Saccharomyces cerevisiae DNA-dependent RNA polymerase III: a zinc metalloenzyme.

Yeast nuclear RNA polymerase III was purified by batch adsorption to phosphocellulose, followed by ion-exchange chromatography on DEAE-Sephadex and affinity chromatography on DNA-Sepharose. Polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band which contained polymerase activity. The molecular weight estimated by sedimentation velocity centrifugation in a glycerol gradient was 380 000. Enzyme activity was inhibited 50% at 0.1 mM 1,10-phenanthroline and 100% of 1.0 mM, but was restored when 1,10-phenanthroline was removed by dialysis. Enzyme activity was not inhibited by 7,8-benzoquinoline, a nonchelating structural analogue of 1,10-phenanthroline. These results strongly suggest that inhibition of enzyme activity occurs by the formation of a reversible enzyme-zinc-phenanthroline ternary complex. The zinc content, measured by atomic absorption spectroscopy, was 2 g-atoms per mol of enzyme. Zinc was not removed from the enzyme by gel filtration on Sephadex G-25, by passage through Chelex-100 resin, or by dialysis against buffer containing 1,10-phenanthroline. Enzyme-bound zinc was removed by dialysis after denaturation of the enzyme with heat and sodium dodecyl sulfate. Enzyme-bound zinc did not exchange with free zinc. These results establish yeast nuclear RNA polymerase III as a zinc metalloenzyme.     

Occurrence of a regulatory deficiency in purine biosynthesis among purA mutants of Salmonella typhimurium

Summary A defect in the repression of the de novo purine biosynthetic enzymes was detected among purA mutants of Salmonella typhimurium. We suggest that the defect is caused by an altered purine regulation gene (purR) which affects the response level of at least five of the de novo enzymes to repression by excess adenine. Thus the unlinked genes controlling these enzymes constitute a regulation controlled wholly or in part by a purR gene product. The regulation of the guanine operon is regulated by some other mechanism independent of purR.     

Detection of EEG abnormalities with feedback stimulation

A feedback method for testing the reactivity of the occipital-parietal EEG in selected brain-lesioned patients revealed abnormalities of(a) insufficient reactivity,(b) bilateral differences in reactivity, and(3) asynchrony. These abnormalities were more evident during feedback stimulation than in the baseline conditions. The utility of feedback method for detecting EEG abnormalities rests on the increased stability or decreased noisy variation in the EEG during feedback. The EEG becomes more predictable even to the on-line human observer. This makes it easier to detect aberrations or deviations from normal effects. Some effects can only be seen with feedback such as the bilateral differences which occur when the left side controls the feedback compared to when the right side controls it. The results show that feedback EEG is a useful tool in clinical research and indicate that a clinical diagnostic test could be developed with more research. However, the feedback EEG method is not yet a proven diagnostic technique.The assistance of Eric Peper, Sylvia Runnals, Rosemary Billingslea, and Thomas McLaughlin in the EEG recording and data analysis was indispensible to this study.     

Characterization of a monoclinic crystal form of an enzyme of steroid metabolism, Δ5-3-ketosteroid isomerase

A monoclinic crystal form ($\text{P2}{}_1\text{, a = 140.4}\text{A}\text{?}\text{, b = 85.0}\text{A}\text{?}\text{, c = 94.5}\text{A}\text{?}$, β= 130.1 °) of Δ⁵-3-ketosteroid isomerase from Pseudomonas testosteroni (EC 5.3.3.1), grown at pH 7.0, has been characterized. Crystal-density measurements show that the asymmetric unit contains 12 protomers (M_r = 13,394).     

On the mechanism of protein-synthesis inhibition by abrin and ricin. Inhibition of the GTP-hydrolysis site on the 60-S ribosomal subunit.

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell-free systems from rabbit reticulocytes and Krebs II ascites cells. In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl-tRNA, demonstrating that the toxins act at some point after the charging of tRNA. Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti-toxins were added. Recombination experiments between toxin-treated ribosomes and untreated supernatant factors and vice versa showed that the toxin-treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis. Recombination experiments between toxin-treated isolated 40-S subunits and untreated 60-S subunits and vice versa showed that only when the 60-S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited. Both the EF-1-catalyzed and the EF-2-catalyzed ability of the ribosomes to hydrolyze [gamma-32P]GTP was inhibited by abrin and ricin. An 8-S complex released from the 60-S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8-S complex were inhibited by abrin and ricin. The present data indicate that there is a common site on the 60-S subunits for EF-1- and EF-2- stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.     

A 25,000 molecular weight protein constituent of human amyloid fibrils related to amyloid protein AA

Amyloid fibrils from a patient with diffuse amyloid disease are dissociated in 6 m guanidine hydrochloride and fractionated by gel chromatography. Two major components are separated on Sepharose 6B. Both proteins are characterized by chromatography, immunodiffusion, discontinuous gel electrophoresis, amino acid tryptic peptide mapping and amino acid sequence analysis. The smaller of the two components is typical of the known protein AA by size (8400 daltons), amino acid composition and a 30-residue N-terminal sequence. The larger of the components (25,000 daltons) undergoes electrophoresis as a single band and appears unaffected by thiol reduction. It differs from protein AA in amino acid content and by its tryptic peptide map, although it contains an N-terminal amino acid sequence identical to protein AA when carried to 20 residues. Treatment of this larger component by mild acid hydrolysis results in the release of the 8400-dalton protein AA. Fractionation after guanidine hydrochloride treatment of this particular amyloid fibril preparation is compared to the fractionation of a typical secondary amyloid preparation that contains only protein AA as the major component. The origin and relationship of the 8,400- and 25,000-dalton protein components is discussed.     

The binding of azide to human methemoglobin A0. Error analysis for the interpolative and noninterpolative methods.

The binding azide to human methemoglobin A0 has been studied at 6 degrees, pH 7, and I = 0.2 by three spectroscopic methods: (1) the conventional interpolative method, (2) an interpolative dialysis technique, and (3) a noninterpolative method. The interpolative methods assume that the fractional spectral change equals the fraction of heme sites bound by ligand, while the noninterpolative method measures the extent of binding directly, i.e., without the interpolative assumption. Both experiment and error analysis show that method 1 has low precision, and consequently, gives an inherently unreliable binding isotherm. Method 2 achieves high experimental and intrinsic precision. However, method 3, which also has high precision, clearly proves that the interpolative assumption of method 2 is incorrect. That is, the true fractional extent of binding becomes equal to the fractional spectral change only after about 97% of heme sites have been bound with ligands.     

Nitrogen and bolus closing volumes: differences after histamine-induced bronchoconstriction.

Studies in normal subjects have shown that there is little difference in the size of the closing volume when measured by either the nitrogen methods or a bolus method. In this study we have examined the changes in closing volume following histamine-induced bronchoconstriction. In five normal subjects histamine resulted in a reduction in the vital capacity, an increase in the residual volume, and an increase in the airway resistance. The size of the closing volume measured by a bolus method increased after induced bronchoconstriction (0.52 +/- 0.15 1 to 0.74 +/- 0.17 1). With the nitrogen method the closing volume became smaller (0.51 +/- 0.19 1 to 0.17 +/- 0.17 1). Similar differences between the two methods are demonstrated in patients with asthma. The suggested explanation for these differences lies in the different methods used to establish a concentration gradient of gas in the lung. If there is "air trapping" the nitrogen method may fail to establish a concentration gradient.