Investigating the entire course of telithromycin binding to Escherichia coli ribosomes

Applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to Escherichia coli ribosomes in a two-step process. During the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (KT = 500 nM), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl–aryl side chain of the drug inserts a groove formed by nucleotides A789 and U790 of 23S rRNA. During the second step, telithromycin shifts slowly to a high-affinity site (KT* = 8.33 nM), in which the lactone ring remains essentially at the same position, while the side chain interacts with the base pair U2609:A752 and the extended loop of protein L22. Consistently, mutations perturbing either the base pair U2609:A752 or the L22-loop hinder shifting of telithromycin to the final position, without affecting the initial step of binding. In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding. Polyamines disfavor both sequential steps of binding. Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes

Partial characterization of an abnormal lactate dehydrogenase isoenzyme, LDH-1ex, in serum from a patient with hepatocellular carcinoma.

Abstract Serum from a patient with hepatocellular carcinoma contained an abnormal isoenzyme of lactate dehydrogenase (LDH; EC 1.1.1.27), LDH-1ex, that on electrophoresis on 10-g/L agarose gel migrated anodally to the LDH-1 band. This isoenzyme was partly purified by ultrafiltration and preparative electrophoresis. Gel chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies of the resulting LDH-1ex preparation suggested that this isoenzyme is probably a tetramer made up of four single polypeptide chains (monomers), each having a molecular mass of about 32,000 Da. LDH-1ex was heat stable and reacted more readily with 2-hydroxybutyrate than did the slower migrating LDH-4 and LDH-5 isoenzymes. LDH-1ex showed no activity when lactate was omitted from the substrate solution or replaced by ethanol.</jats:p

Lactate dehydrogenase isoenzyme pattern in sera of patients with malignant diseases.

Abstract Total lactate dehydrogenase (LDH; EC 1.1.1.27) activity and the percentage distribution of LDH isoenzymes were determined in 127 patients with malignant diseases. A shift in the isoenzyme patterns was observed toward the M-type, with an increase in the percentage of LDH-4 and LDH-5 isoenzymes and a slight increase in total LDH activity of all patients. Serum samples from 68 of the patients contained an abnormal isoenzyme of LDH, "LDH-1 ex," that, on agarose gel electrophoresis at pH 8.6, migrated between albumin and LDH-1 isoenzyme. Chemotherapy, radiotherapy, or surgical removal of the tumor was accompanied by disappearance of this abnormal isoenzyme. The heat stability of LDH-1 ex isoenzyme appears to be similar to that of LDH-1 but greater than that of the other LDH isoenzymes. Statistical analysis of these data demonstrated a significant correlation between malignancy and the appearance of LDH-1 ex isoenzyme (P less than 0.001). In contrast, the relationship between LDH-1 ex isoenzyme and metastasis or anatomical location of the malignancy is not statistically important (P less than 0.1).</jats:p

Clindamycin binding to ribosomes revisited: foot printing and computational detection of two binding sites within the peptidyl transferase center

Clindamycin is a semi-synthetic lincosamide, active against most Gram-positive bacteria and some protozoa. It binds to the 50S ribosomal subunit and inhibits early peptide chain elongation. By kinetic analysis it has been shown that clindamycin (I) competitively interacts with the A-site of translating ribosomes (C) to form the encounter complex Cl, which then slowly isomerizes to a tighter complex, termed C*I. As the final complex is capable of synthesizing peptide bonds with decreased velocity, it was assumed that in C*I complex the drug is fixed near the P-site of the ribosome. In the present study, two series of chemical foot printing experiments were carried out. In the first series, clindamycin and ribosomal complex C were incubated for 1 s and then DMS or kethoxal was added (Cl probing). In the second series, complex C was preincubated with clindamycin for 1 min before the addition of DMS or kethoxal (C*I probing). It was found that clindamycin in Cl complex protects A2451 and A2602 from chemical probing, both located within the A-site of the catalytic center. In contrast, it strongly protects G2505 in C*I complex, which is a discrete foot print of peptidyl-tRNA bound to the P-site. In both Cl and C*I complexes, clindamycin also protects nucleotides A2058 and A2059, located next to the entrance of the exit-tunnel where the nascent peptide leaves the ribosome. Polyamines negatively affect the protection of G2505, but favor the protection of A2451 and A2602 nucleotides. Structure modeling confirms the kinetic and chemical foot printing results and suggests that clindamycin mode of action is more complex than a simple competitive inhibition of peptide bond formation