ABSTRACT
Hydroxylation of C-12 is one of the final steps in the biosynthesis of erythromycin A (ErA). A point of uncertainty in the erythromycin pathway has been whether the C-12 hydroxylase operates on each of two possible substrates, erythromycin B (ErB) and erythromycin D (ErD). Stassi et al. have cloned the gene, designated eryK, which encodes the P-450 monooxygenase responsible for erythromycin C-12 hydroxylation in Saccharopolyspora erythraea [Stassi, D., Donadio, S., Staver, M. J., & Katz, L. (1993) J. Bacteriol. 175, 182-189]. We report the overproduction of EryK in Escherichia coli as insoluble inclusion bodies; the solubilization, refolding, and reconstitution of active holo-EryK; and kinetic confirmation of a 1200-1900-fold preference of the enzyme for ErD over the alternative C-12 hydroxylase substrate ErB. Our results indicate that ErB is a shunt metabolite in the erythromycin biosynthetic pathway.
Subject(s)
Bacterial Proteins , Cytochrome P-450 Enzyme System/metabolism , Gene Expression , Mixed Function Oxygenases/metabolism , Amino Acid Sequence , Base Sequence , Chromatography, High Pressure Liquid , Cloning, Molecular , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/genetics , Erythromycin/biosynthesis , Erythromycin/chemistry , Escherichia coli/enzymology , Escherichia coli/genetics , Kinetics , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Molecular Sequence Data , Molecular Structure , Polymerase Chain Reaction , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Solubility , SpectrophotometryABSTRACT
Aged, memory-impaired rats do not learn an 8-arm radial maze task but differ in their performance along testing. The aim of this study was to determine whether any of the systems that govern calcium homeostasis in synaptosomes may be related to that difference in performance. A negative correlation between initial (5 s) K(+)-stimulated 45Ca2+ uptake and the behavioral scores from the last testing sessions was obtained K(+)-stimulated 45Ca2+ uptake showed also a negative correlation with an improvement score that evaluates the progress made by the rat along testing. The results support the notion that calcium inflow through synaptosomal voltage gated calcium channels in old rats is inversely correlated with their behavior. This may explain the beneficial effects of organic calcium channel blockers on behavioral performance in aged animals.
Subject(s)
Aging/psychology , Calcium Channels/metabolism , Memory Disorders/metabolism , Memory Disorders/psychology , Memory, Short-Term/physiology , Synaptosomes/metabolism , Animals , Calcium/metabolism , Calcium Radioisotopes , Homeostasis/physiology , In Vitro Techniques , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Learning/physiology , Potassium/pharmacology , Rats , Synaptosomes/drug effectsABSTRACT
Aging is associated with alterations in different systems that govern neuronal calcium homeostasis. This study was designed to determine whether any of these alterations may contribute to the decline in spatial working memory that is observed in old rats. Several parameters [initial (5 s) and steady state (15 min) 45Ca2+ uptake, FCCP-releaseable 45Ca2+, [Ca2+]i levels, depolarization-induced phosphoprotein (P97, PP65, P42) dephosphorylation and acetylcholine levels and release) involved in calcium homeostasis/signaling were determined in whole brain synaptosomes derived from adult (9-month-old) and old (24-month-old) rats that were evaluated for spatial memory performance in the eight-arm radial maze. The neurochemical analysis indicated that both the 9- and 24-month-old rats were impaired with respect to 3-month-old animals. When learners (animals reaching criterion; RC) were compared to memory impaired rats (MI), it was found that the FCCP-releaseable 45Ca2+ of synaptosomes, that reflects mitochondrial calcium, was lower in the MI than the RC rats and was correlated with the behavioral performance of the rats in their first testing sessions. The results suggest that the loss of calcium uptake capacity in synaptic mitochondria during aging may be associated with impaired working memory in old animals.
Subject(s)
Aging/metabolism , Calcium/metabolism , Homeostasis/physiology , Memory Disorders/metabolism , Memory, Short-Term , Synaptosomes/metabolism , Acetylcholine/metabolism , Aging/psychology , Animals , Calcium Radioisotopes , Cytosol/metabolism , Male , Mitochondria/metabolism , Phosphorylation , Rats , Rats, Wistar , Space Perception/physiologyABSTRACT
The entire nucleotide sequence of human T-cell lymphotropic virus type II (HTLV-II) from a previously described isolate of patient NRA (HTLV-IINRA) was determined. Clones encoding the 5' LTR and gag, pol, env and tax/rex open reading frames were subcloned and sequenced on both strands. The provirus consisted of 8957 nucleotides and showed 95.2% homology with the HTLV-IIMo prototype at the nucleotide level. Less than 5% amino acid variation between HTLV-IINRA and HTLV-IIMo was observed for coding regions. Although isolate HTLV-IINRA had an additional 25 amino acids at the 3' end of tax/rex, this region was 96% homologous with the 5' end of HTLV-IIMo 3' LTR. To further investigate HTLV-II variability, a portion of the env gp46 gene derived from 9 HTLV-II infected persons was amplified by polymerase chain reaction and sequenced. Sequence was obtained for 320 nucleotides corresponding to HTLV-IIMo positions 5291 to 5610. Isolates similar to the HTLV-IIMo and HTLV-IINRA prototypes were identified, and sequences were highly conserved.
