Subunit location of the iron-sulfur clusters in fumarate reductase from Escherichia coli

The subunit location of the [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters in Escherichia coli fumarate reductase has been investigated by EPR studies of whole cells or whole cells extracts of a fumarate reductase deletion mutant with plasmid amplified expression of discrete fumarate reductase subunits or groups of subunits. The results indicate that both the [2Fe-2S] and [3Fe-4S] clusters are located entirely in the iron-sulfur protein subunit. Information concerning the specific cysteine residues that ligate these clusters has been obtained by investigating the EPR characteristics of cells of the deletion mutant amplified with a plasmid coding for the flavoprotein subunit and a truncated iron-sulfur protein subunit. While the results are not definitive with respect to the location of the [4Fe-4S] cluster, they are most readily interpreted in terms of this cluster being entirely in the flavoprotein subunit or bridging between the two catalytic domain subunits. These new results are discussed in light of the amino acid sequences of the two subunits and the sequences of structurally well characterized iron-sulfur proteins containing [2Fe-2S], [3Fe-4S], and [4Fe-4S] centers.     

Mutagenesis of the regulatory subunit of yeast cAMP-dependent protein kinase. Isolation of site-directed mutants with altered binding affinity for catalytic subunit

Oligonucleotide-directed mutagenesis was used to produce mutants in the hinge region of the regulatory subunit (R) of the Saccharomyces cerevisiae cAMP-dependent protein kinase. The mutant proteins were expressed in Escherichia coli, purified, urea treated to produce cAMP-free regulatory (R), and analyzed in vitro for catalytic (C) subunit inhibitory activity in the presence and absence of cAMP. When assayed in the absence of cAMP, wild type R dimer inhibited C with an IC50 of 40 nM. Replacement of amino acid residue Ser-145 (the autophosphorylation site of yeast R) with Ala or Gly produced mutants which were 2-10-fold better inhibitors of C, while replacement with Glu, Asp, Lys, or Thr produced mutants which were 2-5-fold worse inhibitors of C relative to wild type R. When assayed in the presence of cAMP, all R subunits had a decreased affinity for C subunit, with Ser-145 and Thr-145 undergoing autophosphorylation. These results suggest that the amino acid at position 145 of R contributes to R-C interaction and therefore influences the equilibrium of yeast protein kinase subunits in vitro.     

The complete primary structure of the cellular retinaldehyde-binding protein from bovine retina

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol and 11-cis-retinaldehyde as endogenous ligands and may be a functional component of the visual cycle. The complete amino acid sequence of CRALBP from bovine retina has been determined by direct microanalysis of the protein. Bovine CRALBP contains 316 residues in a single amino-terminal-blocked chain corresponding to a molecular weight of 36,421, inclusive of the blocking group. Overlapping peptides were generated by cleavage of lysyl, arginyl, methionyl, glutamyl, and one tryptophanyl bond and sequenced by gas-phase Edman degradation. Analysis of amino-terminal arginyl and methionyl peptides by fast atom bombardment mass spectrometry identified the N alpha-blocking group as an acetyl moiety, and tandem mass spectrometry provided the sequence of the first 9 residues. Comparison of CRALBP with other known protein sequences reveals no significant structural relatedness. The present results provide a basis for relating CRALBP domains with physiological function and for the future development of a more detailed three-dimensional model of the interaction of 11-cis-retinaldehyde with protein.     

Studies on compound I formation of the lignin peroxidase from Phanerochaete chrysosporium

Ligninase, isolated from the wood-destroying fungus Phanerochaete chrysosporium, catalyzes the oxidation of lignin and lignin-related compounds. Ligninase reacts with H2O2 to form the classical peroxidase intermediates Compounds I and II. We have determined the activation energy of ligninase Compound I formation to be 5.9 kcal/mol. The effect of pH and ionic strength on the rate of ligninase Compound I formation was studied. In contrast to all other peroxidases, no pH effect was observed. This is despite homology of active-site amino acids residues (Tien, M., and Tu, C.-P. D. (1987) Nature 326, 520-523) which are proposed to affect the pH profile of Compound I formation. Ligninase Compound I formation can also be supported by organic peroxides. The second-order rate constants with the organic peroxides are lower, suggesting that H2O2 is the preferred substrate.     

Altered turnover of allelic variants of hypoxanthine phosphoribosyltransferase is associated with N-terminal amino acid sequence variation

The results of our previous studies suggested that differences in the primary structures of the hypoxanthine phosphoribosyltransferase (HPRT) A and B proteins (EC 2.4.2.8) of mice are associated with altered turnover of these proteins in reticulocytes. On the basis of nucleotide sequence comparisons of their corresponding cDNAs, we show here that the HPRT A and B proteins differ at two positions; there is an alanine/proline substitution at amino acid position 2 and a valine/alanine substitution at amino acid position 29 (HPRT A/B proteins, respectively; total protein length, 218 amino acids). On the basis of results obtained from sequencing of the N termini of the purified HPRT A and B proteins, we also show that these amino acid substitutions are associated with differences in processing of the proteins; HPRT B, which is encoded as N-terminal Met-Pro, has a free N-terminal proline residue; HPRT A, which is encoded as N-terminal Met-Ala, lacks a free N-terminal alpha-amino group and is presumed to be acetylated following removal of the N-terminal methionine (i.e. AcO-Ala). These observations are discussed in reference to the idea that the N terminus of a protein plays a role in determining the rate at which the protein is degraded in erythroid cells.     

Calcitonin gene-related peptide in human fetal lung and in neonatal lung disease

The ontogeny of calcitonin gene-related peptide immunoreactivity (CGRP-IR) was evaluated immunohistochemically in 67 human fetal or newborn lungs previously analyzed for calcitonin immunoreactivity (CT-IR). CGRP-IR was present by 10 weeks of gestation in rare, solitary neuroendocrine (NE) cells of developing conducting airways in two of eight first-trimester lungs. During the second trimester, cells with CGRP-IR were found consistently (21/23 fetuses). However, the numbers of positively staining cells did not appear to increase in these fetuses or in third-trimester infants dying of non-pulmonary causes. The highest concentrations of CGRP-IR cells were seen in lungs of premature infants with advancing chronic lung disease associated with bronchopulmonary dysplasia (BPD). CGRP-IR was seen earlier in gestation and in greater numbers of NE cells than was calcitonin immunoreactivity (CT-IR) reported previously in these same fetal lungs (Lab Invest 52:52, 1985). Its presence paralleled that of CT-IR in postnatal chronic lung disease.     

Microdistribution and local dosimetry of 226Ra in trabecular bone of the beagle

Sections of lumbar vertebral bodies of young adult beagle dogs have been analyzed autoradiographically to characterize and quantify the local distribution of 226Ra by means of a scanning microscope photometer. The animals received a single injection of 355 kBq/kg body weight and were serially sacrificed at 5 to 1381 days postinjection. Hotspot concentrations decreased from about 51 kBq/g bone at 5 days to 20 kBq/g at 1381 days postinjection. The diffuse concentration changed from 8.3 to 1.9 kBq/g. The mean 226Ra concentration in the trabecular areas scanned was initially higher and at the end of the observation period lower than the average calculated for the whole lumbar vertebral column. Density and area of, and fraction of bone activity in, hotspots virtually remained constant. With time hotspots tended to become translocated into bone volume. Mean dose rates to lining cells from both hotspots and diffuse labels decreased from about 210 mGy/d at early postinjection times to 105 mGy/d. This corresponds to 2.5 to 1.1 times the average skeletal dose rate. A discussion of the level of irradiation in terms of hit frequencies shows that osteoblasts in the initial phase of hotspot formation receive about 60 hits to their nucleus for the duration of bone formation. After about 6 months, however, the 226Ra concentration in new bone and the corresponding hit frequency appears to be low enough that interference with bone formation is unlikely. Morphometric measurements showed that abnormal bone accretion and thickening of trabeculae occurred. This was interpreted as an imbalance between bone formation and resorption. Both formation and resorption seem to be substantially lowered compared to control animals.     

Cutaneous laser-Doppler flowmetry: influence of underlying muscle blood flow

To find whether the measurement of skin blood flow (SkBF) by laser-Doppler flowmetry (LDF) is influenced by blood flow to underlying skeletal muscle, five subjects performed mild forearm exercise to induce a metabolic hyperemia in muscle in both forearms. This exercise consisted of alternative opening and closing of both hands at a frequency of approximately 1/s for a duration of 3 min. This exercise was performed twice by each subject. Forearm blood flow (FBF) by plethysmography increased from 2.64 +/- 0.49 (rest) to 31.11 +/- 9.95 ml.100 ml-1.min-1 (immediately after exercise) (P less than 0.001). No statistically significant postexercise increase was observed in LDF measured on the dorsal (110 +/- 21 to 105 +/- 21 mV) or ventral surface (266 +/- 113 to 246 +/- 77 mV) of the forearm. LDF measured from the chest also showed no significant change, indicating that the exercise was too mild to have reflex effects on SkBF. Moreover, the slope of the logarithmic linear regression and the half-time for recovery during the postexercise period for FBF were not reflected in LDF measurements from any of the three sites. We conclude that LDF measured from the skin surface is not influenced by blood flow to underlying skeletal muscle.     

Allometric shape change and heterochrony in the freeliving coral Trachyphyllia bilobata (Duncan)

Regression analysis has been used to study the relationship between age, size, shape, and surface area in two ancestral-descendant populations of the Neogene Caribbean coral Trachyphyllia bilobata. Analyses of the relationship between size and age show that the relationship is isometric and that little difference occurs between populations in mean corallite length or height and in their rates of growth. Onset of columella growth is significantly earlier, however, in the descendant population. Studies of the relationship between size and shape show that growth is allometric, with shape change occurring in both corallum elongation and pinching of the corallite wall during ontogeny. In the descendant population, pinching and elongation initiate earlier in the ontogeny of the coral. These results suggest that the evolutionary development of the meandroid form in freeliving corals has been accomplished by heterochrony, involving a complex set of disassociated peramorphic changes in ontogeny accompanied by paedomorphic changes in astogeny. Further analyses show that the observed heterochronic changes serve to decrease corallum surface area which may in turn enhance sediment removal and nutrition in unstable habitats.