Cyclic AMP-dependent protein kinase of Neurospora crassa

$\text{Neurospora}\text{crassa}$ was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type $\text{Neurospora}$ extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs.     

Isolation of poly(A)-containing RNA on synthetic fluorophlogopite (Mica).

Synthetic fluorophlogopite, an aluminosilicate of the same structure as naturally occurring mineral mica in which potassium ions on the basal surface have been replaced by aluminum ions, has the ability to retain polynucleotides irreversibly. This property of Al³⁺-mica was used for irreversible adsorption of poly(U) and subsequent selective adsorption of poly(A)-containing RNA from rabbit reticulocyte polysomes at high salt concentration and its elution by 50% dimethylsulfoxide. The properties of RNA isolated on poly(U)-Al³⁺-mica were studied by sucrose density gradient centrifugation and by stimulation of globin synthesis in an in vitro protein synthesizing system from wheat germ and from Krebs II-ascites cells. The preparation contained 9s RNA species which corresponds to rabbit globin messenger RNA, and under optimal conditions it stimulated protein synthesis more than 100-fold. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate showed that synthesized product was identical with rabbit globin.     

The structure of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone, an effective analog of colchicine

-Methoxy-5-(2',3',4'-trimethoxyphenyl) tropone is an active analog of colchicine, a mitotic spindle inhibitor, which is missing the middle "B" ring. This compound crystallizes in the triclinic system, space group P1, with Z = 2; a = 10.135(2), b = 10.166 (4), and c = 7.863(2) A; alpha = 82.15(3), beta = 103.49(3), and gamma = 107.16(2); degrees and V = 750.7(4) A. The structure was solved by direct methods and refined by full-matrix least-squares to a final R = 0.063, using 2503 observed reflections and 271 parameters. Despite the absence of the middle ring, the conformation of the molecule is similar to that of colchicine, isocolchicine , and their derivatives. The troponoid ring is dissimilar to the phenyl ring in that it is not aromatic and does have alternating short and long bond lengths. The dihedral angle between the least-squares planes of the two rings is -57.4 degrees. Van der Waals surface representations of the analog and colchicine are presented to demonstrate the similarity and differences of these two molecules . The structural information of the analog is consistent with the interpretation of thermodynamic parameters which govern the interactions between brain tubulin and the analog.     

Localization of the 3' end of Escherichia coli 16 S RNA by electron microscopy of antibody-labelled subunits.

The 3′ end of 16 S RNA is localized on the 30 S subunit of Escherichia coli ribosomes by immune electron microscopy. It is located in the groove between the side “ledge” and the “head” of the subunit on the level of the ledge top. Thus, we have localized the 30 S subunit functional site which is believed to be responsible for binding of the specific messenger RNA sequence preceding the initiation codon. The localization of the 3′ end of 16 S RNA has been done by a new approach in immune electron microscopy. It is based on the covalent binding of low molecular weight ligands, containing the residue of phenyl-β-d-lactoside hapten, to certain points of RNA and the localization of the binding site of the antibody specific to this hapten by electron microscopy. The advantages of this approach in comparison with conventional methods of immune electron microscopy are discussed.     

In vitro reconstitution of nitrate reductase activity of the Neurospora crassa mutant nit-1: specific incorporation of molybdopterin

The reduced, metal-free pterin of the molybdenum cofactor has been termed molybdopterin. Oxidation of any molybdopterin-containing protein in the presence or absence of iodine yields oxidized molybdopterin derivatives termed Form A and Form B, respectively. Application of these procedures to whole cells and cell extracts has demonstrated the presence of molybdopterin in wild-type Neurospora crassa, and its absence in the cofactor-deficient mutant nit-1. In order to demonstrate that the reconstitution of nitrate reductase activity in nit-1 extracts results from the incorporation of molybdopterin into the apoprotein, active molybdopterin, free of contaminating amino acids or peptides, was isolated from chicken liver sulfite oxidase and used in the reconstitution system. The results show that, during reconstitution, exogenous molybdopterin is specifically incorporated into the nitrate reductase protein, confirming the role of molybdopterin as the organic moiety of the molybdenum cofactor.     

Degradation of poly(adenosine diphosphate ribose) by homogenates of various normal tissues and tumors of rats.

The extent of increase in the activity of phenylalanine ammonia-lyase upon illumination for 15 hr of cultured Petroselinum hortense cells was greatly dependent upon the age of the culture. Two distinct peaks in specific activity were observed during the growth cycle, one occurring at the beginning and the other at the end of the period of increase in cell fresh weight. High yields in both cell fresh weight and enzyme activity were obtained with the second peak shortly before the stationary phase of the culture was reached. Cells were harvested at this stage and stored at ?20 °C.The enzyme was purified from the frozen cells to apparent homogeneity by precipitation with (NH₄)₂SO₄, chromatography on DEAE-cellulose, Sephadex G-200 and hydroxyapatite columns, and preparative polyacrylamide gel electrophoresis. An over-all yield of 16% was achieved with a 440-fold increase in the specific activity by purification of the enzyme through the hydroxyapatite step.Upon analytical polyacrylamide gel electrophoresis either in the presence or in the absence of sodium dodecyl sulfate, the purified protein migrated essentially as a single band. Molecular weights of about 330,000 and 83,000, respectively, were estimated for the enzyme and for its protein subunits. Thus, the enzyme molecule seems to be composed of four probably identical protein subunits. Two Michaelis constants for l-phenylalanine (K_m^L, 3.2 × 10^?5 M, and K_m^H, 2.4 × 10^?4 M) and a Hill coefficient of h = 0.6 were obtained. This suggests that the enzyme is subject to regulation of its catalytic properties by negative cooperativity of the protein subunits.     

Effect of chromium(III) on poly(dG-dC) conformation

The interaction of chromium(III) with poly(dG-dC) inhibits the B to Z transition and results in the condensation of the polymer at high Cr/nucleotide ratios. At low Cr/nucleotide ratios chromium(III) enhanced the ability of ethanol to induce the B to Z transition of poly(dG-dC). The effects of chromium(III) on the conformation of DNA may be related to the carcinogenicity of chromium compounds.     

Reductive activation of methanol: 5-hydroxybenzimidazolylcobamide methyltransferase of Methanosarcina barkeri

Methanol: 5-hydroxybenzimidazolylcobamide methyltransferase (MT1) from Methanosarcina barkeri, which is one of the enzymes responsible for the transmethylation from methanol to coenzyme M, was found to be activated in the presence of hydrogenase and ferredoxin. This activation was shown to involve a reduction of the bound corrinoid to the Co (I) level, and was demonstrated by spectrophotometry and chemical conversion of reduced MT1 to its methylated form. The reducing system of hydrogenase and ferredoxin was able to reduce dithiols, like dithiodiethanesulfonate and cystine to their monomers, in the presence of a corrinoid, which acts as an electron carrier. The ferredoxin was purified 133-fold and was tentatively identified on the basis of spectral properties and iron content of 3.8-4.0 atoms iron per molecule ferredoxin (12,000 daltons).     

Regulation of methionine biosythesis in Neurospora crassa.

The regulation of serine hydroxymethyltransferase, methylenetetrahydrofolate reductase, and methyltetrahydropteroylpolyglutamate:homocysteine methyltransferase was investigated in Neurospora crassa. Adding choline to the medium decreased the specific activity of these enzymes. Methionine potentiated the choline effect, but, when added alone, was without effect. Neither choline, methionine, nor S-adenosylmethionine appears to be the immediate corepressor of synthesis of these enzymes.Several nonallelic mutants were examined for the enzymes of methionine methyl group synthesis. The formate-requiring mutant for lacks serine hydroxymethyltransferase. The methionine-requiring mutants me-1 and me-8 lack, respectively, the reductase and the methyltransferase. The methionine-requiring mutants me-1, me-6 (folate polyglutamate synthetase deficient) and me-8 were found to have significantly higher serine hydroxymethyltransferase specific activities than did the wild-type strain.     

Complete turnover of poly(A) on maternal mRNA of sea urchin embryos

Measurement of the incorporation of radioactive adenosine into precursor pools and into poly(A) of fertilized sea urchin eggs showed that the amount of adenosine incoporated into poly(A) after a 2 hr incubation approximated the total poly(A) content of the embryos. This was observed whether the incubation was begun at fertilization when the poly(A) content is tripling or at 2.5 hr after fertilization when the poly(A) levels are not changing, and thus indicates that poly(A) turns over continually and completely. The turnover appears to take place on polysomal mRNA, since after either 10 or 120 min of incubation, 75% of the ³H-adenosine incorporated into poly(A) is on polysomes. Poly(A) lengths before and after fertilization are not significantly different, indicating that the increase in poly(A) content reflects the addition of poly(A) sequences onto mRNA molecules which previously contained no poly(A) sequences or only short poly(A) sequences. Both the new as well as the preexisting poly(A) tracts must turn over to produce the incorporation we observe. The radioactive poly(A) tracts measured by alkaline release of adenosine begin as short sequences and gradually extend their lengths until they have reached a size consistent with the idea that the poly(A) sequences have become fully radioactive. This labeling pattern shows that the poly(A) is turning over from the 3′ end terminal probably by a shortening and lengthening mechanism.     

Purification of glutathione reductase from porcine erythrocytes by the use of affinity chromatography on 2', 5'-ADP-Sepharose 4B and crystallization of the enzyme.

Glutathione reductase has been purified to homogeneity from porcine erythrocytes by use of affinity chromatography on 2′,5′-ADP-Sepharose 4B. The enzyme was crystallized from an ammonium sulfate solution. Some of the physical and kinetic properties of the purified enzyme are reported.     

Characterization of Neurospora crassa mutant stratins deficient in adenylate cyclase activity.

Mycelial extracts from Neurospora crassa strains having any one of five different mutations of the cr-1 allele (“crisp”) exhibited an adenylate cyclase specific activity 2 to 3% of that found in wild-type strains. The enzyme deficiency seemed to be specific for the cr-1 mutation but not for the “crisp” morphology and recessive in heterocaryons carrying mutated and wild-type cr-1 alleles. The reduced adenylate cyclase activity detected in extracts from cr-1 mutants was not due to an impairment in the extraction of membranes, to a preferential inactivation of the enzyme after extraction, or to the presence of inhibitors.     

Cellulase and ethanol production from cellulose by Neurospora crassa

Two strains of Neurospora crassa have been identified which utilize cellulase and produce extracellular cellulase [see 1,4-(1,3; 1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21]. The activities were detected as early as 48 h in the culture broth. These cultures also fermented d-glucose, d-xylose and cellulosic materials to ethanol as the major product of fermentation. The conversion of cellulose to ethanol was >60%, indicating the potential of using Neurospora for the direct conversion of cellulose to ethanol.     

Improved synthesis of 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 26(27)-tetrol isomers from cholic acid

Synthesis of a mixture of the 25(R) and 25(S) isomers of 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 26(27)-tetrol from cholic acid in four steps, including a Wittig reaction, is described.