Structural changes of proteins in fish red blood cells after copper and mercury treatment.
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Animals↗
Biomedical subjects
K Gwozdzinski
Publications and source records attributed to K Gwozdzinski.
Direct evidence for the alterations in protein structure and conformation upon in vitro nonenzymatic glycosylation.
The formation of nonenzymatic glycosylation products appears to be a link between chronic hyperglycaemia and long-term diabetic complications. However, little is known concerning the glycation-induced modifications in the structure and conformation of proteins, which possibly underlie their altered functional characteristics. This study conveys a direct evidence for and compares the glucose-induced modifications in the conformation of three proteins with various half-lives: bovine serum albumin, human haemoglobin and bovine tendon collagen. These proteins incubated in vitro with glucose in various media containing optionally EDTA and Fe2+ ions contained up to 4-10 times as much attached glucose as did their relevant controls, and the extent of glycation was the highest in the samples incubated under air or in the absence of EDTA. The fluorescence and ESR data indicate that the Trp in albumin molecule, given albumin glycation-induced structural modifications, became more exposed to water surrounding solution whereas the Trp residues of haemoglobin remained shielded from water; also collagen fluorescence derived from the supposedly newly formed covalent crosslinks is vastly increased, and particularly when collagen was glycated under air or in the presence of Fe2+ ions. Possible mechanisms underlying the increased mobility of selected protein domains and glycation-mediated alterations in protein conformation are considered and discussed.
Animals↗
Enzymatic sulfation of mucus glycoprotein in gastric mucosa. Effect of ethanol.
A sulfotransferase activity that catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate to carbohydrate chains of gastric mucus glycoprotein has been demonstrated in the antral and body mucosa of rat stomach. Subcellular fractionation studies revealed that the enzyme is associated with Golgi-rich membrane fraction. The sulfotransferase activity of this fraction in antral mucosa was about 35% lower than that in the body. Optimum enzyme activity was obtained with 0.5% Triton X-100 and 30 mM NaF at a pH of 6.8 using desulfated mucus glycoprotein substrate. The enzyme was equally capable of sulfation of the proteolytically degraded and reduced forms of the desulfated glycoprotein, but the acceptor capacity of the intact mucus glycoprotein was about 60% lower than that of the desulfated preparation. The enzyme preparation also catalyzed the transfer of sulfate to galactosylceramide. The sulfation of mucus glycoprotein, however, was not affected by the presence of this glycolipid, suggesting that the sulfotransferase involved in mucus glycoprotein sulfation is different from that responsible for the synthesis of sulfatoglycosphingolipid. The mucus glycoprotein sulfotransferase activity was inhibited by ethanol. The rate of inhibition was proportional to the concentration of ethanol up to 0.3 M and was of the competitive type. The apparent Km value of the enzyme for mucus glycoprotein was 10.5 X 10(-6) M (21 mg/ml), and the KI in the presence of ethanol was 4.7 x 10(-1) M. The 35S-labeled mucus glycoprotein product of the enzyme reaction gave in CsCl density gradient a band in which the 35S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein led to the liberation of the label into reduced acidic oligo-saccharide fraction. Most of the label was found incorporated in three oligosaccharides. These were identified as tri-, tetra-, and pentasaccharides, each carrying a labeled sulfate ester group on the terminal N-acetyl-glucosamine residue. Based on the results of structural analyses, the most abundant oligosaccharide was characterized as SO3H----6GlcNAc beta 1----3Gal beta 1----3GalNAc-ol.
Animals↗
Gastric mucin hydrophobicity: effects of associated and covalently bound lipids, proteolysis, and reduction.
The hydrophobic properties of gastric mucus glycoprotein were investigated using the fluorescent probe, bis(8-anilino-1-naphthalenesulfonate). The glycoprotein was subjected to removal of associated and covalently bound lipids, peptic degradation, and disulfide bridge reduction. Fluorescence titration data revealed the presence of 55 hydrophobic binding sites in the intact mucin molecule, 71 binding sites in the glycoprotein devoid of associated lipids, and 53 binding sites in the glycoprotein devoid of associated lipids and covalently bound fatty acids. Proteolytic digestion of the glycoprotein with pepsin essentially abolished the probe binding, while reduction of disulfide bridges resulted in glycoprotein subunits whose combined number of binding sites was about 3 times greater than that of the mucin polymer. The binding of the probe to mucus glycoprotein varied with the pH of the medium, being highest at pH 2.0 and lowest at pH 9.0. The results indicate that lipids contribute to the hydrophobic character of gastric mucin and that hydrophobic binding sites reside on the nonglycosylated regions of the glycoprotein polymer buried within its core.
Animals↗
In vitro fatty acid acylation of mucus glycoprotein from sublingual salivary glands.
The enzyme activity which catalyzes the transfer of palmitic acid from palmitoyl-coenzyme A to sublingual gland mucus glycoprotein has been demonstrated in the detergent extracts of the microsomal fraction of rat sublingual and parotid salivary glands. The acyltransferase activity of this fraction was similar in both types of glands. Further subcellular fractionation performed on sublingual glands revealed that the enzyme is associated with the Golgi-rich membrane fraction. Optimum enzymatic activity for fatty acylation of mucus glycoprotein was obtained using 0.5% Triton X-100, 2 mM dithiothreitol, 25 mM NaF, and 10 mM MgCl2 at a pH of 7.4. Higher concentrations of NaF, MgCl2 and dithiothreitol, however, were inhibitory. The apparent Km of the sublingual glands microsomal enzyme for mucus glycoprotein was 0.55 mg/ml and for palmitoyl-CoA, 3.5 X 10(-5) M. A 15% decrease in the acyltransferase activity was obtained with the reduced and alkylated mucus glycoprotein and it showed no activity towards the proteolytically degraded glycoprotein. The 14C-labeled product of the enzyme reaction gave in CsCl density gradient a band at the density of 1.49 in which the 14C label coincided with the glycoprotein. The 14C label in this glycoprotein was susceptible to deacylation with hydroxylamine, and the released labeled material was identified as palmitate.
Acylation↗
Lipid composition and viscosity of parotid saliva in Sjögren syndrome in man.
Extraction of the dialysed and lyophilized saliva of patients with this syndrome by chloroform-methanol yielded 15.9 +/- 2.4 mg of lipid/100 ml of secretion, a level 2-times higher than in normal individuals. The saliva of such patients also had 3-times more glycolipid and 20-times more phospholipid than normal, but differences in the content of neutral lipids were less apparent. The neutral lipids, however, had higher proportions of glycerides, and lower proportions of cholesterol and cholesteryl esters than normal. Viscosity measurements, made with a cone/plate viscometer at shear rates between 1.15 and 230 s-1, revealed similarities between the saliva of normal individuals and Sjögren's syndrome.
Cholesterol↗
Tooth surface-pellicle lipids and their role in the protection of dental enamel against lactic-acid diffusion in man.
The lipid content and composition of the enamel pellicle from caries-resistant (CR) and caries-susceptible (CS) subjects and their effect on its ability to retard the diffusion of lactic acid were investigated. Lipids accounted for 22.2 per cent of the dry weight of CR pellicle and 23.7 per cent of CS pellicle. The content of glycolipids in both groups was similar but CR pellicle contained 42 per cent less neutral lipids and 31 per cent less phospholipids. CR lipids had a higher content of cholesterol, cholesterol esters and sphingomyelin, whereas CS pellicle was richer in free fatty acids and phosphatidylethanolamine. Retardation of lactic-acid diffusion by CR pellicle was 45 per cent higher than by CS. Removal of lipids caused 50 per cent reduction in retardation by CR pellicle and 35 per cent by CS pellicle.
Adult↗
Effect of cupric ions on the permeability of erythrocyte membrane to non-electrolyte spin labels.
The addition of cupric ions caused decreased permeability to hydrophilic molecules and increased permeability to hydrophobic molecules. These results suggest that TEMPOL penetrates the erythrocyte in a different way than TEMPO. Penetration of TEMPOL is controlled by-SH groups, while TEMPO probably diffuses through the lipid bilayer. Cupric ions increase the permeability of erythrocyte membranes to both non-electrolytes in vivo.
Animals↗
Effect of adenine nucleotides and gamma radiation on the transport of TEMPOL across the erythrocyte membrane.
External adenine compounds bring about changes in the transport of hydrophilic molecules across control and irradiated bovine erythrocyte membranes. Changes in the transport induced by incubation of erythrocytes with nucleotides depend on the type of nucleotide and its concentration. The range of nucleotide concentrations over which the stimulatory effect on the transport occurs is established.
Adenine Nucleotides↗
Hyperthermic modification of bleomycin--DNA interaction detected by electron spin resonance.
Electron spin resonance spectra of DNA labeled with each of four spin-labeling compounds have been studied to detect interaction between the antibiotic bleomycin and DNA. Only one of these labels, compound IV, resulted in a modified spectrum when bound to DNA and the latter was subjected to bleomycin. This property has been used to monitor DNA-bleomycin interactions under physiological and hyperthermic conditions. Bleomycin produced an increase in rotational correlation time of the residue bound to DNA at 37 degrees C and a significantly higher increase at 43 degrees C. Some effect was still detected with bleomycin at 37 degrees C after preheating at 43 degrees C. Parallel studies have revealed enhanced binding of 59Fe-bleomycin to DNA during and after hyperthermic treatment.
Bleomycin↗
Aging of the erythrocyte XIII. Decrease in nitroxide reducing capacity.
A method is proposed for the determination of nitroxide-reducing capacity (NRC) of erythrocytes, and other cells, based on measurements of reduction of a nitroxide stable free radical in cell suspensions by ESR spectroscopy. Comparison of different age fractions of bovine erythrocytes by this method demonstrates a decrease in NRC during cell aging in vivo.
Animals↗