Glucose-C14 metabolism of dormant and activated ascospores of Neurospora

Budd, Kenneth (The University of Michigan, Ann Arbor), Alfred S. Sussman, and Frederick I. Eilers. Glucose-C(14) metabolism of dormant and activated ascospores of Neurospora. J. Bacteriol. 91:551-561. 1966.-Dormant and activated ascospores of Neurospora tetrasperma, incubated in C(14)-labeled glucose, absorb and metabolize this sugar. At the same time, up to 55% of the CO(2) production from endogenous substrates is quenched, whereas total CO(2) production is unchanged. Glucose-carbon appears in CO(2), lipids, and ethyl alcohol-soluble and -insoluble material in both dormant and activated ascospores, although the proportions entering these fractions differ in the two groups of spores. With few exceptions, the identifiable intermediates of glucose metabolism are the same in dormant and activated ascospores, indicating that the principal pathways may be identical. During glucose metabolism, dormant ascospores accumulate a nondialyzable, ethyl alcohol-soluble polymer, or polymers, which is either absent from activated spores or present in much smaller amounts. This material contains glucose, ribose, and at least nine amino acids, and may represent precursors of more complex cell material which accumulate because of an enzymatic deficiency in the dormant spore. Radioactivity is incorporated into all fractions of the dormant spores and into CO(2) without a noticeable lag, indicating that most, if not all, of the enzymes for glucose utilization are present. A lag in incorporation is observed in the activated spores, which most probably is due to rapid endogenous production of glucose from trehalose, resulting in dilution of lable. After absorption of labeled glucose, two pools of trehalose are found in dormant spores, one of which is extractable without breaking the spores, and the other, only after the spores are disintegrated. The widely differing specific radioactivity of the two pools indicates that these are separated in the intact spore.     

Solubilization of the receptor for N-1-naphthylphthalamic Acid

A receptor protein for the auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), has been solubilized from corn coleoptile membranes using Triton X-100. [³H]NPA binding activity of the receptor was compared in soluble and membrane-bound states. Both activities are abolished by treatment with trypsin. Differences between the two are observed in pH optima and rates of heat inactivation.     

Synthesis of recA protein and induction of bacteriophage lambda in single-strand deoxyribonucleic acid-binding protein mutants of Escherichia coli.

We investigated the capacity of Escherichia coli mutants defective in the single-strand deoxyribonucleic acid (DNA)-binding protein to amplify the synthesis of the recA protein, induce prophage lambda, and degrade their DNA after treatment with ultraviolet radiation, mitomycin C, or bleomycin. The thermosensitive ssbA1 strain induced recA protein and lambda phage normally at 30 degrees C, but no induction was observed at 42 degrees C when ultraviolet radiation or mitomycin C was used. The lexC113 mutant did not amplify recA protein synthesis or induce phage lambda at either 30 or 42 degrees C with those agents. Bleomycin was able to elicit induction of recA and phage lambda in both mutants at any temperature. After induction with ultraviolet radiation at the elevated temperature, no DNA degradation was observed for 40 min, but at later times there was increased degradation in the lexC113 strain, compared with the wild type, and even greater degradation in the ssbA1 mutant. We discuss the role of single-strand DNA-binding protein in induction and the possibility that the lexC product may exert its influence on recA and lambda induction at the level of the single-strand DNA gap.     

Distribution and variation of hemagglutinating activity in the hemolymph of Biomphalaria glabrata

A sensitive hemagglutination assay utilizing glutaraldehyde-fixed trypsinized calf erythrocytes (GTC) is described to test for agglutinin levels in hemolymph and albumen gland extracts from nine populations of Biomphalaria glabrata, and from B. straminea and B. obstructa. High levels of GTC-reactive hemagglutinin were found in all snail populations. There was no correlation between hemagglutinin titer and innate resistance of B. glabrata strains to Schistosoma mansoni. However, an increase in hemagglutinin titer occurs in B. glabrata M-RLc snails infected with Echinostoma lindoense and in snails sensitized and reexposed to this parasite.     

Cobalt: an effector of E. coli recA protein activity.

Studies of cation requirements in the recA-catalyzed proteolysis of lambda repressor and strand assimilation reactions have demonstrated that Co2+ significantly enhances both activities. In the presence of 4mM MgCl2, the optimal concentration of CoCl2 for proteolysis was 1mM. 2mM Co2+ increased the rate and extent of D-loop formation as measured by membrane filtration. Cobalt did not replace Mg2+ for the ssDNA-dependent ATPase activity of recA, and did not affect the rate of hydrolysis of ATP, measured over a wide range of DNA concentrations. Cobalt did prevent the Mg-dependent ssDNA renaturation catalyzed by recA protein. Membrane filter binding assays established that Co2+ increases the affinity of recA protein for ssDNA with ATP, dATP, or ATP gamma S as cofactors. The dissociation of recA protein from ssDNA-nucleoside triphosphate complex was much slower with CoCl2. This metal provides an excellent tool for dissecting the various activities inherent in recA protein.     

The plasma membrane H+-ATPase gene family in Arabidopsis: genomic sequence of AHA10 which is expressed primarily in developing seeds

The plasma membrane H+-ATPases in Arabidopsis thaliana represent the largest family of cation translocating P-type ATPases identified in plants or animals. We report here seven new isoforms, which were identified by polymerase chain reaction (PCR) amplification of genomic DNA. Amplifications were performed with degenerate primers corresponding to two short conserved sequence motifs (CSDK and GDGV) found in most P-type ATPases. A comparison was made of three CSDK-side primers, which were used either as totally degenerate mixtures or rendered less degenerate by substitution with deoxyinosine or fluorodeoxyuridine. Amplified genomic fragments were cloned, partially sequenced and shown to correspond to Arabidopsis genes by Southern blot analysis with gene-specific probes. One newly identified isoform, AHA10, was isolated as a cosmid clone and sequenced. The 5 and 3 ends of the gene were determined by comparison with the AHA10 cDNA sequence. AHA10 is the most divergent isoform characterized in the Arabidopsis family. AHA10 appears to be expressed primarily in developing seeds, as indicated by Northern blot analysis of AHA10 mRNA and by the analysis of transgenic plants expressing a -glucuronidase (GUS) reporter gene fused to an AHA10 promoter. Our results indicate that one function of this unusually large H⁺-ATPase gene family is to allow for expression of different isoforms in different cell types.     

Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.     

Electrooptical measurements demonstrate a large permanent dipole moment associated with acetylcholinesterase.

Acetylcholinesterase (AChE) from krait (Bungarus fasciatus) venom is a soluble, nonamphiphilic monomer of 72 kDa. This snake venom AChE has been analyzed by measurements of the stationary and the transient electric dichroism at different field strengths. The stationary values of the dichroism are consistent with the orientation function for permanent dipoles and are not consistent with the orientation function for induced dipoles. The permanent dipole moment obtained by least-squares fits for a buffer containing 5 mM MES is 1000 D, after correction for the internal directing field, assuming a spherical shape of the protein. The dipole moment decreases with increasing buffer concentration to 880 D at 10 mM MES and 770 D at 20 mM MES. The dichroism decay time constant is 90 ns (+/- 10%) which is clearly larger than the value expected from the size/shape of the protein and indicates contributions from sugar residues attached to the protein. The dichroism rise times observed at low field strengths are larger than the decay times and, thus, support the assignment of a permanent dipole moment, although it has not been possible to approach the limit where the energy of the dipole in the electric field is sufficiently low compared to kT. The experimental value of the permanent dipole moment is similar to that calculated for a model structure of Bungarus fasciatus AChE, which has been constructed from its amino and acid sequence, in analogy to the crystal structure of AChE from Torpedo californica.