Volume changes in the binding of lanthanides to peptide analogues of loop II of calmodulin

The solution expansion accompanying coordination of lanthanide ions to synthetic peptide analogues of a metal-binding loop in calmodulin was determined by a density method. This study was designed to further test the hypothesis that the nonlinear expansions observed upon sequential addition of Ca2+ to intracellular calcium-binding proteins reflect principally upon the coordination event at specific binding sequences. Three peptides of 13 residues each were synthesized as analogues of binding loop II in mammalian calmodulin: Peptide I was the native analogue; peptide II contained an aspartyl in place of an asparaginyl residue at position 5 from the N-terminus; for peptide III, the aspartyl residue in position 3 of the native analogue was interchanged with the asparaginyl residue in position 5. Thus, the number of charged-oxygen donor atoms for coordination was the same in I and in III, but the latter peptide could permit two pairs of acidic groups to converge toward the metal ion as in some loops of these proteins. The observed expansions with different lanthanide ions to the same peptide varied appreciably, suggesting dissimilar structures [Gariépy et al. (1983) Biochemistry 22, 1765-1772]; coordination to the simpler tetracarboxylate sequestrants, on the other hand, generated an expansion profile approximately as expected from the properties of the lanthanide series. The largest expansions were generated with peptide II (having the additional acidic group) for all lanthanides tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Perturbation in liver mitochondrial Ca2+ homeostasis in experimental iron overload: a possible factor in cell injury

The functional state of isolated mitochondria and specifically the integrity of the inner membrane, were investigated in the liver of rats made siderotic by dietary supplementation with carbonyl iron. The concentration of iron in the hepatic tissue increased progressively up to nearly 40 days and reached a steady-state level. When the iron content reached a threshold value (higher than 90 nmol/mg protein) the occurrence of in vivo lipid peroxidation in the mitochondrial membrane was detected. This process did not result in gross alterations in the mitochondrial membrane, as indicated by electron microscopy, phosphorylative capability and membrane potential measurements. On the contrary, the induction of lipoperoxidative reaction appeared to be associated with the activation of Ca2+ release from mitochondria. This was shown to occur as a consequence of rather subtle modifications in the inner membrane structure via a specific efflux route, which appeared to be linked to the oxidation level of mitochondrial pyridine nucleotides. The induction of this Ca2+ release from iron-treated mitochondria resulted in enhancement of Ca2+ cycling, a process which dissipates energy to reaccumulate into mitochondria the released Ca2+. The perturbation in mitochondrial Ca2+ homeostasis reported here may be a factor in the onset of cell damage in this experimental model of hepatic iron overload.     

Secondary structure prediction of 52 membrane-bound cytochromes P450 shows a strong structural similarity to P450cam

The secondary structure of 52 aligned cytochrome P450 sequences, all of which are membrane bound, is predicted and collectively compared with the crystal structure of the soluble cytochrome P450cam. Ten of 13 helical regions, 6 of 7 beta-pair regions, and beta-structure corresponding to a known beta-bulge near the active site of P450cam are predicted to exist in the membrane-bound P450s. Three turns associated with beta-structure in the soluble enzyme are also predicted for the membrane-bound forms. A strong structural similarity is evident between membrane P450s and the soluble P450cam. Consequently, a multitransmembrane structure involving much of P450 seems highly unlikely. A structure with two N-terminal transmembrane segments is compatible with these observations.     

UDP-galactose 4-epimerase. Phosphorus-31 nuclear magnetic resonance analysis of NAD+ and NADH bound at the active site

The phosphorus atoms of NAD+ bound within the active site of UDP-galactose 4-epimerase from Escherichia coli exhibit two NMR signals, one at delta = -9.60 +/- 0.05 ppm and one at delta = -12.15 +/- 0.01 ppm (mean +/- standard deviation of four experiments) relative to 85% H3PO4 as an external standard. Titration of epimerase.NAD+ with UMP causes a UMP-dependent alteration in the chemical shifts of the resulting exchange-averaged spectra, which extrapolate to delta = -10.51 ppm and delta = -11.06 ppm, respectively, for the fully liganded enzyme, with an interconversion rate between epimerase.NAD+ and epimerase.NAD+.UMP of at least 490 s-1. Conversely, the binding of 8-anilinonaphthalene-1-sulfonate, which is competitive with UMP, causes a significant sharpening of the epimerase.NAD+ resonances but very little alteration in their chemical shifts, to delta = -9.38 ppm and delta = -12.16 ppm, respectively. UMP-dependent reductive inactivation by glucose results in the convergence of the two resonances into a single signal of delta = -10.57 ppm, with an off-rate constant for UMP dissociation from the epimerase.NADH.UMP complex estimated at 8 s-1. Reductive inactivation by borohydride under anaerobic conditions yields a single, broad resonance centered at about delta = -10.2 ppm. The data are consistent with, and may reflect, the activation of NAD+ via a protein conformational change, which is known from chemical studies to be driven by uridine nucleotide binding. Incubation of epimerase.NAD+ with UMP in the absence of additional reducing agents causes a very slow reductive inactivation of the enzyme with an apparent pseudo-first-order rate constant of 0.013 +/- 0.001 h-1, which appears to be associated with liberation of inorganic phosphate from UMP.     

Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier M?ssbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.     

A major single-stranded DNA binding protein from ovaries of the frog, Xenopus laevis, is lactate dehydrogenase

The most abundant single-stranded DNA binding protein (SSB) found in ovaries of the frog, Xenopus laevis, was purified to electrophoretic homogeneity. Under physiological conditions, the purified SSB lowered the Tm of poly[d(A-T)] and stimulated DNA synthesis by the homologous DNA polymerase DNA primase alpha complex on single-stranded DNA templates. These properties are characteristic of a bona fide single-stranded DNA binding protein. The Stokes radius of native SSB was calculated to be 45 A, corresponding to a molecular mass of about 140 kDa. On SDS polyacrylamide gels, the SSB migrated as a single band with a molecular mass of 36 kDa. We assumed, therefore, that the SSB was a tetramer of 36 kDa subunits. We subsequently discovered that the SSB was LDH, D-lactate dehydrogenase, EC 1.1.1.28. Purified SSB has high LDH specific activity. Following electrophoresis on SDS polyacrylamide gels, the 36 kDa subunits were renatured and exhibited LDH activity. The amino-acid composition of X. laevis SSB/LDH was similar to that of LDH from other species and to other reported single-stranded DNA binding proteins. Mammalian SSB/LDH also preferentially bound single-stranded DNA. Mammalian SSB/LDH bound to RNA as demonstrated by affinity chromatography on poly(A)-agarose and by its effect on translation of mRNA in vitro.     

Opposing effects of strychnine and brucine on the germination and growth of lettuce seeds

Strychnine and brucine, two related alkaloids that occur in plants belonging to theStrychnos species, were shown to have opposing effects on the elongation of the radicle of lettuce seeds. Strychnine was found to be inhibitory, whereas brucine was found to be stimulating to radicle elongation. Alkaloids, generally, are more commonly known for their inhibitory effects on plant growth rather than on their stimulating effects.