Binding of a spin-labelled chlorpromazine analogue to calmodulin.

The binding of a spin-labelled derivative of chlorpromazine to calmodulin was investigated by e.s.r. spectrometry. The completion of the spectroscopic changes requires the presence of 4 Ca2+ ions per calmodulin molecule. The influences of various physicochemical factors (pH, ionic strength) are discussed in relation to the nature (hydrophobic and polar) of the interactions that hold the drug-calmodulin complex together.     

Valine-sensitive acetohydroxy acid synthases in Escherichia coli K-12: unique regulation modulated by multiple genetic sites.

Summary Spontaneous mutants (146) of Escherichia coli K-12 were selected that were resistant to inhibition of growth by 1.2 mM L-valine (Val^r). The Val^r isolates, containing acetohydroxy acid synthase resistant to feedback inhibition by L-valine (AHAS^r), were classed according to cotransduction of the mutation with leu. Several mutations resulting in an AHAS^r phenotype were found to be cotransducible with glyA. However, no mutations causing a Val^r phenotype were linked to ilv. AHAS activity was more closely examined in representatives of three classes of mutants with Val^r linked to leu, labeled ilv-660, ilv-661, and ilv-662. The ilvE503 allele in E. coli K-12, known to cause a two- to three-fold derepression of AHAS, was found to affect regulation of synthesis of both valine-sensitive AHAS (AHAS^s) and AHAS^r in the mutants containing ilv-660 and ilv-661, whereas it affected repression of AHAS^s, only, in the mutant containing ilv-662. Further, both AHAS^s and AHAS^r in the ilv-661 mutant were repressed by valine, whereas valine did not repress AHAS^r synthesis in the strain carrying ilv-660 and only partially repressed AHAS^r in the strain carrying ilv-662. Unexpectedly, AHAS^r synthesis in strains carrying ilv-660 or ilv-662 was repressible by leucine. The ilv-660 locus appears to be similar in position to ilvH and encodes a product that confers valine-sensitivity upon AHAS activity in the wild-type E. coli K-12. The ilv-660 and ilv-662 loci may normally encode products that influence both the feedback sensitivity of AHAS and control of AHAS biosynthesis.     

Purification and characterization of two isoenzymes of lipoxygenase from soybeans

A chromatographic procedure for the purification of two lipoxygenase isoenzymes (linoleate: O₂ oxidoreductase, EC 1.13.11.12.) from soybean is described. The procedure for the purification of isoenzyme L-1 includes optimalized extraction, ammonium sulfate fractionation, heat treatment and gradient elution from a CM-Sephadex C-50 column. The purification of L-2 includes ammonium sulfate fractionation, gelfiltration on Sephadex G-150 and gradient elution from a DEAE-cellulose column. Both isoenzymes L-1 and L-2 appear homogeneous after Disc-PAGE. The isoelectric points are 5.6 for L-1 and 5.8 for L-2. Molecular weights are estimated as 100,000 for L-1 as well as L-2 applying three different methods. Both isoenzymes contain 0.9 mol iron per mol protien. The estimated turn over numbers are 8,200 mol linoleate per mol enzyme and min for L-1 and 3,100 for L-2. Amino acid compositions determined after acid hydrolysis show marked differences between L-1 and L-2, particularly with respect to the amino acids Lys, Phe, Ser, Gly and Leu. L-1 posesses a total of 9 cysteine molecules, 6 of which are present as disulfide bonds. L-2 posesses a total of 8 cysteine molecules with only one disulfide bond.These results have been presented in part at the 13th ISF Congress in Marseille on 2nd September 1976     

The genetic significance of accessory bisatellited marker chromosomes

Ten new cases of accessory bisatellited marker chromosomes examined in different laboratories are reported. As a basis for genetic counseling in the context of prenatal diagnosis a cytogenetic categorization of such marker chromosomes is proposed and an estimation of the genetic risk associated with each category is carried out. The results are as follows: There is no increased risk for offspring with abnormal phenotype born to a healthy carrier of an accessory bisatellited marker chromosome with either a single or two closely adjacent C-bands (Category AI or AII). The unbiased sample of cases with de novo accessory bisatellited marker chromosomes of categories AI and AII is too small to allow a satisfactory estimation of the actual risk that, in case of such a prenatal finding, the foetus may not show a normal phenotype as a consequence of the marker chromosome. There is, however, evidence that this risk may be lower than 10%. Accessory bisatellited marker chromosomes showing a discrete pattern of G- and R-bands situated between two distant C-bands (Category AIII) usually indicate a chromosomal imbalance giving rise to an abnormal phenotype. Mosaic carriers of such dicentric marker chromosomes may, however, present a normal phenotype.     

Soluble inhibitory factor (SIF) in normal human serum

We have previously noted that dividing T cells release soluble inhibitory factor (SIF) into culture fluids. SIF was distinguished from most other suppressor factors by consisting of two components: a protein and a glycolipid, lipid suppressor substance (LSS). We had noted large quantities of LSS in serum of a patient with a cutaneous lymphoma. This prompted the present study in which SIF was found in normal human serum in a fraction derived by ethanol precipitation. The SIF complex reduced uptake of [³H]thymidine into mixed leukocyte culture (MLC) by 77%. SIF from serum (SIF-serum) resembled SIF in culture fluids (SIF-sup) by chromatography and function at all stages of purification. LSS was extracted from SIF-serum, as measured by an 88% reduction of [³H]thymidine incorporation into phytohemagglutinin-stimulated lymphocytes. The apparent MW of SIF was between 100, 000 and 150, 000. LSS from serum was purified by two-dimensional thin-layer chromatography to apparent homogeneity. The presence of SIF in normal human serum suggests that it may have an in vivo role in immune regulation.     

Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro

Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation ara cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbonyl-14C-labeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this compound is activated by microsomal enzymes. However, only about 5% of this metabolite can be accounted for as protein-bound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole.     

Maximal activities of hexokinase, 6-phosphofructokinase,oxoglutarate dehydrogenase,and carnitine palmitoyltransferase in rat and avian muscles

The maximum activities of 6-phosphofructokinase and oxoglutarate dehydrogenase in muscle provide quantitative indices of the maximum capacities of anaerobic glycolysis and the Krebs cycle (i.e. the aerobic capacity) respectively. These activities were measured in red, white, and cardiac muscle of birds and the rat. The activities in the white pectoral muscle of the domestic fowl suggest that the Krebs cycle plus electron transfer could provide only about 1% of the rate of ATP production provided by anaerobic glycolysis whereas in pigeon pectoral muscle the predicted maximal rates from the two processes are similar. In contrast to domestic-fowl pectoral muscle, the white rat muscle, epitrochlearis, contains a significant activity of oxoglutarate dehydrogenase, which indicates that the Krebs cycle could provide about 12% of the maximum rate of ATP formation. This may be explained by a higher proportion of type-I and -IIA fibres in the rat muscle compared to the avian muscle. In the aerobic muscles of the rat the maximum activities of carnitine palmitoyl transferase indicate that fatty-acid oxidation could provide a high rate of ATP formation.