Electron paramagnetic resonance and optical evidence for interaction between siroheme and Fe4S4 prosthetic groups in complexes of Escherichia coli sulfite reductase hemoprotein with added ligands

Janick & Siegel [Janick, P. A., & Siegel, L. M. (1982) Biochemistry 21, 3538-3547] showed that the EPR spectrum of the reduced Fe4S4 center (S = 1/2) in fully reduced native ("unligated") Escherichia coli NADPH-sulfite reductase hemoprotein subunit (SiR-HP) is perturbed by interaction with paramagnetic ferrous siroheme (S = 1 or 2) to yield several novel sets of EPR signals: one set with all g values between 2.0 and 2.8, termed "S = 1/2" type, and two sets with the lowest field g value between 4.7 and 5.4, termed "S = 3/2" type. The present study has shown that EPR spectra of fully reduced SiR-HP are nearly quantitatively converted to the classical "g = 1.94" type typical of S = 1/2 Fe4S4 clusters when the heme has been ligated by strong field ligands such as CO, CN-, S2-, and AsO2-, converting the ferroheme to S = 0. However, the exact line shapes and g values of the g = 1.94 differ markedly when different ligands are bound to the heme. Also, optical difference spectra taken between enzyme species in which the heme is kept in the same (Fe2+) oxidation state while the Fe4S4 center is reduced or oxidized show that the optical spectrum of the ligated siroheme is sensitive to the oxidation state of the Fe4S4 cluster. These results indicate that the heme-Fe4S4 interaction of native SiR-HP persists even when the heme Fe is bound to exogenous ligands. We have also found that the g values of the exchange-coupled S = 1/2 and S V 3/2 type signals of native reduced SiR-HP can be significantly shifted by addition of potential weak field heme ligands--halides and formate--or low concentrations of certain chaotropic agents--guanidinium salts and dimethyl sulfoxide--to the fully reduced enzyme. Such agents can also promote interconversion of the S = 1/2 and S = 3/2 type signals. These effects are reversed on removal of the agent. Treatment of reduced SiR-HP with relatively large concentrations of chaotropes, e.g., 60% dimethyl sulfoxide or 2 or 3 M urea, leads to abolition of the S = 1/2 and S = 3/2 EPR signals and their replacement by signals of the g = 1.94 type.     

Polyribosome disaggregation and cell-free protein synthesis in preparations from cerebral cortex of hyperphenylalaninemic rats

Abstract— Seven-day-old rats were injected intraperitoneally with l -phenylalanine (1 g/kg) and the time course of brain polyribosome disaggregation and changes in brain levels of phenylalanine, tryptophan and tyrosine were determined. Disaggregation of brain polyribosomes preceded the increase in levels of phenylalanine in brain, and followed the same time course as depletion of tryptophan from brain. The effects of several metabolites of phenylalanine (which are formed in phenylketonuria) on protein synthesis in vitro was determined for brain and liver systems. None of the compounds tested was inhibitory at concentrations below 10 mM and in all cases hepatic protein synthesis was more sensitive to inhibition than was the corresponding system from brain. Ribosomal dimers, formed in brain after injection of phenylalanine, were incapable of supporting high levels of protein synthesis in vitro, a finding that suggested that the inhibition of protein synthesis in vitro in cell-free systems of brain tissue after injection of phenylalanine into young rats was mediated by disaggregation of brain polyribosomes associated with tryptophan deficiency in brain.     

Effects of prenatal X-irradiation: studies on the mechanism of X-irradiation-induced inhibition of microsomal enzyme development in rat liver

Exposure of pregnant rats to 25 R of x-irradiation on the fourteenth gestation day has produced in the male offspring an impairment of the development of the hepatic microsomal enzyme system which metabolizes hexobarbital. However, irradiation did not suppress the increase of enzyme activity brought about by the administration of chemical inducers (pheno-barbital). Actinomycin, on the other hand, inhibited to varying degrees both the ontogenic and phenobarbital-induced increases in enzyme activity. The effects on the enzyme system have been supported by in vivo measurements of the duration of hexobarbital hypnosis. The ontogenic increase in enzyme activity is hormone-dependent, while that following phenobarbital administration is independent of hormonal regulation as evidenced by the response in hypophysectomized or sexually immature animals. It is concluded from these results that the inhibitory effect of x-irradiation on the hepatic enzyme system is mediated through an action on the hormonal regulation of enzyme activity. Evidence for this hypothesis is discussed.     

Mutator Gene of Escherichia coli B

An azaserine-resistant derivative of Escherichia coli B/UV, AZA/R(1), was found to carry a mutator gene. This gene, designated mutS1, was mapped by means of conjugation and P1kc-mediated transduction. The mutS1 gene was cotransduced with argB at a frequency of 2.4%; the gene order in this region of the chromosome is thy argB mutS1. To determine whether a relationship commonly exists between azaserine resistance and the mutator property, 12 additional azaserine-resistant derivatives of B/UV were developed and tested for the mutator phenotype. None of the twelve was a mutator strain. The level of azaserine resistance was not increased over that of the recipient parent when mutS1 was transduced to an azaserine-susceptible strain. Reversion studies indicated that mutS1 induced adenosine-ribosylthymine to guanosine-cytidine and guanosine-cytidine to adenosine-ribosylthymine transitions. Because such mutational changes are suppressible with deoxynucleosides when induced by base analogues, an attempt was made to suppress the mutator activity of mutS1 by the addition of deoxyribonucleosides to the medium. No suppression was found. Recombinants were prepared containing mutS1 and the Treffers mutator gene of E. coli K-12. The effect of the mutator genes appears to be additive.     

Comparative Evaluation of Rheumatic Activity—A Study of Relationship Between Histologic Changes and Serologic Test Results in Cardiac Surgical Patients

Atrial or ventricular myocardium from patients with surgically corrected rheumatic valvular disease was studied for rheumatic lesions in 86 cases. Histologically active Aschoff bodies were found in 20 per cent of the cases. A slight, but statistically not significant relationship was demonstrated in comparison of elevated serologic tests for rheumatic activity with the presence of Aschoff bodies.     

SYNTHETIC ACTIVITIES DURING SPERMATOGENESIS IN THE LOCUST

Isolated testes of the locust Schistocerca gregaria were immersed in solutions of tritiated thymidine, cytidine, uridine, or arginine for short periods to study nucleic acid and protein synthesis during spermatogenesis. DNA synthesis in this tissue is completed prior to initiation of meiosis. Protein synthesis continues throughout the whole meiotic cycle as well as during spermatid development. Meiotic cells, except those in metaphase through early telophase, and early spermatids are also actively synthesizing RNA. The heteropycnotic X-chromosome does not produce RNA at any stage of spermatogenesis. The rates of protein and particularly RNA synthesis decrease as chromosome condensation progresses. Depression of RNA synthesis, however, is not always accompanied by cytologically detectable condensation of chromatin, since very little or no RNA is synthesized in spermatids in which chromatin condensation has barely begun.     

Biexponential Kinetics of (R)-α-[3H]Methylhistamine Binding to the Rat Brain H3 Histamine Receptor

The H3 histamine receptor is a high-affinity receptor reported to mediate inhibition of CNS histidine decarboxylase activity and depolarization-induced histamine release. We have used (R)-alpha-[3H]methylhistamine, a specific, high-affinity agonist, to characterize ligand binding to this receptor. Saturation binding studies with rat brain membranes disclosed a single class of sites (KD = 0.68 nM; Bmax = 78 fmol/mg of protein). Competition binding assays also yielded an apparently single class of sites with a rank order of potency for ligands characteristic of an H3 histamine receptor: N alpha-methylhistamine, (R)-alpha-methylhistamine greater than histamine, thioperamide greater than impromidine greater than burimamide greater than dimaprit. In contrast, kinetic studies disclosed two classes of sites, one with fast, the other with slow on-and-off rates. Density of (R)-alpha-[3H]methylhistamine binding followed the order: caudate, midbrain (thalamus and hippocampus), cortex greater than hypothalamus greater than brainstem greater than cerebellum. These data are consistent with an H3 histamine receptor, distinct from H1 and H2 receptors, that occurs in two conformations with respect to agonist association and dissociation or with multiple H3 receptor subtypes that are at present pharmacologically undifferentiated.     

Identification, purification, and characterization of truncated forms of the human nerve growth factor receptor

We report the presence of truncated forms of the nerve growth factor receptor (NGFRt) in the conditioned medium of the human melanoma cell line A875 and in human urine and amniotic fluid. Radioiodinated nerve growth factor (125I-NGF) specifically bound to NGFRt was chemically cross-linked. After immunoprecipitation, labeled receptor species were visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NGFRts were purified from human adult male urine or a mixture of human amniotic fluid and infant urine by using a combination of either ion exchange chromatography (adult) or ammonium sulfate precipitation (infant) and immunoaffinity chromatography. Typical yields were about 1 microgram/liter of adult urine and 75 micrograms/liter of amniotic fluid/infant urine. The purified proteins, with molecular masses of 45, 40, and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%), were confirmed to be NGFRts by amino-terminal sequencing and were designated NGFRt-1, NGFRt-2, and NGFRt-3, respectively. The isoelectric points of these three species ranged from 3.3 to 3.95 and displayed intraspecies heterogeneity; subsequently, amino acid residues covalently modified with sialic acid-containing carbohydrates were documented. The binding affinities of these species for nerve growth factor were comparable to that of the low affinity cell surface receptor. The potential to isolate milligram quantities of human NGFRts allows for model studies of the physicochemical structure of the intact receptor and the generation of polyclonal antibodies to study the biological functions of the NGF receptor.