Effect of substrate and pH on the oxidative half-reaction of phenol hydroxylase

The oxidative half-reaction of phenol hydroxylase has been studied by stopped-flow spectrophotometry. Three flavin-oxygen intermediates can be detected when the substrate is thiophenol, or m-NH2, m-OH, m-CH3, m-Cl, or p-OH phenol. Intermediate I, the flavin C(4a)-hydroperoxide, has an absorbance maximum at 380-390 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate III, the flavin C(4a)-hydroxide, has an absorbance maximum at 365-375 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate II has absorbance maxima of 350-390 nm and extinction coefficients of 10,000-16,000 M-1 cm-1 depending on the substrate. A Hammett plot of the logarithm of the rates of the oxygen transfer step, the conversion of intermediate I to intermediate II, gives a straight line with a slope -0.5. Fluoride ion is a product of the enzymatic reaction when 2,3,5,6-tetrafluorophenol is the substrate. These results are consistent with an electrophilic substitution mechanism for oxygen transfer. The conversions of I to II and II to III are acid-catalyzed. A kinetic isotope effect of 8 was measured for the conversion of II to III using deuterated resorcinol as substrate. The conversion of III to oxidized enzyme is base-catalyzed, suggesting that the reaction depends on the removal of the flavin N(5) proton. Product release occurs at the same time as the formation of intermediate III, or rapidly thereafter. The results are interpreted according to the ring-opened model of Entsch et al. (Entsch, B., Ballou, D. P., and Massey, V. (1976) J. Biol. Chem. 251, 2550-2563).     

A Yersinia pseudotuberculosis protein which cross-reacts with HLA-B27

The most-debated question in the investigation of the spondyloarthropathies has been whether there is molecular mimicry between host HLA-B27 antigens and the arthritis-causing pathogens. We have generated a monoclonal anti-HLA-B27 antibody in our laboratory and have used a radioimmunoassay to screen a panel of bacterial species. Two strains of Yersinia pseudotuberculosis were found to be highly reactive. The cross-reactive Yersinia component was identified by Western blot to be a 19,000 component. A preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis chromatography apparatus was constructed to isolate milligram quantities of this component. To verify that the component carried the HLA-B27-specific epitope, rabbits were hyperimmunized with the purified materials. Affinity-purified antibodies from one of the immunized rabbits indeed carried anti-HLA-B27 activity. Last, antibodies generated against synthetic peptides derived from the HLA-B27.1 amino acid sequence were tested against the Yersinia component. Positive reactivity was found with antibodies generated against a peptide spanning residues 69-83 of the HLA-B27.1 protein. Since this resides in the segment responsible for the allotypic specificity of the antigen, these experiments establish the presence of molecular mimicry to a high degree of confidence.     

Escherichia coli RecBC pseudorevertants lacking chi recombinational hotspot activity

Pseudorevertants of an Escherichia coli exonuclease V (RecBC enzyme)-negative mutant have been isolated after ethyl methane sulfonate mutagenesis of a recC73 (presumed missense) mutant. The remedial mutations in each of the four pseudorevertants studied in detail map and complement as recC mutations. By several criteria, such as recombination proficiency, support of phage growth, RecBC nuclease activity, and cell viability, the pseudorevertants appear to have regained partially or completely various aspects of RecBC activity. However, chi recombinational hotspots, which stimulate exclusively the RecBC pathway of recombination, have no detectable activity in lambda vegetative crosses in the pseudorevertants. The properties of these mutants, in which the RecBC pathway of recombination is active yet in which chi is not active, are consistent with the hypothesis that wild-type RecBC enzyme directly interacts with chi sites; alternatively, the mutants may block or bypass the productive interaction of another recombinational enzyme with chi.     

Coordinate regulation of ribosomal protein mRNA level in regenerating rat liver. Study with the corresponding mouse cloned cDNAs.

Cloned mouse ribosomal protein (rp) cDNAs exhibit extensive homology with the corresponding rat sequences. The size of the rp-mRNAs and complexity of the rp-genes are very similar in the two species. Using the mouse rp-recombinant DNAs we find that the relative abundance of rat L7, L13, L18, L30, L32/33 and S16 mRNAs increases after partial hepatectomy. Their maximal level is about twice that of normal rat liver, and is achieved 12-18 h after the operation, while the relative abundance of albumin mRNA decreases to half the normal values 12 h after partial hepatectomy. This concomitant increase in the relative content of these rp-mRNAs indicates coordinate regulation of their level in the rat. The dissimilar behavior of L10 and L19 rp-mRNA suggests additional control mechanisms of rp-mRNA levels in the regenerating rat liver.     

Properties and serum levels of pregnancy-associated variant of human transcortin

The amino acid composition, N- and C-terminal amino acid sequences, and the basic physicochemical and immunochemical properties of the recently discovered pregnancy-associated molecular variant of human transcortin (Strel'chyonok, O.A., Avvakumov, G.V. and Akhrem, A.A. (1984) Carbohydr. Res. 134, 133-140) have been found to be identical to those of transcortin from normal donor serum. This suggests the identity of polypeptide moieties of the two glycoproteins. The transcortin variant has a lower isoelectric point (3.5-4.1) than normal transcortin (3.6-4.2), and different electrophoretic mobility in low-porosity polyacrylamide gel (one band versus two for normal transcortin). These differences can be reasonably explained by different organization of the carbohydrate moieties of these glycoproteins due to diverse post-translational modification of a single polypeptide chain. The levels of transcortin variant in the maternal venous serum throughout normal gestation (447 donors in all) and on the fifth day after delivery, as well as in umbilical cord serum and extracts of term placenta, have been measured by a radioimmune assay. Analysis of the data obtained allowed us to conclude that the biosynthesis of pregnancy-associated transcortin variant occurs in some organ of the maternal organism rather than in the feto-placental system, and it is a characteristic of pregnancy as a unique physiological state of the female organism rather than a phenomenon caused by individual features of certain women. We assume that the transcortin variant takes part in the guided transport of corticosteroids and/or progestins into some tissues that develop in the course of gestation.     

Experimental investigations in prolonged myocardial ischaemia. Note I. Biology of the myocardial fiber after transient myocardial fiber after transient myocardial ischaemia

The first ten days' evolution of post-ischaemic lesions of the premonitory or angina pectoris syndrome type was experimentally studied by the challenge of a short-term (10 and 15 min) ischaemia, of an adaptation to ischaemia and an adaptation followed by prolonged ischaemia (20 and 35 min). Worthy of note was the persistence of reversible lesions after short-term ischaemia and adaptation, and the progressive evolution towards cytolysis and cicatrization of some pancicellular foci after adaptation followed by prolonged ischaemia. The role of mitochondrial lesions, of lysosomal hydrolases, the inefficiency of renewed circulation, as well as problems of diagnosis are discussed.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.