Characterization of three-subunit chloroplast coupling factor

The delta- and epsilon-polypeptides were removed from chloroplast coupling factor 1 (CF1). The resulting enzyme, CF1(-delta, epsilon), is a stable active ATPase containing only alpha-, beta-, and gamma-polypeptides. The dependence of the steady-state kinetics of ATP hydrolysis catalyzed by CF1(-delta, epsilon) on the concentrations of ATP and ADP was found to be essentially the same as by activated CF1. Nucleotide binding studies with CF1(-delta, epsilon) revealed three binding sites: a nondissociable ADP site (site 1), a tight MgATP binding site (site 2), and a site that binds ADP and ATP with a dissociation constant in the micromolar range (site 3). Similar results have been obtained with CF1. For both CF1 and CF1(-delta, epsilon), the binding of MgATP at site 2 is tight only in the presence of Mg2+. Fluorescence resonance energy transfer was used to map distances between the gamma-sulfhydryl ("dark" site) and gamma-disulfide and between the gamma-sulfhydryl and the three nucleotide sites. These distances are within 5% of the corresponding distances on CF1. These results indicate that removal of the delta- and epsilon-polypeptides from CF1 does not cause significant changes in the structure, kinetics, and nucleotide binding sites of the enzyme.     

Correlation of enzymatic activities and aggregation state in chicken liver fatty acid synthase

The relationships between the aggregation state and the enzymatic activities of chicken liver fatty acid synthase have been explored by monitoring the changes in light scattering, fluorescence, and the overall, beta-ketoacyl synthase, beta-ketoacyl reductase and enoyl reductase activities during dissociation and reassociation of the enzyme. The data obtained indicate that the enzyme dissociates at low temperature in both 0.1 M potassium phosphate (pH 7.0), 1 mM EDTA, and 5 mM Tris(hydroxymethyl)aminomethane, 35 mM glycine (pH 8.3) and 1 mM EDTA, but the extent of dissociation is less in the phosphate buffer. The assay conditions influence the assessment of the degree of dissociation and association: high temperatures, phosphate (high salt), NADPH and acetoacetyl-coenzyme A promote association of the monomeric enzyme, whereas dilution in the Tris-glycine buffer (low salt) and low temperature promote dissociation. Both the rate and extent of association and dissociation are altered by substrates. The monomeric enzyme does not possess beta-ketoacyl synthase and beta-ketoacyl reductase activities. Results obtained with the 1,3-dibromo-2-propanone cross-linked enzyme, which lacks beta-ketoacyl synthase activity, indicate that the NADPH-binding site of beta-ketoacyl reductase is disrupted at low ionic strength. In contrast, changes in ionic strength have little effect on the enoyl reductase activity. The dimer is stabilized by both electrostatic and hydrophobic interactions, with the former being of special importance for maintenance of the beta-ketoacyl reductase active site. site.     

Fluorescence studies of chicken liver fatty acid synthase. Segmental flexibility and distance measurements

The 4'-phosphopantetheine of chicken liver fatty acid synthase was specifically labeled with the fluorescent substrate analog coenzyme A 6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminohexanoate at low salt concentrations. A serine at the active site of the thioesterase was specifically labeled with the fluorescent compounds 6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminopentylmethylphosphono fluoridate and/or pyrenebutyl methylphosphonofluoridate. Dynamic anisotropy measurements indicate the thioesterase has considerable segmental flexibility, whereas the fluorescent labeled 4'-phosphopantetheine does not display detectable local or segmental flexibility. Fluorescence resonance energy transfer measurements indicate that the distance between the fluorescent label at the end of the 4'-phosphopantetheine and NADPH bound to the beta-ketoacyl reductase or enoyl reductase site on the same polypeptide chain is essentially the same, approximately 38 A. The two types of reductases were distinguished by specifically blocking enoyl reductase with pyridoxal 5'-phosphate. No significant energy transfer occurs between sites on different polypeptide chains so that the distances must be greater than 55 A. The distance between the serine on the thioesterase and the 4'-phosphopantetheine on the same polypeptide is 48 A; again no interpolypeptide chain energy transfer was observed. The distance between the serines of the two thioesterases within a fatty acid synthase molecule is greater than 56 A. The monomeric enzyme obtained at 1 degree C does not have beta-ketoacyl synthase and reductase activities. Also fluorescent titrations indicate NADPH is not bound to beta-ketoacyl reductase in monomeric enzyme. The addition of potassium phosphate to the monomers at 1 degree C rapidly dimerizes the enzyme and restores the beta-ketoacyl reductase activity. The beta-ketoacyl synthase activity is slowly restored when the dimer is raised to room temperature. The results obtained suggest that relatively large conformational changes may be part of the catalytic cycle.     

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: beta-ketoacyl reductase and enoyl reductase

The following reactions catalyzed by chicken liver fatty acid synthase have been studied with the stopped-flow method in 0.1 M potassium phosphate (pH 7.0) and 1 mM ethylenediaminetetraacetic acid at 25 degrees C by monitoring the change in NADPH fluorescence: the transfer of acetoacetyl from acetoacetyl coenzyme A to the enzyme, reduction of the enzyme-bound acetoacetyl by NADPH (beta-ketoacyl reductase), and reduction of enzyme-bound D-hydroxybutyryl/crotonyl by NADPH (enoyl reductase). The first two reactions were studied by mixing enzyme-NADPH with acetoacetyl-CoA under conditions where the kinetics can be analyzed as two consecutive pseudo-first-order processes: a mechanism consistent with the aceto-acetyl-CoA dependence of the pseudo-first-order rate constant associated with formation of the aceto-acetyl-enzyme is a relatively rapid binding of substrate to the enzyme, with a dissociation constant of 650 microM, followed by formation of covalently bound acetoacetyl, with a rate constant of 10.2 s-1. The aceto-acetyl-enzyme is reduced by enzyme-bound NADPH with a rate constant of 20 s-1, and the NADPH binding is characterized by a dissociation constant of 5.3 microM. Reduction of the D-hydroxybutyryl-/crotonyl-enzyme was studied by mixing NADPH with enzyme that was equilibrated with D-hydroxybutyryl-CoA or crotonyl-CoA; the rate constant for reduction of an equilibrium mixture of D-hydroxybutyryl- and crotonyl-enzyme is 36.6 s-1. Steady-state kinetic studies of the reduction of acetoacetyl-CoA and crotonyl-CoA by NADPH also have been carried out.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: reduced nicotinamide adenine dinucleotide phosphate binding and formation and reduction of acetoacetyl-enzyme

The kinetics of reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding to fatty acid synthase from chicken liver and of the reduction of enzyme-bound acetoacetyl by NADPH (beta-ketoacyl reductase) and the steps leading to formation of the acetoacetyl-enzyme have been studied in 0.1 M potassium phosphate-1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0, at 25 degrees C by monitoring changes in NADPH fluorescence with a stopped-flow apparatus. Improved fluorescence detection has permitted the use of NADPH concentrations as low as 20 nM. The kinetics of the binding of NADPH to the enzyme is consistent with a simple bimolecular binding mechanism and four equivalent sites on the enzyme (presumably two beta-ketoacyl reductase sites and two enoyl reductase sites). The bimolecular rate constant is 12.7 X 10(6) M-1 s-1, and the dissociation rate constant is 76.7 s-1, which gives an equilibrium dissociation constant of 6.0 microM. The formation of the acetoacetyl-enzyme and its subsequent reduction by NADPH could be analyzed as two consecutive pseudo-first-order reactions by mixing enzyme-NADPH with acetyl-CoA and malonyl-CoA under conditions where [acetyl-CoA], [malonyl-CoA] much greater than [enzyme] much greater than [NADPH]. From the dependence of the rate of reduction of aceto-acetyl-enzyme by NADPH on enzyme concentration, an independent estimate of the equilibrium dissociation constant for NADPH binding to the enzyme of 5.9 microM is obtained, and the rate constant for the reduction is 17.5 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of peptidoglycan in Gaffkya homari: role of the peptide subunit of uridine diphosphate-N-acetylmuramyl-pentapeptide

The incorporation of N-acetylmuramyl (MurNAc)-peptides from nucleotide-activated precursors (reference: uridine diphosphate [UDP]MurNAc-Ala(1)-dGlu(2)-Lys(3)- dAla(4)-dAla(5)) with incomplete or modified peptide subunits into peptidoglycan was studied with membrane preparations from Gaffkya homari. The effectiveness of their utilization at low and high concentrations was compared on the basis of the values of V(max)/K(m) and V(max), respectively. At low concentration, replacement of alanine by glycine in position 5 has a small effect on the activity of the peptidoglycan synthesizing system, whereas it has a significantly larger effect in positions 1 and 4. The importance of d-alanine in position 4 at low substrate concentrations is also observed with the incomplete UDP-MurNAc-peptides. For UDP-MurNAc-tripeptide and -tetrapeptide, V(max)/K(m) is 0.06 and 0.55, respectively, of the value for the -pentapeptide. At high substrate concentration, replacement of d-alanine by glycine in either position 1 or 5 decreases the activity to 0.37 of the value for the reference nucleotide, whereas replacement in position 4 has a smaller effect (0.74). The profiles established from V(max) and V(max)/K(m) with UDP-MurNAc-tripeptide, -tetrapeptide, and -pentapeptide show good correlation. At low concentration the specificity profiles of phospho-MurNAc-pentapeptide translocase, catalyzing the initial membrane reaction, are similar to those for the peptidoglycan synthesizing system; at high concentration, however, the profiles differ. The translocase appears to provide a primary specificity barrier at high substrate concentration for UDP-MurNAc-Ala-dGlu-Lys-dAla-dAla and UDP-MurNAc-Ala-dGlu-Lys-Gly-dAla, and at low concentration for UDP-MurNAc-Ala-dGlu-Lys and UDP-MurNAc-Ala-dGlu-Lys-Gly-dAla. Moreover, it is suggested that an additional specificity barrier exists in the peptidoglycan synthesizing system for certain nucleotides. Thus, the cytoplasmic enzymes and the membrane-associated enzyme(s) cooperate to insure the formation of functioning peptidoglycan in this organism.     

Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes.

BACKGROUND: Chronic diabetes causes structural changes in the retinal capillaries of nearly all patients with a disease duration of more than 15 years. Acellular occluded vessels cause hypoxia, which stimulates sight-threatening abnormal angiogenesis in 50% of all type I diabetic patients. The mechanism by which diabetes produces acellular retinal capillaries is unknown. MATERIALS AND METHODS: In this study, evidence of programmed cell death (PCD) was sought in the retinas of early diabetic rats, and the effect of nerve growth factor (NGF) on PCD and capillary morphology was evaluated. RESULTS: Diabetes induced PCD primarily in retinal ganglion cells (RGC) and Muller cells. This was associated with a transdifferentiation of Muller cells into an injury-associated glial fibrillary acidic protein (GFAP)-expressing phenotype, and an up-regulation of the low-affinity NGF receptor p75NGFR on both RGC and Muller cells. NGF treatment of diabetic rats prevented both early PCD in RGC and Muller cells, and the development of pericyte loss and acellular occluded capillaries. CONCLUSIONS: These data provide new insight into the mechanism of diabetic retinal vascular damage, and suggest that NGF or other neurotrophic factors may have potential as therapeutic agents for the prevention of human diabetic retinopathy.