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Biomedical subjects

A Aitken

Publications and source records attributed to A Aitken.

At least 127 records · Page 7Linked to original sources
PubMed1987

Amino acid sequence of beta-galactoside-binding bovine heart lectin. Member of a novel class of vertebrate proteins.

A variety of animal tissues contain beta-galactoside-binding lectins with molecular masses in the range 13-17 kDa. There is evidence that these lectins may constitute a new protein family although their function in vivo is not yet clear. In this work the major part of the amino acid sequence of the 13 kDa lectin from bovine heart muscle has been determined. Comparison of this sequence with the cDNA-deduced sequence published for the chick embryo skin lectin showed 58% homology. Comparison of the bovine lectin sequence with partial sequences from two cDNA clones from a human hepatoma library and partial amino acid sequences of human lung lectin showed 70, 40 and 85% homology, respectively. The sequences of these vertebrate lectins are thus clearly related, supporting earlier results of immunological cross-reactivity within this group of proteins. Computer searching of protein sequence databases did not detect significant homologies between the bovine lectin sequence and other known proteins.

Amino Acid Sequence↗
PubMed1987

Embryonal carcinoma-derived growth factor activates protein kinase C in vivo and in vitro.

We have recently reported that a polypeptide mitogen, the embryonal carcinoma-derived growth factor (ECDGF), induces phosphorylation of the epidermal growth factor (EGF) receptor in intact C3H 10T 1/2 mouse fibroblasts with concomittant loss of high affinity EGF binding sites. This phenomenon appears to be mediated through an activation of protein kinase C. Several groups have described an acidic 80,000 dalton protein substrate of protein kinase C. In this paper, we demonstrate that the addition of ECDGF or the phorbol ester TPA to intact C3H 10T 1/2 cells results in the enhanced phosphorylation of this 80 kd protein in vivo. Furthermore, this response is demonstrable in vitro. Thus the addition of ECDGF, the phorbol ester TPA, protein kinase C or phosphoinositidase C to crude membranes prepared from C3H 10T 1/2 cells resulted in the enhanced phosphorylation of this protein. Data obtained by phosphopeptide mapping of the 80 kd protein show that the ECDGF-induced activation of protein kinase C in our membrane preparations is comparable with that obtained in vivo. The availability of an in vitro system in which this response is preserved should now allow a detailed biochemical analysis of the steps between binding of a mitogen to its receptor and the activation of protein kinase C.

Animals↗
PubMed1987

The binding of an aminoazo dye carcinogen to a specific methionine residue in rat liver alcohol dehydrogenase in vivo.

On the administration of 3'-methyl-N,N-dimethyl-4-aminoazobenzene to rats pure aminoazo dye-bound alcohol dehydrogenase accounting for 45% of the total soluble protein bound aminoazo dye is isolated from the liver soluble supernatant. Tryptic digestion of that purified aminoazo dye-bound enzyme yields an aminoazo dye-bound nonapeptide which has a sequence identical to amino acids 301-309 in the known sequence of alcohol dehydrogenase (H. Jornvall and O. Markovic, Eur. J. Biochem., 29 (1972) 167-174) with the exception of methionine 306 which is replaced by an aminoazo dye modified amino acid. The nature of the aminoazo dye adduct was determined by studying the structure of the related tetrapeptide obtained by Pronase B digestion and shown by proton NMR spectroscopy and fast atom bombardment mass spectroscopy to have the structure 3-(Val. Asn. Pro. Homocystein-S-yl)-4-methylamino-3'-methylazobenzene. This carcinogen-protein adduct is assumed to arise from attack of the ultimate carcinogenic metabolite, N-sulphonyloxy-4-methylamino-3'-methylazobenzene (FF. Kadlubar, J.A. Miller and E.C. Miller, Cancer Res., 36 (1976) 2350-2359) at the sulphur of methionine 306 followed by spontaneous S-demethylation. This highly specific reaction of carcinogen with alcohol dehydrogenase lowers its Vmax and increases its Km with cyclohexanone thereby reducing its catalytic efficiency for this substrate. This highly specific reaction of the carcinogen with alcohol dehydrogenase may be regarded as a major detoxication reaction.

Alcohol Dehydrogenase↗
PubMed1987

Toxic phorbol esters from Chinese tallow stimulate protein kinase C.

Phorbol esters were isolated from the seeds of Chinese tallow (Sapium sebiferum L. Roxb.). These compounds were based on the tigliane nuclei, 4-deoxyphorbol, 12-deoxyphorbol and 4,20-dideoxy-5-hydroxyphorbol. The pro-inflammatory activity (ID50) of the pure compounds was between 0.042 and 2.6 nmoles per ear. Protein kinase C activation assays were carried out on samples of enzyme purified from mammalian brain and the activities (Ka) were in the range 76-176 nM. The 4,20-dideoxy-5-hydroxy analogue was inactive in both tests. Chinese tallow, which is used as a substitute for linseed oil, may represent an industrial toxic hazard in terms of both pro-inflammatory and tumour-promoting effects.

Animals↗
PubMed1987

Sapintoxin A. A fluorescent phorbol ester that is a potent activator of protein kinase C but is not a tumour promoter.

In this communication we report on the activity of the naturally occurring, highly fluorescent phorbol ester Sapintoxin A (12-o-[2-methylaminobenzoate]-4-deoxyphorbol 13-acetate). This compound potently activates the enzyme protein kinase C (PKC) (Ka 76 nM) but is neither a complete nor second-stage tumour promoter in traditional Berenblum tests. Sapintoxin A has properties in common with promoters such as 12-o-tetradecanoylphorbol 13-acetate (TPA) in that it will induce erythema in vivo, induce lymphocyte mitogenesis in vitro and cause aggregation of human and rabbit platelets. Accordingly, Sapintoxin A is a suitable negative control compound for biochemical studies concerning the involvement of PKC in tumour promotion and cell proliferation.

Animals↗
PubMed1986

Preferred sites on cytochrome c for electron transfer with two positively charged blue copper proteins, Anabaena variabilis plastocyanin and stellacyanin.

Rate constants for the reactions of horse cytochrome c (E'0 of +260 mV) with the copper proteins Anabaena variabilis plastocyanin (E'0 of +360 mV) used as oxidant and stellacyanin (E'0 of +187 mV) used as reductant have been determined at 25 degrees C, pH 7.5 and 7.0, respectively, and an ionic strength of 0.10 M (NaCl). These rate constants were also measured with eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse cytochrome c derivatives, modified at lysine-7, -13, -25, -27, -60, -72, -86, or -87 and with the trinitrophenyl (TNP) derivative modified at lysine-13. The influence of the modifications on the bimolecular rate constants for these reactions defines the region on the protein that is involved in the electron-exchange reactions and demonstrates that the preferred site is at or near the solvent-accessible edge of the heme prosthetic group on the "front" surface of the molecule. Both reactions are strongly influenced by the lysine-72 modification to the left of the exposed heme edge and, to this extent, behave similar to the earlier studied reaction with azurin. These effects span only an order of magnitude in rate constants and are thus many times smaller than those for the physiological protein redox partners of cytochrome c. While the preferred sites of reaction on the surface of cytochrome c for small inorganic complexes appear to be dependent only on the net charge of the reactants, with the copper proteins additional factors intervene. These influences are discussed in terms of hydrophobic patches and the distribution of charges on the surface of the four copper proteins so far examined.

Binding Sites↗
PubMed1986

Characterization of the phosphorylation of rat mammary ATP-citrate lyase and acetyl-CoA carboxylase by Ca2+ and calmodulin-dependent multiprotein kinase and Ca2+ and phospholipid-dependent protein kinase.

ATP-citrate lyase and acetyl-CoA carboxylase purified from lactating rat mammary gland are phosphorylated stoichiometrically by the calmodulin-dependent multiprotein kinase from rabbit skeletal muscle. The reactions are completely dependent on the presence of both Ca2+ and calmodulin. ATP-citrate lyase and acetyl-CoA carboxylase are also phosphorylated stoichiometrically by the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) purified from bovine brain. Phosphorylation of these substrates is stimulated 6-fold and 40-fold respectively by Ca2+ and phosphatidylserine. The calmodulin-dependent and phospholipid-dependent protein kinases phosphorylate the same serine residue on ATP-citrate lyase that is phosphorylated by cyclic-AMP-dependent protein kinase. The sequence of the tryptic peptide containing this site on the mammary enzyme is identical with the sequence of the peptide containing the site on ATP-citrate lyase that is phosphorylated in isolated hepatocytes in response to insulin and/or glucagon. The calmodulin-dependent, phospholipid-dependent and cyclic-AMP-dependent protein kinases phosphorylate distinct sites on acetyl-CoA carboxylase. However, one of the three phosphorylated tryptic peptides derived from enzyme treated with the phospholipid-dependent kinase is identical with the major phosphopeptide (T1) derived from enzyme treated with cyclic-AMP-dependent protein kinase. Phosphorylation of acetyl-CoA carboxylase by the phospholipid-dependent protein kinase inactivates acetyl-CoA carboxylase in a similar manner to cyclic-AMP-dependent protein kinase. With either protein kinase slightly greater phosphorylation and inactivation is seen after pretreatment of acetyl-CoA carboxylase with protein phosphatase-2A, but the effects of the protein phosphatase treatment are not completely reversed. Inactivation by the phospholipid-dependent protein kinase is Ca2+- and phospholipid-dependent, is reversed by protein phosphatase-2A, and correlates with the degree of phosphorylation. The relevance of these findings to insulin- and growth-factor-promoted phosphorylation of ATP-citrate lyase and acetyl-CoA carboxylase in intact cells is discussed.

ATP Citrate (pro-S)-Lyase↗
PubMed1986

The protein phosphatases involved in cellular regulation. Primary structure of inhibitor-2 from rabbit skeletal muscle.

The complete primary structure of inhibitor-2, a specific inhibitor of protein phosphatase-1, has been determined. The protein consists of a single polypeptide chain of 203 residues, and has a relative molecular mass of 22835 Da. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The threonyl residue phosphorylated by glycogen synthase kinase-3 is located at position 72. The molecule is very hydrophilic, lacks cysteine residues and the single tryptophanyl and phenylalanyl residues are at positions 46 and 139, respectively. The N-terminal alanyl residue is N-acetylated. Digestion with Staphylococcus aureus V8 proteinase, trypsin, or cleavage with cyanogen bromide, destroyed the biological activity of inhibitor-2, demonstrating that many large fragments (e.g. 1-49, 49-92, 67-101, 108-134, 142-182 and 163-197) are inactive. Digestion with clostripain generated a peptide comprising residues 25-114 which retained 2% of the inhibitory potency of the parent molecule. There is no sequence homology between inhibitor-2 and inhibitor-1.

Amino Acid Sequence↗
PubMed1985

Amino acid sequence around the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase.

Two thermolytic peptides containing the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase have been isolated and sequenched by Edman degradation and fast atom bombardment mass spectrometry. The sequence (V-A-G-Y-S-Y-G) contains the motif G-X-S-X-G found around the reactive serine residue of all known serine proteinases and esterases.

Amino Acid Sequence↗
PubMed1985

A simple and rapid method of quantitative analysis of phosphoamino acids by high-performance liquid chromatography.

A high-performance liquid chromatographic system for the separation of nonradiolabeled phosphoamino acids and orthophosphate by ion-pair reverse-phase chromatography has been developed. By the use of low-ionic-strength phthalate buffers at pH 6.3, the phosphoamino acids can be visualized by virtue of this uv-active eluant. The technique is sensitive to 200 pmol of phosphoamino acid and has been shown to be directly applicable to the analysis of isolated phosphoproteins.

Chromatography, High Pressure Liquid↗
PubMed1984

Regulation of bovine kidney branched-chain 2-oxoacid dehydrogenase complex by reversible phosphorylation.

Bovine kidney mitochondrial branched-chain 2-oxoacid dehydrogenase complex is inactivated by covalent phosphorylation catalysed by a specific protein kinase intrinsic to the complex. It has been shown previously [Cook, K.G., Lawson, R. and Yeaman, S.J. (1983) FEBS Lett. 157, 59-62] that tryptic digestion of phosphorylated complex releases three phosphopeptides, indicative of multisite phosphorylation. In this communication we report several findings. (a) These three tryptic peptides contain only two sites of phosphorylation which are closely grouped on the alpha subunit of the E1 component of the complex. (b) The amino acid sequence of the phosphorylated region has been determined. (c) Conditions have been developed which allow investigation of the phosphorylation and dephosphorylation of the two sites. (d) Both sites can be dephosphorylated at significant rates in vitro by two cytosolic protein phosphatases, namely phosphatases 2A and 2C. Dephosphorylation of one site correlates closely with re-activation of the complex.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1984

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.

Amino Acid Sequence↗
PubMed1984

Purification and characterization of thioredoxin from the N2-fixing cyanobacterium Anabaena cylindrica.

Thioredoxin has been purified to homogeneity from the cyanobacterium Anabaena cylindrica. The protein consists of a single polypeptide chain with a relative molecular mass of about 11 680 which has two cysteine residues (residues 31 and 34) in the sequence-Cys-Gly-Pro-Cys- and an isoelectric point at pH 4.55. The N-terminal amino acid sequence of 39 residues shows distinct homologies with the sequences of Escherichia coli and Corynebacterium nephridii thioredoxins. Anti-(A. cylindrica thioredoxin) antiserum was used to quantify the thioredoxin which constituted about 0.22% of the soluble protein in cell-free extracts of N2-fixing, NO3- -grown or NH4+-grown A. cylindrica. Activation of fructose-1,6-bisphosphatase of A. cylindrica, activation of glutamine synthetase and NADP+-dependent malate dehydrogenase of the green alga Scenedesmus obliquus but not of A. cylindrica, and deactivation of glucose-6-P dehydrogenase of the cyanobacterium Anabaena variabilis were all achieved using the same thioredoxin species. No other thioredoxin species were detected in extracts of A. cylindrica when examined for the activation of these enzymes.

Amino Acid Sequence↗
PubMed1984

The structure of the B subunit of calcineurin.

The complete primary structure of the B subunit of calcineurin (protein phosphatase 2B) has been determined by automated sequence analysis. The protein consists of a single polypeptide chain of 168 residues, relative molecular mass 19200. The structure shows 35% identity with the sequence of calmodulin and 29% with troponin C. Homology is mainly confined to the regions of the four putative Ca2+-binding loops. The results demonstrate that the B subunit is a new member of this family of Ca2+-binding proteins. The N-terminal glycine residue is blocked with the C14-saturated fatty acid myristic acid and the first four residues are very similar to those of the catalytic subunit of cyclic-AMP-dependent protein kinase which also contains a myristoyl blocking group.

Amino Acid Sequence↗
PubMed1983

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 alpha 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.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1983

The multifunctional polypeptide chain of rabbit mammary fatty acid synthase contains a domain homologous with the acyl carrier protein of Escherichia coli.

The phosphopantetheine thiol of rabbit mammary fatty acid synthase was specifically alkylated using chloro[14C]acetyl-CoA and a radioactive fragment generated by limited elastase digestion of the modified protein was purified by gel filtration. We have previously mapped this fragment to an internal location in the 250 000-Mr polypeptide adjacent to the thioesterase domain [Eur. J. Biochem. 130, 185-193 (1983)]. The purified fragment had apparent molecular weights of 23 000 by gel filtration and 10 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, while amino acid analysis indicated a minimal molecular weight of 10 400. We have determined the amino acid sequence of the first 64 residues of the fragment. The phosphopantetheine moiety is esterified to a serine at residue 38 in the sequence. When the sequences of the rabbit acyl carrier fragment and the 8847-Mr acyl carrier protein of Escherichia coli are aligned, 17 out of 64 residues are identical. These results suggest that the limited proteolysis delineates an internal acyl carrier domain within the rabbit protein and provide the first clear evidence that multifunctional fatty acid synthases have arisen by fusion of ancestral monofunctional proteins.

Acyl Carrier Protein↗