peptides derived from the middle section of the polypeptide chain were found to contain [32P]phosphate; in these cases, however, the exact localization of the phosphate could not be determined, due to the low yield of radioactivity. Two glutamine residues, Gln328 and Gln366, in this portion of the A alpha-chain take part in the cross-linking of fibrin.