Acylation of endogenous myelin proteolipid protein with different acyl-CoAs

Fatty acyltransferase activity that catalyzes the transfer of palmitic acid from palmitoyl-CoA to the endogenous myelin proteolipid protein has been demonstrated in isolated rat brain myelin. Optimum enzyme activity for the acylation of proteolipid protein was obtained in 0.1% Triton X-100, 2 mM MgCl2, and 1 mM dithiothreitol at a pH of 7.5 and at 37 degrees C. Other detergents had little or no effect on the reaction whereas acylation was completely abolished by sodium dodecyl sulphate (0.1%). Pulse-chase experiments indicated that the reaction involves the net addition of fatty acid to the protein and not a rapid fatty acid exchange. The rate of acylation was linear up to 30 min, indicating that the concentration of endogenous protein acceptor was constant. Under these conditions and at short time periods, the enzyme activity versus acyl-CoA concentration showed a hyperbolic curve. The apparent Km and Vmax for palmitoyl-CoA was 41 microM and 115 pmol/mg protein/min. Similar values were obtained for stearoyl and oleoyl-CoA, whereas myristoyl-CoA showed a lower specificity for the enzyme. The acyl-CoA specificity was also studied in competition experiments using several saturated and unsaturated fatty acid-CoAs. The product of the reaction was identified as myelin proteolipid protein and the fatty acid was shown to be attached to the protein via an ester linkage. Limited proteolysis and peptide mapping showed that the same sites on the proteolipid protein were acylated when the reaction was carried out in isolated myelin preparations or in brain tissue slices, suggesting physiological importance for the in vitro acylation of endogenous myelin proteolipid protein.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Haemopoiesis in the beagle foetus after in utero irradiation

On day 33 of gestation, foetal beagles were irradiated in utero (0.9 Gy of 60Co gamma-irradiation, 0.4 Gy/min). Foetal haematocytopoiesis was studied during the third trimester of gestation (days 42-55). Peripheral blood nucleated cell counts were 33 per cent lower than normal on day 44 and continued to be lower until day 49, when values became higher than normal. Splenic cellularities of irradiated pups on day 44 were more than 3 times those of the nonirradiated, but thereafter they were similar to normal. Differences in haemopoietic progenitor cell activity between irradiated and normal foetuses were observed. In comparison with the other foetal tissues, the foetal liver appeared to experience greater radiation injury. For example, on day 44, the irradiated liver BFU-E, CFU-E, and GM-CFC per 10(5) cells were almost fivefold lower than normal values. Spleens of irradiated foetal beagles contained a marked increase in all haemopoietic progenitor cells (BFU-E, CFU-E, and GM-CFC) and recognizable proliferative granulocytic cells and nucleated erythroid cells. The haemopoietic activity of the irradiated bone marrow during days 42-44 was similar to that of the irradiated spleen, and compensated for the damaged liver. However, unlike the irradiated spleen, the irradiated bone marrow had decreased BFU-E activity compared with the values for the nonirradiated bone marrow during days 48-55. Until day 50, the irradiated marrow contained fewer recognizable proliferative granulocytic cells but more nucleated erythroid cells.     

Adenosine discriminates between the caffeine and adenine nucleotide sites on the sheep cardiac sarcoplasmic reticulum calcium-release channel

Calcium-release channels of sheep cardiac sarcoplasmic reticulum were incorporated into phosphatidylethanolamine bilayers and single channel currents were recorded under voltage-clamp conditions. The effect of adenosine on single channel conductance and gating was investigated, as were the interactions between adenosine and caffeine and adenosine and ,-methylene ATP.Addition of adenosine (0.5–5 mm) to the cytosolic but not the luminal side of the membrane increased the open probability of single calcium-activated calcium-release channels by increasing the frequency and duration of open events, yielding an EC₅₀ of 0.75 mm at 10 m activating Ca²⁺.Addition of 1 mm caffeine potentiated the effects of adenosine at 10 or 100 m-activating cytosolic calcium, but had no effect on the inability of adenosine to activate the channel at 80 pmcalcium, suggesting discrete sites of action on the calcium-release channel for adenosine and caffeine. In contrast, addition of 100 m ,-methylene-ATP decreased single channel open probability in the presence of adenosine, suggesting that these compounds act on the same site on the channel.Activation of single channel opening by adenosine, or by adenosine together with caffeine, had no effect on single channel conductance or the Ca²⁺/Tris⁺ permeability ratio. Channels activated by adenosine were characteristically modified by ryanodine and blocked by m ruthenium red or mm magnesium.These results show that adenosine activates the sheep cardiac sarcoplasmic reticulum Ca²⁺-release channel by increasing the frequency and duration of open events in a Ca²⁺-dependent manner. The receptor site on the channel for adenosine is distinct from that for caffeine but probably the same as that for adenine nucleotides.This work was supported by the British Heart Foundation.     

NADPH-dependent sulfite reductase flavoprotein adopts an extended conformation unique to this diflavin reductase

This is the first X-ray crystal structure of the monomeric form of sulfite reductase (SiR) flavoprotein (SiRFP-60) that shows the relationship between its major domains in an extended position not seen before in any homologous diflavin reductases. Small angle neutron scattering confirms this novel domain orientation also occurs in solution. Activity measurements of SiR and SiRFP variants allow us to propose a novel mechanism for electron transfer from the SiRFP reductase subunit to its oxidase metalloenzyme partner that, together, make up the SiR holoenzyme. Specifically, we propose that SiR performs its 6-electron reduction via intramolecular or intermolecular electron transfer. Our model explains both the significance of the stoichiometric mismatch between reductase and oxidase subunits in the holoenzyme and how SiR can handle such a large volume electron reduction reaction that is at the heart of the sulfur bio-geo cycle.