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A Watkinson

Publications and source records attributed to A Watkinson.

64 records · Page 4Linked to original sources
PubMed1987

Partial purification and characterization of an insulin-like material from spinach and Lemna gibba G3.

The existence in invertebrates, unicellular eukaryotes, and prokaryotes of materials that resemble several vertebrate peptide hormones led to the suggestion that these peptide messengers may have arisen earlier in evolution than had previously been thought. Consistent with this hypothesis, we describe here material in two plants, spinach and Lemma gibba G3, that is very similar to mammalian insulin, yet distinctive. In each of the early purification steps, which consisted of acidic methanol chloroform extraction and sequential chromatography on C-18 hydrophobic resin, Sephadex G-50, CM-Sepharose, and a short C-3 high performance liquid chromatography column, the immunoactive material from plants resembled the common vertebrate insulins. The protein nature of the material was suggested by its destruction by Pronase but not by the inactivated enzyme. In addition, on TSK chromatography it eluted in a position similar to that of insulin, i.e. equivalent to a protein of 6000 daltons. Using an isocratic high performance liquid chromatography system, the plant immunoactivity eluted earlier, and thus was more hydrophilic, than most of the common mammalian insulins, including pork insulin. The interaction of the plant material with anti-insulin antibodies in a radioimmunoassay was confirmed by using an affinity column of anti-insulin antibodies which adsorbed the plant immunoactivity at neutral pH, and released the material with acid elution. Using a quantitative double radioimmunoassay, the plant insulin-like material was distinguished immunologically from chicken insulin. Although the plant insulin-like material is clearly distinct from pork insulin chromatographically, and from chicken insulin immunologically, it resembles vertebrate insulins in its overall configuration. The plant insulin-like material bound to insulin receptors on IM-9 lymphocytes and stimulated glucose oxidation and lipogenesis in isolated adipocytes from young rats. The bioactivity was neutralized in the presence of anti-insulin antibodies, but not in the presence of normal guinea pig IgG. The role of this insulin-like material in plants is unknown but its existence is consistent with an early evolutionary origin of the insulin messenger peptide family. Alternatively we cannot exclude a later convergent development of this family or introduction of vertebrate DNA into plants.

Biological Assay↗
PubMed1987

Myristyl and palmityl acylation of the insulin receptor.

The presence of covalently bound fatty acids in the insulin receptor has been explored in cultured human (IM-9) lymphocytes. Both alpha (Mr = 135,000) and beta (Mr = 95,000) subunits of the receptor incorporate [3H]myristic and [3H]palmitic acids in a covalent form. The effects of alkali and hydroxylamine on the labeled subunits indicate the existence of two different kinds of fatty acid linkage to the protein with chemical stabilities compatible with amide and ester bonds. The alpha subunit contains only amide-linked fatty acid while the beta subunit has both amide- and ester-linked fatty acids. Analysis by high performance liquid chromatography after acid hydrolysis of the [3H]myristate- and [3H]palmitate-labeled subunits demonstrates the fatty acid nature of the label. Furthermore, both [3H]myristic and [3H]palmitic acids are found attached to the receptor subunits regardless of which fatty acid was used for labeling. The incorporation of fatty acids into the insulin receptor is dependent on protein synthesis and is also detectable in the Mr = 190,000 proreceptor form. Fatty acylation is a newly identified post-translational modification of the insulin receptor which may have an important role in its interaction with the membrane and/or its biological function.

Acylation↗
PubMed1986

Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits.

The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the alpha- and beta-subunits.

Cells, Cultured↗
PubMed1986

The evolutionary origins of intercellular communication and the Maginot Lines of the mind.

By extending the evolutionary age of the vertebrate hormones from the vertebrates to include the metazoans, we expand their phyletic distribution about 30-fold. By tracing these molecules into the unicellular range including both eukaryotes and prokaryotes, the distribution of these molecules becomes very wide indeed. While "universal" or "ubiquitous" is probably not yet warranted, their recognition as "cosmic" molecules rather than "parochial" molecules does seem appropriate. Interestingly, the breakdown of the barriers for the hormonal molecules between the vertebrates and the rest of the metazoans, between the metazoans and the unicellular organisms, between the eukaryotes and prokaryotes, or the eubacteria and archebacteria is concordant with findings in multiple other systems. For example, hemoglobin or myoglobin is present in higher plants, Protozoa, and insects. The photosynthetic proteins of higher plants have their homologues in the photosynthetic bacteria, and the heat shock proteins of eukaryotes have their equivalents in the prokaryotes as well.

Animals↗
PubMed1985

Evolutionary origins of neuropeptides, hormones, and receptors: possible applications to immunology.

Immune function requires intercellular communication. The vocabulary includes messenger molecules closely linked to the immune system as well as more widely acting messengers such as hormones and neuroactive substances. To try to bring these together, we have used an evolutionary approach. Materials that resemble hormonal peptides and neuropeptides, previously thought to be restricted to multicellular animals, are present in protozoa, bacteria, and higher plants. There is also evidence for substances in microbes that bind hormones and other messengers, which resemble receptors of vertebrates. Therefore, we suggest that the molecules of intercellular communication probably arose much earlier in evolution than the endocrine, nervous, and immune systems. This insight provides new understanding of messenger systems in vertebrates, as applied to the immune system, as well as new insights into possible disease mechanisms, including those that involve autoimmunity.

Allergy and Immunology↗
PubMed1984

The insulin-like action of beta-cell-tropin on glucose and lipid metabolism in adipocytes.

The insulin-like activity of the pituitary pars-intermedia insulin secretagogue beta-cell-tropin, ACTH22-39, has been studied on rat adipocytes. The peptide was prepared by tryptic digestion of synthetic human CLIP, ACTH18-39. beta-Cell-tropin stimulated the incorporation of 3H2O into total lipids. The 50% maximal activity concentration was 5 X 10(-2) ng/ml-1 about 2.5 X 10(-11) M. Iodination of tyrosine, the penultimate amino-acid of the N-terminal, eliminated lipogenic activity, and acetylation of the N-terminal valine reduced activity. ACTH and CLIP (ACTH18-39) had no lipogenic action on the adipocyte system studied. beta-Cell-tropin stimulated the oxidation of glucose and the conversion of glucose into saponified fatty acids and glyceride glycerol. The influence of beta-cell-tropin and insulin on the incorporation of glucose into total lipids, saponified fatty acids and glyceride glycerol was not additive. The results suggest that beta-cell-tropin is either a potent lipogenic hormone, stimulating the conversion of glucose into lipids or that it activates endogenous insulin. The biological activity is associated with the N-terminal amino-acids of the peptide. The possible significance of beta-cell-tropin in obesity is discussed.

Adipose Tissue↗
PubMed1984

Efficacy of a food supplement in correcting riboflavin deficiency in pregnant Gambian women.

Pregnant women living in rural Gambian villages, whose natural riboflavin intake is about 0.5 mg/d, have abnormal biochemical riboflavin status and signs of clinical deficiency. A vitamin-fortified food supplement given in one village which increased the riboflavin intake to about 1.3 mg/d was followed by a substantial improvement in biochemical status, although seasonally-related variations in status somewhat complicated the picture. It was calculated that the amount of riboflavin needed to satisfy the requirement, for normal biochemical status, of the majority of pregnant women throughout pregnancy and throughout the year, is about 2.6 mg/d. Clinical signs associated with riboflavin deficiency, especially atrophic lingual papillae, showed significantly reduced incidence in the supplemented, compared with an unsupplemented, village. Cord blood values of the activation coefficient of erythrocyte glutathione reductase were in the abnormal range for 84 per cent of infants before introduction of the supplement, but were abnormal for only one of 12 infants after its introduction. Thus even a suboptimal maternal riboflavin intake of about 1.3 mg/d appears to be sufficient to prevent biochemical deficiency in cord blood, and to reduce considerably the incidence of clinical deficiency signs during pregnancy.

Adult↗
PubMedDate not supplied

Differential accumulation of catecholamines, proenkephalin- and chromogranin A-derived peptides in the medium after chronic nicotine stimulation of cultured bovine adrenal chromaffin cells.

We have used an antiserum to a synthetic peptide fragment of bovine chromogranin A (ChrgA)[Tyr0] bovine ChrgA (306-313): YLSKEWEDA, together with antibodies to proenkephalin-derived peptides, to measure the release of immunoreactive peptides from nicotine-stimulated cultured bovine adrenal chromaffin cells. Over a period of 6 hr the accumulation of YLSKEWEDA immunoreactivity and Met-enkephalin Arg6Gly7Leu8 (MERGL) immunoreactivity in the medium of 10 microM nicotine-stimulated cells was shown to be biphasic; the initial phase occurred in the first 15-30 min and the second phase reached a peak after 4 hr. In contrast, catecholamine release occurred monophasically over the initial 15-30 min. Investigation of the second phase of peptide accumulation revealed that it was due in part to nicotine-evoked exocytosis and in part to extracellular processing of high molecular weight precursor proteins.

Adrenal Medulla↗