Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Modification of a tyrosine protected by phosphate

Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg²⁺-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca²⁺-ATPase activity of the soluble coupling factor from chromatophores (R. rubrum F₁) was inactivated by NBD-Cl with kinetics resembling those described for Mg²⁺-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD-R. rubrum F₁ fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca²⁺-ATPase activity of R. rubrum F₁ were fully protected by 5 mM P_i against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca²⁺-ATPase activity by P_i indicated that NBD-Cl and P_i are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F₁ underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.     

Cross-linking of mitochondrial matrix proteins in situ

Different cross-linkers (10 mM) of varying specificity and arm length were found to cross-link mitochondrial matrix proteins in situ in 2 min at pH 7.4. As seen by SDS-polyacrylamide electrophoresis, the disappearance of individual protein bands was accompanied by concomitant appearance of polymeric aggregates that failed to enter the 4% spacer gel. The disorganization of the mitochondrial matrix infrastructure either by swelling or sonication of the mitochondria resulted in a decrease in the rate of cross-linking. Leakage of citrate synthase, malate dehydrogenase and fumarase was found to be reduced when cross-linked mitochondria were made permeable with toluene. On lysing the cross-linked mitochondria, a major part of the matrix protein (75%) was found to sediment with the membrane fraction. The activities of citrate synthase, malate dehydrogenase and fumarase in rat liver mitochondria were also found to increase in the precipitates with a concomitant decrease in their activities in the soluble matrix fraction. These results indicate that the cross-linker enters the mitochondria and cross-links matrix proteins including Krebs cycle enzymes either to the mitochondrial membranes, or to themselves resulting in very large molecular weight complexes. These results are interpreted to mean that in liver mitochondria, the Krebs cycle enzymes are preferentially located near the membrane.     

Characterization and Location of Met5-Enkephalin-Arg6-Phe7 Stored in Various Rat Brain Regions

A specific antiserum against met⁵-enkepha-lin-arg⁶-phe⁷ was raised and used to study the distribution and characterization of met⁵-enkephalin-arg⁶-phe⁷-like immunoreactive material in rat brains by radioimmunoassay and immunohistochemical procedures. The antiserum appears to be directed to the COOH-terminus of the peptide, as it fails to cross-react with met⁵-enkeph-alin, met³-enkephalin-arg⁶, met⁵-enkephalin-arg⁶-arg⁷, met⁶-enkephalin-lys⁶, and leu-enkephalin. However, it cross-reacts with phe-met-arg-phe by about 10% and with phe-met-arg-phe-NH₂ to an insignificant degree. The highest content of met⁵-enkephalin-arg⁶-phe⁷ was found in the striatum, which contains a dense network of immunoreactive varicose fibers and terminals, as well as immunoreac tive cell bodies. The met⁵-enkephalin-arg⁶-phe⁷ in striatum can be released in a Ca²⁺-dependent manner by a depolarizing concentration of KC1, raising the possibility of a neu-roregulatory role for met⁵-enkephalin-arg⁶-phe⁷. Characterization of the immunoreactive material by gel filtration and high pressure liquid chromatography revealed the presence of multiple forms of immunoreactive material in some brain regions.     

Levonorgestrel as a new radioligand for determination of sex hormone binding globulin in human plasma

Levonorgestrel which binds with high affinity to SHBG, is suggested as a new radioligand for estimation of SHBG in human plasma. Using 3H-levonorgestrel as a ligand, a number of samples from men, women pregnant and non-pregnant were analysed. The SHBG content was lowest in men, low in women and rose to higher level during pregnancy. The results of the present study suggest that levonorgestrel appears to be a better ligand than dihydrotestosterone for measuring SHBG.     

Identification of natural tricetin-derived C-glycosides through synthesis

C-Glycosylation of 5,7-dihydroxy-3′,4′,5′-trimethoxyflavone was carried out with acetobromo-α-d-glucose, -α-d-galactose, -α-d-xylose, -β-l-arabinose and -αt-L-rhamnose. The respective 6-C-glycosides and 6, 8-di-C-glycosides (excepted for galactose) were isolated and permethylated. MS and TLC comparison confirmed the proposed structures of five natural tricetin-derived C-glycosides.     

The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells

The majority of the high (12-fold elevated) baseline sister-chromatid exchanges (SCEs) that occur in the CHO mutant line EM9 appear to be a consequence of incorporated BrdUrd, and they arise during replication of DNA containing BrdUrd in a template strand. In normal CHO cells the alkaline elution patterns of DNA newly replicated on a BrdUrd-containing template are significantly altered compared with those seen during the replication on an unsubstituted template. The nascent DNA synthesized on such an altered template is delayed in reaching mature size, possibly because replication forks are temporarily blocked at sites occurring randomly along the template. Transient blockage of replication forks may be a prerequisite for SCE. The delay in replication on BrdUrd-substituted templates was greater in EM9 cells than in parental AA8 cells and was also greater in AA8 cells treated with benzamide, an inhibitor of poly(ADPR) polymerase, than in untreated AA8 cells. Under these conditions, treatment with benzamide also produced a 7-fold increase in SCEs in AA8. An EM9-derived revertant line that has a low baseline SCE frequency showed less delay in replication on BrdUrd-substituted templates than did EM9. However, under conditions where the template strand contained CldUrd, which was shown to produce 4-fold more SCEs than BrdUrd in AA8 cells, the replication delay in AA8 was not any greater in the CldUrd-substituted cells. Thus, other factors besides the delay appear to be involved in the production of SCEs by the template lesions resulting from incorporation of the halogen-substituted pyrimidine molecules.     

Possible roles of calcium and ammonium in the development of bitter pit in apple

Apple trees ( Malus pumila Mill . var. domestica Fuji/ Malus prunifolia rootstock) showed a high susceptibility to bitter pit when supplyed with ammonium salt instead of nitrate (control) in the nutrient solution. When apple fruit was affected by bitter pit, a lower calcium as well as a higher nitrogen and ammonium-nitrogen contents was observed in the fruit flesh near the calyx end. The activity of the mitochondrial Ca²⁺-uptake of the fruit flesh near the calyx end was higher when the tree was grown with ammonium salt than when grown with nitrate. Both the activities of succinate: cytochrome c oxidoreductase and the mitochondrial Ca²⁺-uptake per g of tissue were higher in affected fruit than in healthy fruit. Each of chlorpromazine, N-(6-aminohexyl)-5-chloro-l-napthalenesulfonamide (W-7) and N-(6-aminohexyl)-l-naphthalenesulfonamide (W-5), calmodulin antagonists, was infiltrated into the fruit for 20 min under reduced pressure (about 1 × 10⁴ Pa). Few days later, numerous bitter pit-like spots were observed in both fruit treated with W-7 and chlorpromazine, while only a few spots were observed after the infiltration with W-5, a less potent calmodulin antagonist. A possible mechanism for the occurrence of bitter pit is discussed.     

Precocene causes male determination in the aphid Myzus persicae

When adult apterous viviparous females of Myzus persicae, reared in short night conditions at 21–23°C, are treated with the precocene analogue 6-methoxy-7-ethoxy-2,2-dimethylchromene, they deposit males towards the end of their reproductive lives. The first-born normal daughters of the treated females also deposit some males at various times in their reproductive lives. Karyotypic analysis was used to investigate the sequence of male and female embryos in the ovarioles of precociously metamorphosed aphids. The experiments support the hypothesis (Mittler et al., 1979) that juvenile hormone level controls the sex determination process in aphids. Since male aphids have an XO sex chromosome constitution, this implies that juvenile hormone level influences the behaviour of the X-chromosomes at or before the single maturation division of the egg. At this division one X-chromosome is eliminated from eggs which will develop as males. Aphids provide the first example of a specific endocrine influence on chromosome behaviour in sex determination.     

Thiolated nucleotides in yeast transfer RNA

By culturing Saccharomyces cerevisiae in growth medium containing Mg³⁵SO₄, we have determined the extent and variation of tRNA thiolation in this yeast. We find that 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U)¹ is the major, if not only, thiolated derivative in S. cerevisiae tRNA. In addition, a comparison of the chromatographic mobility of mcm⁵s²Up on cellulose thin layers with those reported for unknown uridine derivatives found in purified yeast tRNA digests, leads to the conclusion that at least two of these tRNAs contain this modification.     

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K⁺ and 3 mM Mg²⁺ retards the reaction, while 20 mM K⁺ and 2 mM Mg²⁺ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300–400 mol/mol per min. in each case. The K_m for ribosomes is 1 μM in the presence of low salt and 0.2 μM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pK_a value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.