ESR study of copper(II) retention by entire cell, cells walls, and protoplasts of Saccharomyces cerevisiae

1987 ◽  
Vol 33 (9) ◽  
pp. 777-782 ◽  
Author(s):  
Jean Claude Kihn ◽  
Michèle M. Mestdagh ◽  
Paul G. Rouxhet
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Energy Source ◽  
Plasma Membrane ◽  
Binding Sites ◽  
Cell Walls ◽  
Outer Face ◽  
Whole Cells ◽  
Entire Cell ◽  
Acidic Conditions ◽  
Isolated Cell

Copper retention by whole cells, protoplasts, and isolated cell walls of Saccharomyces cerevisiae was investigated in the absence of any energy source in the medium. The cell walls accounted only for a small fraction of the cation retention by whole cells. ESR results showed that copper was not bound only at the outer face of the plasma membrane, but it was also distributed in the plasma membrane and (or) in the cytoplasm. ESR studies also showed that, in all three systems, copper was chelated by peptides or proteins. The binding sites were formed by an amide and a strongly complexing ligand such as an amine. Their configuration depended upon pH: in slightly acidic conditions, copper was bound by the oxygen of the amide; at basic pH, NHCO became deprotonated and the negatively charged nitrogen bound to the metal.

scholarly journals Ultrastructural visualization of selective peanut agglutinin binding sites in rat osteoclasts.

1988 ◽  
Vol 36 (1) ◽  
pp. 95-101 ◽  
Author(s):  
M Takagi ◽  
H Yagasaki ◽  
T Baba ◽  
H Baba
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Cell Surface ◽  
Binding Sites ◽  
Peanut Agglutinin ◽  
Coated Pits ◽  
Cytoplasmic Surface ◽  
Coated Membranes ◽  
Entire Cell ◽  
Ruffled Border

We investigated the distribution of concanavalin A (ConA)-reactive alpha-D-mannosyl and alpha-D-glucosyl groups and peanut agglutinin (PNA)-reactive beta-D-galactose-(1----3)-N-acetyl-D-galactosamine residues on the surface of osteoclasts with pre-embedment ultrastructural lectin cytochemistry after aldehyde fixation of the metaphyses of the rat tibiae. By routine morphology, the plasma membrane of the ruffled border of the osteoclast was distinguished from the rest of the cell membrane, with the exception of the membrane of coated pits, by its characteristic thick coat at its cytoplasmic surface. Cytochemistry, using ConA in combination with horseradish peroxidase (ConA-HRP) and PNA conjugated to HRP, showed that binding of ConA was distributed over the entire cell surface of osteoclasts. In contrast, intense binding of PNA was limited to the membranes of the ruffled border and coated pits, whereas the remainder of the cell membrane stained weakly or not at all. These results demonstrate that preferential PNA binding sites of the cell surface correspond to coated membranes associated with osteoclastic endocytosis.

Transglutaminase-like activity participates in cell wall biogenesis in Saccharomyces cerevisiae

Biologia ◽  
2007 ◽  
Vol 62 (2) ◽  
Author(s):  
Marián Mazáň ◽  
Vladimír Farkaš
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Cell Walls ◽  
Insoluble Fraction ◽  
Amide Bond ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Wall Biogenesis ◽  
Cross Links ◽  
Wall Construction ◽  
Isolated Cell

AbstractTransglutaminases (TGases) catalyze the cross-linking between protein molecules by formation of an amide bond between γ-carboxyamide group of glutamine and the ε-amine group of lysine under deamination of glutamine. We have demonstrated the participation of transglutaminase-like activity in the isolated cell walls and in the process of cell wall regeneration in protoplasts of the yeast Saccharomyces cerevisiae. A radioactive TGase substrate [3H]putrescine was incorporated into the isolated cell walls and into the TCA-insoluble fraction in regenerating protoplasts. The incorporation was increased by adding exogenous artificial substrate of TGase N,N’-dimethylcasein and was inhibited by TGase inhibitor cystamine and/or EDTA. These results suggest the existence of a TGase-type reaction involved in the formation of covalent cross-links between glycoprotein molecules during cell wall construction in S. cerevisiae.

scholarly journals Na+ coordination at the Na2 site of the Na+/I− symporter

2016 ◽  
Vol 113 (37) ◽  
pp. E5379-E5388 ◽  
Author(s):  
Giuseppe Ferrandino ◽  
Juan Pablo Nicola ◽  
Yuly E. Sánchez ◽  
Ignacia Echeverria ◽  
Yunlong Liu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Sodium Iodide ◽  
The Other ◽  
Similar Function ◽  
Sodium Iodide Symporter ◽  
Whole Cells ◽  
Thyroid Hormone Biosynthesis ◽  
Hormone Biosynthesis ◽  
Medical Relevance

The sodium/iodide symporter (NIS) mediates active I− transport in the thyroid—the first step in thyroid hormone biosynthesis—with a 2 Na+: 1 I− stoichiometry. The two Na+ binding sites (Na1 and Na2) and the I− binding site interact allosterically: when Na+ binds to a Na+ site, the affinity of NIS for the other Na+ and for I− increases significantly. In all Na+-dependent transporters with the same fold as NIS, the side chains of two residues, S353 and T354 (NIS numbering), were identified as the Na+ ligands at Na2. To understand the cooperativity between the substrates, we investigated the coordination at the Na2 site. We determined that four other residues—S66, D191, Q194, and Q263—are also involved in Na+ coordination at this site. Experiments in whole cells demonstrated that these four residues participate in transport by NIS: mutations at these positions result in proteins that, although expressed at the plasma membrane, transport little or no I−. These residues are conserved throughout the entire SLC5 family, to which NIS belongs, suggesting that they serve a similar function in the other transporters. Our findings also suggest that the increase in affinity that each site displays when an ion binds to another site may result from changes in the dynamics of the transporter. These mechanistic insights deepen our understanding not only of NIS but also of other transporters, including many that, like NIS, are of great medical relevance.

scholarly journals Cytochalasin B as a probe for the two hexose-transport systems in rat L6 myoblasts

1988 ◽  
Vol 251 (1) ◽  
pp. 63-72 ◽  
Author(s):  
S R Chen ◽  
T C Y Lo
Keyword(s):  
Plasma Membrane ◽  
Transport System ◽  
Binding Sites ◽  
Cytochalasin B ◽  
Transport Systems ◽  
Scatchard Analysis ◽  
Hexose Transport ◽  
Whole Cells ◽  
High Affinity ◽  
Binding Data

We have recently demonstrated that two hexose-transport systems are present in undifferentiated rat L6 myoblasts: D-glucose and 2-deoxy-D-glucose are preferentially transported by the high-affinity system, whereas 3-O-methyl-D-glucose is transported primarily by the low-affinity system. Mutant D23 is found to be defective only in the high-affinity hexose-transport system. The low-affinity transport system is much more sensitive to inhibition by cytochalasin B (CB). The present study examines the identity, properties and regulation of the CB-binding sites by measuring CB binding to both whole cells and plasma membrane. Scatchard analysis of the binding data revealed the presence of two CB-binding sites, namely CBH and CBL. These two sites differ not only in their affinity for CB, but their levels can also be differentially altered by various biochemical, physiological and genetic manipulations. CBL resembles the high-affinity hexose-transport system in that it is absent in mutant D23 and is present in larger quantities in glucose-starved cells. Moreover, CB binding to this site is inhibited by D-glucose and 2-deoxy-D-glucose, the preferred substrates of the high-affinity hexose-transport system. On the other hand, CBH is found to be unaltered in mutant D23, which also retains the normal low-affinity hexose-transport system. CBH also resembles the low-affinity transport system in that it is not elevated in glucose-starved cells. Furthermore, binding of CB to this site can be inhibited by 3-O-methyl-D-glucose, the preferred substrate of the low-affinity transport system. It should be noted that 2-deoxy-D-glucose does not have much effect on CBH, and vice versa. Studies with purified membrane preparations indicate that both CB-binding sites are present in similar ratios in the plasma membrane and the low-density microsomal fraction. Plasma-membrane studies also reveal that D-glucose 6-phosphate, but not 2-deoxy-D-glucose 6-phosphate, is very effective in activating CB binding. Data presented suggest that CB binding may be regulated by sugar analogues in an allosteric manner.

scholarly journals The purification and properties of two staphylolytic enzymes from Streptomyces griseus

1968 ◽  
Vol 106 (1) ◽  
pp. 69-76 ◽  
Author(s):  
J. B. Ward ◽  
H R Perkins
Keyword(s):  
Molecular Weight ◽  
Cell Walls ◽  
Teichoic Acid ◽  
Streptomyces Griseus ◽  
Low Molecular Weight ◽  
Whole Cells ◽  
Ph Values ◽  
Purification And Properties ◽  
Soil Isolate ◽  
Isolated Cell

1. Two staphylolytic enzymes have been purified from cultures of a soil isolate of Streptomyces griseus. 2. The purified enzymes were shown to be basic proteins of low molecular weight. Each enzyme released N-acetylmuramic acid reducing groups from the cell walls of Staphylococcus aureus. 3. The enzymes lysed whole staphylococci best at higher pH values and lower ionic strengths than when the substrate was isolated cell walls or purified mucopeptide. 4. Added teichoic acid did not inhibit the enzymes, but it formed an ethanol-precipitable complex with them. 5. The possibility that teichoic acid on the surface of whole cells prevents the access of the enzymes to their mucopeptide substrate is discussed.

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