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A I Magee

Publications and source records attributed to A I Magee.

At least 73 records · Page 4Linked to original sources
PubMed1987

Dynamic fatty acylation of p21N-ras.

To study the acylation of p21N-ras with palmitic acid we have used cells which express the human N-ras gene to high levels under control of the steroid-inducible MMTV--LTR promoter. Addition of [3H]palmitate to these cells resulted in detectable incorporation of label into p21N-ras within 5 min, which continued linearly for 30-60 min. Inhibition of protein synthesis for up to 24 h before addition of [3H]palmitate had no effect on acylation of p21N-ras, suggesting that this can occur as a late post-translational event. Acylated p21N-ras with a high SDS--PAGE mobility is found only in the membrane fraction, whereas approximately 50% of the [35S]methionine-labelled p21N-ras is cytoplasmic and has a lower mobility. Conversion of the acylated high mobility form to a deacylated form of slightly lower mobility can be achieved with neutral hydroxylamine, which is known to cleave thioesters. This treatment also results in partial removal of p21N-ras from the membranes. A remarkably high rate of turnover of the palmitate moiety can be demonstrated by pulse--chase studies (t1/2 approximately 20 min in serum-containing medium) which cannot be attributed to protein degradation. The data suggest an active acylation--deacylation cycle for p21N-ras, which may be involved in its proposed function as a signal transducing protein.

Acylation↗
PubMed1987

Calcium-induced changes in cytoskeleton and motility of cultured human keratinocytes.

In normal epidermis keratinocytes migrate upward from the basal layer as they undergo terminal differentiation, yet they also have the capacity for lateral movement during wound healing. The purpose of our experiments was to investigate these two types of movement by manipulating the calcium ion concentration of the medium so that keratinocytes formed monolayers (0.1 mM calcium) or stratified sheets (2.0 mM calcium). Time-lapse video recording indicated that keratinocytes in low-calcium medium were laterally more motile than keratinocytes in normal medium. This was consistent with the ultrastructural appearance of the cells and the lack of desmosomal junctions, determined by scanning and transmission electron microscopy. During calcium-induced stratification keratinocytes moved upward from the basal layer by gliding over their neighbors and forming contacts with other suprabasal cells. Keratinocytes in low-calcium medium migrated into wounds made in the cultures, a process which was inhibited by monensin; however, stratified keratinocytes in normal medium did not enter wounds. Cytochalasin D caused rapid cell rounding and disruption of actin filaments in keratinocytes grown in low-calcium but not in normal medium, indicating more rapid treadmilling of actin and consistent with the greater motility of keratinocytes in low-calcium medium. Our results suggest that desmosome formation may place constraints on the movement of individual keratinocytes and that the actomyosin cytoskeleton is involved in lateral migration.

Actins↗
PubMed1987

Fatty acid acylation of proteins in Physarum polycephalum.

We have investigated the occurrence of protein-fatty acid acylation by metabolic incorporation of [3H]myristic and [3H]palmitic acids in Physarum polycephalum. We show that this organism contains fatty acylated proteins with mainly myristic acid covalently attached in alkali-stable linkages, probably amides. We find no evidence for ester-linked fatty acids, in contrast to the situation in vertebrate cells.

Acylation↗
PubMed1987

Structure and assembly of desmosome junctions: biosynthesis, processing, and transport of the major protein and glycoprotein components in cultured epithelial cells.

Extracts of metabolically labeled cultured epithelial cells have been analyzed by immunoprecipitation followed by SDS-PAGE, using antisera to the major high molecular mass proteins and glycoproteins (greater than 100 kD) from desmosomes of bovine muzzle epidermis. For nonstratifying cells (Madin-Darby canine kidney [MDCK] and Madin-Darby bovine kidney), and A431 cells that have lost the ability to stratify through transformation, and a stratifying cell type (primary human keratinocytes) apparently similar polypeptides were immunoprecipitated with our antisera. These comprised three glycoproteins (DGI, DGII, and DGIII) and one major nonglycosylated protein (DPI). DPII, which has already been characterized by others in stratifying tissues, appeared to be absent or present in greatly reduced amounts in the nonstratifying cell types. The desmosome glycoproteins were further characterized in MDCK cells. Pulse-chase studies showed all three DGs were separate translation products. The two major glycoprotein families (DGI and DGII/III) were both found to be synthesized with co-translational addition of 2-4 high mannose cores later processed into complex type chains. However, they became endo-beta-N-acetylglucosaminidase H resistant at different times (DGII/III being slower). None of the DGs were found to have O-linked oligosaccharides unlike bovine muzzle DGI. Transport to the cell surface was rapid for all glycoproteins (60-120 min) as demonstrated by the rate at which they became sensitive to trypsin in intact cells. This also indicated that they were exposed at the outer cell surface. DGII/III, but not DGI, underwent a proteolytic processing step, losing 10 kD of carbohydrate-free peptide, during transport to the cell surface suggesting a possible regulatory mechanism in desmosome assembly.

Adenocarcinoma↗
PubMed1987

Structure and assembly of desmosome junctions: biosynthesis and turnover of the major desmosome components of Madin-Darby canine kidney cells in low calcium medium.

Neither stratifying (primary keratinocytes) nor simple (Madin-Darby canine kidney [MDCK] and Madin-Darby bovine kidney [MDBK]) epithelial cell types from desmosomes in low calcium medium (LCM; less than 0.1 mM), but they can be induced to do so by raising the calcium level to physiological concentrations (standard calcium medium [SCM], 2 mM). We have used polyclonal antisera to the major bovine epidermal desmosome components (greater than 100 kD) in a sensitive assay involving immunoprecipitation of the components from metabolically labeled MDCK cell monolayers to investigate the mechanism of calcium-induced desmosome formation. MDCK cells, whether cultured in LCM or SCM, were found to synthesize the desmosome protein, DPI and desmosome glycoproteins DGI and DGII/III with identical electrophoretic mobility, and also, where relevant, with similar carbohydrate addition/processing and proteolytic processing. The timings of these events and of transport of DGI to the cell surface were similar in low and high calcium. Although the rates of synthesis of the various desmosome components were also similar under both conditions, the glycoprotein turnover rates increased dramatically in cells cultured in LCM. The half-lives decreased by a factor of about 7 for DGI and 12 for DGII/III and, consistent with this, MDCK cells labeled for 48 h in SCM had three and six times the amount of DGI and DGII/III, respectively, as cells labeled for 48 h in LCM. The rate of turnover and the levels of DPI were changed in the same direction, but to much lesser extents. Possible mechanisms for the Ca2+-dependent control of desmosome formation are discussed in the light of this new evidence.

Animals↗
PubMed1986

A major role for carbohydrate epitopes preferentially recognized by chronically infected mice in the determination of Schistosoma mansoni schistosomulum surface antigenicity.

A radioimmunoassay that makes use of whole schistosomula and 125I-labeled protein A has been used to characterize and to quantify the binding of antisera to the surface of 3 hr mechanically transformed schistosomula of Schistosoma mansoni. This technique facilitates the determination of epitopes on the schistosomula in addition to those detected by surface labeling and immunoprecipitation. By using this technique, it has been demonstrated that there is a much greater binding to the parasite surface of antibodies from chronically infected mice (CMS) than of antibodies from mice infected with highly irradiated cercariae (VMS), and CMS recognizes epitopes that VMS does not. Treatment of the surface of the schistosomula with trifluoromethanesulphonic acid and sodium metaperiodate has suggested that the discrepancy of the binding between the two sera is due to the recognition of a large number of additional epitopes by CMS, which are carbohydrate in nature. Some of the carbohydrate epitopes are expressed on the previously described surface glycoprotein antigens of Mr 200,000, 38,000, and 17,000.

Animals↗
PubMed1985

Two classes of fatty acid acylated proteins exist in eukaryotic cells.

Labelling of cultured cells with [3H]palmitic and [3H]myristic acids demonstrates that each of these fatty acids modifies a substantially different subset of cellular proteins. Hydroxylamine treatment can be used to differentiate sensitive thioester linkages to palmitate from insensitive amide linkages to myristate. Several palmitoylated proteins are surface-oriented glycoproteins while all of the myristylated proteins appear to be internal. Myristate addition is much more tightly coupled to protein synthesis than palmitoylation, which is able to continue at a reduced level even in the prolonged absence of protein synthesis. Acyl proteins patterns were affected both qualitatively and quantitatively by transformation and growth status. The preferential addition of palmitate to the transferrin receptor and myristate to pp60src, and the absence of these modifications from several other proteins is reported. We propose a nomenclature for fatty acyl proteins based on these observations.

Acylation↗
PubMed1984

Release of fatty acids from virus glycoproteins by hydroxylamine.

The fatty acids bound to the glycoproteins of Sindbis and vesicular stomatitis viruses can be released by treating the protein with 1 M hydroxylamine at pH 8.0, but the rates of release vary greatly among the three proteins. The most labile fatty acyl bonds were in the Sindbis virus PE2/E2 proteins and the most stable were in the E1 protein. Some of the fatty acids in Sindbis virus glycoproteins were reduced to the alcohol after treatment with sodium borohydride, indicating that protein-bound fatty acids could be in thiolester linkage. Sindbis virus PE2/E2 has several cysteine residues near the carboxy terminus, a region of the protein postulated to be localized on the inside (cytoplasmic face) of the bilayer, and protease digestion of microsomal membranes containing E2 protein removed a small portion of this cytoplasmic tail as well as significant amounts of the fatty acid. For the vesicular stomatitis virus G protein, the sensitivity of fatty acid hydrolysis appeared to depend on the conformation of the protein and a significant fraction of G protein was converted to a disulfide-linked dimer by hydroxylamine. These data implicate cysteinyl groups on these proteins as sites involved in fatty acid acylation.

Animals↗
PubMed1981

Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from alpha-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity.

We report a novel assay method for enterokinase capable of detecting approx. 1 fmol of enzyme. The method depends on quantification of the release of specifically radiolabelled activation peptides from bovine trypsinogen and is unaffected by trypsin inhibitors. The assay is applicable to biological fluids such as serum. The substrate was produced by selective epsilon-amidination of bovine trypsinogen followed by acetylation with [3H]acetic anhydride and deprotection. The assay has been used to study the effects of pH, Ca2+, ionic strength abd glycodeoxycholate on enterokinase activity.

Acetylation↗
PubMed1981

Cleavage of peptide hormones by alpha 2-macroglobulin-trypsin complex and its relation to the pathogenesis and chemotherapy of acute pancreatitis.

Access to the active site of alpha 2-macroglobulin bound proteinases by macromolecular substrates and inhibitors is likely to be influenced by steric favourability and flexibility, as well as by molecular size. Hydrolysis of pure porcine cholecystokinin-pancreozymin 39, a flexible single chain peptide, by alpha 2-macroglobulin-trypsin complex resulted in a rapid up to 6-fold increase in cholecystokinin bioactivity; alpha 2-macroglobulin-trypsin rapidly abolished the bioactivity of endogenous parathormone in human plasma. Inhibition of both reactions was completed by low concentrations of antipain and leupeptin; Trasylol (aprotinin), a single chain peptide with three disulphide bonds, was an ineffective inhibitor even in massive molar excess. These findings may provide an explanation for the disordered calcium homeostasis in severe acute pancreatitis and for the failure of Trasylol to reduce mortality; they suggest that sterically favourable low molecular weight inhibitors may provide effective specific chemotherapy for the disease.

Acute Disease↗
PubMed1981

Further studies on the subunit structure and oligosaccharide moiety of human enterokinase.

Highly purified human enterokinase was found by SDS-polyacrylamide gel electrophoresis to contain three heavily glycosylated subunits of apparent molecular masses 54 000, 102 000 and 140 000. The smallest subunit contained the active site serine residue and the oligosaccharide chains appear to be N-glycosidically linked as inferred from their stability to mild alkaline hydrolysis. Lectin affinity chromatography was used to separate sub-populations of the enzyme, the major one of which appeared to contain terminal alpha -linked N-acetyl galactosamine. Despite the presence of this sugar, no anti-A response was elicited in rabbits immunized with this sub-population. However, this sub-population did bind rabbit antibody directed against human blood group A substance, suggesting the presence of an "A-like" determinant. Studies with immobilized rabbit anti-human blood group A IgG suggest that there is no correlation between the blood group of an individual and the antigenic determinants on the enterokinase produced by the enterocytes of that individual. The study of the molecular properties of this important enzyme may give insights into pathological conditions with which it is linked.

ABO Blood-Group System↗
PubMed1981

Induction and maintenance of mucosal enterokinase activity in proximal small intestine by a genetically determined response to mediated sodium transport.

Mucosal enterokinase activity was established at intervals throughout the small intestine in guinea-pigs; maximum activity was present in the duodenum and proximal jejunum in new born as well as adult animals. Transposition of 5 cm lengths of small gut from the high enterokinase containing proximal region to the distal intestine and vice versa showed that mucosal enterokinase activity in the transposed segments was little changed after several weeks of healthy life. Isolation of proximal jejunal loops from luminal continuity resulted in the fall of mucosal enterokinase activity to minimal levels within 16 hours. Low levels of mucosal enterokinase activity were identified in loops of both proximal and distal jejunum 12 weeks after isolation. Luminal perfusion studies in vivo in proximal jejunal loops 24 hours after isolation showed that mucosal enterokinase activity could be restored to near normal levels within four to six hours by luminal sodium in the presence of active pancreatic endopeptidases, oligopeptides, L-amino acids, or D-glucose but not D-amino acids or D-fructose. Near normal mucosal enterokinase activity persisted in the loops for as long as luminal perfusion with 144 mM sodium and L-lysine or trypsin was maintained (24 hours). The time course of the restoration of mucosal enterokinase activity was compatible with an initial precursor activation as well as biosynthesis. The requirement for luminal sodium appeared to be absolute regardless of the co-substrate and supports the conclusion that mucosal enterokinase activity is dependent on mediated sodium transport. The ability of proximal intestinal enterocytes to respond to sodium flux with an increase in enterokinase activity is a property determined in intrauterine life: distal intestinal enterocytes may have functioning structural genes for enterokinase but appear to be unable to respond.

Aminopeptidases↗
PubMed1980

Fatty acid acylation of proteins in cultured cells.

Addition of [3H]palmitic acid to chick embryo fibroblasts labeled a set of membrane proteins that was distinct from those proteins labeled with [3H]leucine or [3H]mannose when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The palmitate label, but not the mannose or leucine label, was removed from the proteins by treating electropherograms with hydroxylamine prior to fluorographic analysis. This result and other data indicate that the fatty acid labeling of cell proteins was analogous to that recently described for fatty acid acylation of three virus membrane glycoproteins. Mouse and human cultured cell lines show a similar set of protein-bound fatty acid, and we propose that fatty acid acylation is a general cellular activity that modifies proteins destined to become membrane-bound.

Acylation↗
PubMed1979

Identification of a defence mechanism in vivo against the leakage of enterokinase into the blood.

1. The serum proteinase inhibitors alpha 1-antitrypsin, alpha 2-macroglobulin, inter-alpha-trypsin inhibitor and C1-esterase inhibitor were found not to affect the catalytic activity of human enterokinase, whereas bovine trypsin activity was modified essentially as expected. Enterokinase was also not inhibited by Trasylol (trypsin inhibitor from bovine lung) or bovine pancreatic trypsin inhibitor. No other component in human or mouse serum complexing with enterokinase was identified. 2. Human enterokinase administered intravenously into mice was rapidly cleared from the circulation with a half-life of 2.5 min. This removal was not the result of the difference in species, since partially purified mouse enterokinase was cleared at the same rate as the human enzyme. Clearance was mediated by recognition of the carbohydrate portion of enterokinase and not through specific recognition of its catalytic site. Immunofluorescent staining showed that the enzyme accumulated in the liver. Attempts to block the clearance by the simultaneous infusion of competing glycoproteins suggested that enterokinase was taken up by hepatocytes. Of the glycoproteins tested only two, human lactoferrin (terminal fucosyl alpha 1 leads to 3 N-acetylglucosamine) and bovine asialo-fetuin (terminal galactosyl beta 1 leads to 4 N-acetylglucosamine) were weakly competitive. Two inhibitors of endocytosis, Intralipid and Triton WR1339, failed to delay the removal of enterokinase. It is proposed that enterokinase is cleared from the circulation by an as yet uncharacterized hepatocyte receptor.

Animals↗