Alastair Aitken

Identification of the NH2-terminal blocking group of calcineurin B as myristic acid

The NH2‐terminal blocking group of the Ca 2+‐binding B‐subunit of calcineurin (protein phosphatase‐2B) has been identified as myristic acid by fast atom bombardment mass spectrometry and gas chromatography. The sequence, myristyl‐Gly‐Asn‐Glu‐Ala‐, is very similar to that of the catalytic subunit of cyclic AMP‐dependent protein kinase, the only other protein known to contain this fatty acid. This finding, and the elution of all myristyl peptides at 57% acetonitrile on reverse phase HPLC, may facilitate the identification of other proteins with this blocking group.

Multisite Phosphorylation of Glycogen Synthase from Rabbit Skeletal Muscle

Glycogen synthase is a substrate for five distinct protein kinases in skeletal muscle which phosphorylate seven different serine residues on the enzyme. Cyclic-AMP-dependent protein kinase phosphorylates sites 1a, 1b and 2, phosphorylase kinase, site 2, glycogen synthase kinase 3, sites 3a, 3b and 3c, glycogen synthase kinase 4, site 2 and glycogen synthase kinase 5 site 5. Site 2 is seven residues from the N-terminus of glycogen synthase and is located in a cyanogen bromide peptide termed CB1 (apparent Mr = 9000). The other six phosphorylation sites are located in a cyanogen bromide peptide termed CB2 (apparent Mr = 24 000) at the C-terminal end of the molecule. The sequence of the N-terminal 123 residues of peptide CB2, has been completed. Sites 3a, 3b, 3c, 5, 1a and 1b are located at residues 30, 34, 38, 46, 87 and 100 from the N-terminus of CB2 respectively. Site 1a is the next serine residue after site 5. The region surrounding sites 3a, 3b and 3c is very rich in proline residues while that surrounding sites 1a and 1b contains many serine and threonine residues. The 23 residues following site 5 contain 15 aspartic acid and glutamic acid residues, while the region immediately N-terminal to site 1a is very basic. The whole region is remarkably hydrophilic and is the region at which the native enzyme is attacked by proteinases. The sites at which glycogen synthase is cleaved by trypsin, chymotrypsin and thermolysin have been identified. The finding that trypsin cleaves the enzyme C-terminal to site 3c while chymotrypsin cleaves N-terminal to site 3a has formed the basis of a simple procedure for determining the state of phosphorylation of the seven serine residues in vivo [Parker, P.J., Embi, N., Caudwell, F.B., and Cohen, P. (1982) Eur. J. Biochem. 124, 47-55].

Identification of the residues on cyclic GMP-dependent protein kinase that are autophosphorylated in the presence of cyclic AMP and cyclic GMP

Autophosphorylation of cyclic GMP-dependent protein kinase (GMP:protein phosphotransferase, EC 2.7.1.37) in the presence of cyclic AMP and Mg-ATP has already been shown to result in the incorporation of up to 2.6 mol phosphate per mol subunit and decrease the A0.5 for cyclic AMP approx. 10-fold. The major sites of autophosphorylation have now been identified as serine-50, threonine-58, serine-72 and threonine-84. Serine-1 and serine-64 are phosphorylated to a minor extent. Threonine-58, which is initially phosphorylated most rapidly, is also the major site that is phosphorylated in the presence of cyclic GMP and Mg-ATP. Since autophosphorylation in the presence of cyclic GMP does not decrease the A0.5 for cyclic AMP, phosphorylation of serine-50, serine-72, or threonine-84 must be responsible for this effect.

Isolation and characterisation of active fragments of protein phosphatase inhibitor-1 from rabbit skeletal muscle.

Inhibitor1 is a small heat-stable protein first described by Huang and Glinsmann [l] that is a specific inhibitor of protein phosphatase-1 [2,3]. This enzyme is the major protein phosphatase involved in the regulation of glycogen metabolism in skeletal muscle [2-51, but its broad tissue distribution and ability to dephosphorylate enzymes involved in the regulation of glycolysis, fatty acid synthesis, cholesterol synthesis and protein synthesis suggests a wider role in metabolic regulation [3-61. Inhibitor1 is only active after phosphorylation by cyclic AMP-dependent protein kinase [l] on a threonine 171 35 residues form the N-terminus [8]. It is therefore a novel protein in metabolic regulation that can mediate the control of a protein phosphatase by a protein kinase. The phosphorylation of inhibitor-l in mammalian skeletal muscle increases in response to adrenalin [9] and decreases in response to insulin [lo]. The latter is due to suppression of the effects of low concentrations of the /?-adrenergic agonist isoproterenol [ 111. These observations suggest that inhibitor1 may play an important role in the hormonal control of glycogen metabolism and other cellular processes in which protein phosphatase1 participates. The phosphorylated form of inhibitor-l is a non-competitive inhibitor [12] that is effective at nM levels, while the dephosphorylated form is completely inactive [ 121. The physico-chemical properties of inhibitor-l are unusual. It has a very

Amino acid sequence at the site on protein phosphatase inhibitor-2, phosphorylated by glycogen synthase kinase-3

The primary structure surrounding the residue on Inhibitor-2 phosphorylated by glycogen synthase kinase-3 has been determined. The sequence is: Lys-Ile-Asp-Glu-Pro-Ser-Thr(P)-Pro-Tyr-His-Ser. This finding will facilitate studies of the effects of hormones on the phosphorylation state of Inhibitor-2 in vivo.

Amino acid sequence of a region in rabbit skeletal muscle glycogen synthase phosphorylated by cyclic AMP-dependent protein kinase

Glycogen synthase can be phosphorylated in vitro by several protein kinases, producing forms of the enzyme that are more dependent on the allosteric activator glucose 6-phosphate (reviewed in [ 1,2]). One of these glycogen synthase kinases is the enzyme cyclic AMP-dependent protein kinase [3,4] which may underlie the inhibition of glycogen synthase that occurs in vivo in response to adrenaline [5,6]. Cyclic AMP-dependent protein kinase has been shown to phosphorylate glycogen synthase on three serine residues in vitro termed site-l a, site-lb and site-2 [7,8]. The initial rate of phosphorylation of site-l a is 7-lo-fold faster than site-2 and 15-20-fold faster than site-lb; the activity of glycogen synthase is determined by the state of phosphorylation of site-2 as well as site-l a [8]. The phosphorylation of site-lb does not appear to have a direct effect on the activity [7,8]. Site-2, which is located seven residues from the N-terminus of glycogen synthase, is the major site phosphorylated by phosphorylase kinase [9,10]. Here, the amino acid sequence surrounding site-la has been determined. This analysis has surprisingly demonstrated that sites-l a and 1 b are separated by only 13 amino acids in the primary structure of glycogen synthase, which comprises -770 residues [ 1 I].

Daphnane diterpenes of Thymelaea hirsuta

Five 12-hydroxy-daphnane esters were isolated from the leaves and twigs of Egyptian Thymelaea hirsuta. These compounds were identified as gnidicin, gniditrin, genkwadaphnin, the aliphatic C-12 ester, 12-O-heptadecenoyl-5-hydroxy-6,7-epoxy-resiniferonol-9,13,14-orthobenzoate and the novel aliphatic C-12 ester 12-O-butenyl-5-hydroxy-6,7-epoxy-resiniferonol-9,13-14-orthobenzoate.

Amino acid sequence at the major phosphorylation site on bovine kidney branched-chain 2-oxoacid dehydrogenase complex.

Inactivation of branched‐chain 2‐oxoacid dehydrogenase complex correlates with phosphorylation at one site on the α subunit of the E1 component. The amino acid sequence surrounding this phosphorylated serine residue has now been determined. This sequence shows certain similarities with the sequence surrounding phosphorylated residue(s) on pyruvate dehydrogenase complex.

Amino acid sequence around the active serine in the acyl transferase domain of rabbit mammary fatty acid synthase

Rabbit mammary fatty acid synthase was labelled in the acyl transferase domain(s) by the formation of the O‐ester intermediates after incubation with [14C]acetyl‐ or malonyl‐CoA. Elastase peptides containing the labelled acyl groups were isolated using high performance liquid chromatography and sequenced by fast atom bombardment mass spectrometry. An identical peptide (acyl‐Ser‐Leu‐Gly‐Glu‐Val‐Ala) was obtained after labelling with acetyl‐ or malonyl‐CoA. This confirms the hypothesis that, unlike Escherichia coli or yeast, a single transferase catalyses the transfer of both acetyl‐ and malonyl‐groups in the mammalian complex. The sequence at this site is compared with that around the active serine in other acyl transferases and hydrolases.

[13] Protein sequences as taxonomic probes of cyanobacteria

Publisher Summary This chapter outlines studies carried out on primary structures of cyanobacterial proteins and methods used to compare them with the corresponding proteins in eukaryotic and eubacterial cells. This has shed some light on their evolutionary relationships and on the possible origin of chloroplasts. Chloroplasts themselves are semiautonomous organelles with their own prokaryotic type ribosomes and DNA. They have 70 S type ribosomes with the appropriate prokaryotic antibiotic specificities and N-formylated methionine tRNA as the initiator of protein synthesis. A polyphyletic origin of chloroplasts has been proposed on the basis of the similarity in type of chlorophyll and presence of phycobiliproteins in cyanobacteria, Rhodophyta, and Cryptophyta. Cyanobacteria are prokaryotic organisms, and like other prokaryotes, they lack internal compartmentalization of their cells including the membrane-bound nucleus characteristic of eukaryotic cells. They do not undergo mitotic division. Photosynthesis and respiration is carried out on membrane systems in prokaryotes not in separate organelles.