Bror Edlund

Amino acid sequence of a (32P)phosphopeptide from pig liver pyruvate kinase phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase and γ-(32P)ATP

Abstract Pig liver pyruvate kinase (type L) was 32 P-labelled by incubation with ( 32 P)ATP and cyclic 3′,5′-AMP-stimulated protein kinase from the same source. One major ( 32 P)phosphopeptide was isolated from a peptic hydrolysate of the enzyme. Its amino acid sequence was Leu-Arg-Arg-Ala-( 32 P)SerP-Leu.

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 pyruvate kinase

One dominating peptic phosphopeptide, Asx-Thr-Lys-Gly-Pro-Glx-Ile-Glx-Thr-Gly-Val-Leu-Arg-Arg-Ala-(32P)SerP-Val-Ala-Glx-Leu, was obtained from rat liver pyruvate kinase (type L) phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase from the same tissue. The sequence around the phosphorylated serine residue is similar to that of a corresponding but smaller peptic phosphopeptide previously isolated from pig liver (type L) pyruvate kinase, Leu-Arg-Arg-Ala-(32P)SerP-Leu.

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.

Phosphorylation of synthetic peptides by (32P)ATP and cyclic GMP-stimulated protein kinase.

Abstract Cyclic GMP-stimulated protein kinase from pig lung has been shown to phosphorylate synthetic peptides. The rate of phosphorylation was about one order of magnitude higher than that for mixed histones at a comparable concentration, i.e. 0.1 mM. The peptides represented sites, phosphorylatable by cyclic AMP-stimulated protein kinase, in pyruvate kinase type L from rat liver, calf thymus histone H2B and the α-subunit of rabbit muscle phosphorylase b kinase. The shortest pyruvate kinase peptide that could be phosphorylated at a significant rate by cyclic GMP-stimulated protein kinase was Arg-Arg-Ala-Ser-Val-Ala, which is one amino acid residue longer than the minimal substrate of cyclic AMP-stimulated protein kinase. The apparent Km was 0.3 mM which is about 10 times higher than that with cyclic AMP-stimulated protein kinase. The Km was only slightly decreased upon successive extension of the peptide in the N-terminal direction to Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala. Modification of the sequence showed the importance of two adjacent arginyl residues, and substitution of arginine for the C-terminal alanine abolished the measurable activity. Thus, it has been demonstrated that there are both differences and similarities in substrate specificity of the two protein kinases.

Tetrameric structure of nucleoside diphosphate kinase from pea seed.

Nucleoside diphosphate kinase (NDP kinase), (EC 2.7.4.6), catalyzes a transphosphorylation reaction between a nucleoside triphosphate and a nucleoside diphosphate. The enzyme has recently been shown to be phosphorylated during its action 11-51. The enzyme from bovine liver, baker’s yeast and pea seed incorporates 3-4 moles of phosphate per mole of enzyme [6-91, suggesting that the enzyme is composed of four subunits, each with one active site. This view is further supported by the finding by Pedersen [3] that NDP kinase from bovine liver mitochondria contains eight titratable sulfhydryl groups, four of which are accessible in the native enzyme. For these reasons it was of considerable interest to investigate the subunit structure of NDP kinase. This enzyme may be isolated readily and in sufficient amounts from pea seed. The molecular weight of the enzyme is 70,000 [9].

Evidence for an intermediary phosphorylation of nucleoside diphosphate kinase from pea seed

Several nucloside diphosphate kinases (EC 2.7.4.6) have been shown to be phosphorylated by AT32p, among them the enzyme from pea seed [1 ]. The degradation products obtained after alkaline hydrolysis of 32p-labeUed NDP kinases suggest that the phosphoryl group is bound to the N-1 position of an imidazol group in the enzyme [ 1-3]. Kinetic data obtained with NDP kinases from different sources indicate that during catalysis the enzyme exists in two inter.convertable and stable forms, since the mechanism is of the ping-pong type [4-6]. A prerequisite for such a mechanism is that the phosphorylation as well as the dephosphorylation reactions are at least as rapid as the overall reaction. Evidence for this has been presented for the bovine liver enzyme by using a rapid mixing technique [7]. It was, however, of interest to investigate whether this mechanism was valid also for NDP kinases from other sources. In the present work the enzyme from pea seed has been subjected to such an investigation. It has been possible to design the experiments so that all reaction rates studied were determined using the rapid mixing device. The pea seed enzyme was chosen since it represents a phylogenetically different source and can be obtained in reasonable quantities. In the case of the bovine liver enzyme the rate of the partial reactions was determined in a rapid mixing apparatus, while the rat of the overall reaction was measured spectrophotometricaUy by a coupled pyruvate kinase-lactate dehydr0genase method [7]. The rapid mixing experiments were performed in the presence of the components of the coupled assay in order to obtain as similar conditions in the two types of experiment as possible. The results with the bovine liver enzyme suggested, however, that the components of the coupled assay were inhibitory to the enzyme at least under the conditions used. When starting the present investigation this inhibition was also found with the pea seed enzyme. Therefore the overall reaction was measured in the rapid mixing apparatus using no other components than those of importance for the NDP kinase reaction itself. All data including initial velocity analysis of substrate inhibition are in accordance with the view that the phosphorylated enzyme of pea seed NDP kinase is an intermediate in enzyme catalysis

Evidence for identical subunits in pea seed nucleoside diphosphate kinase

All nucleoside diphosphate kinases (NDP kinase) (EC 2.7.4.6) studied have been shown to be phosphorylated by their substrate ATP, incorporating more than 3 moles of phosphate per mole of enzyme [ 13]. The phosphorylated pea seed enzyme has been shown to dissociate into four subunits of equal size when treated with detergent. This suggests that each subunit of this enzyme is phosphorylated [4]. Evidence for this should be at hand if subunits could be shown to be identical. From amino acid analysis of the enzyme and molecular weight of its subunits the average number of lysine and arginine residues in each subunit can be calculated. Provided the subunits are identical, the maximal number of tryptic peptides would be one more than the sum of arginine and lysine residues of each subunit. This possibility has been investigated in the present work.

Phosphorylation of human and rat blood plasma proteins in vitro with cyclic 3′,5′-AMP-stimulated protein kinase and [32P]ATP

The number of enzymes and other proteins known to be reversibly modified by specific phosphorylationdephosphorylation reactions is steadily increasing [ 1 ]. In several cases this phosphate transfer has been shown to result from hormone stimulation and to influence the activity of key enzymes in mammalian metabolism. Phosphorylation might also be involved in the regulation of other processes, e.g., protein synthesis, nervous conduction and secretion [2]. However, for the majority of the proteins modified in this way present knowledge of the function of the phosphorylation seems to be incomplete. Hitherto, interest has been focused on intracellular and on membrane phosphoproteins. With the exception of fibrinogen [3,4] we have found no reports on the phosphate content or on any substantial phosphorylation of blood plasma proteins. We have recently found purified human fibrinogen to be a substrate of cyclic AMP-stimulated protein kinase in vitro, although the possible physiological effects of this phosphorylation are as yet unknown [5]. This report describes the phosphorylation of human and rat plasma and serum protein with the catalytic subunit of cyclic AMP-stimulated protein kinase.

Active Site Phosphopeptides from Pea Seed Nucleoside Diphosphate Kinase

Nucleoside diphosphate kinase from pea seed was incubated with (32P)ATP, inactivated with alkali and digested with trypsin. From the digest the main part of the bound phosphorus was isolated as two phosphopeptides, both containing 14 amino acid residues. These phosphopeptides had an identical amino acid sequence Asx-Val-Ile-His-Gly-Ser-Asx-Ala-Val-Glx-Ser-Ala-Asx-Lys, as determined by dansyl-Edman technique. The only difference found between the two phosphopeptides was a different lability to acid of the phosphoryl bond. The possibility that the appearance of two phosphopeptides was due to a specific migration of the phosphoryl bond within the peptide chain from 1-phosphohistidine to 3-phosphohistidine is discussed.