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M L Pall

Publications and source records attributed to M L Pall.

At least 37 records · Page 2Linked to original sources
PubMed1983

Characterization of an ATP-Mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from Neurospora crassa.

A novel adenylate cyclase activity was found in crude homogenates of Neurospora crassa. The adenylate cyclase had substantial activity with ATP-Mg2+ as substrate differing significantly from the strictly ATP-Mn2+-dependent enzyme characterized previously. Additionally, the ATP-Mg2+-dependent activity was stimulated two- to fourfold by GTP or guanyl-5'-yl-imido-diphosphate (Gpp(NH)p). We propose that the ATP-Mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory component (G component) of the adenylate cyclase which was present in carefully prepared extracts. The adenylate cyclase had a pH optimum of 5.8 and both the catalytic and G component were particulate. The Km for ATP-Mg2+ was 2.2 mM in the presence of 4.5 mM excess Mg2+. Low Mn2+ concentrations had no effect on adenylate cyclase activity whereas high concentrations of Mn2+ or Mg2+ stimulated the enzyme. Maximal Gpp(NH)p stimulation required preincubation of the enzyme in the presence of the guanine nucleotide and the K1/2 for Gpp(NH)p stimulation was 110 nM. Neither fluoride nor any of a variety of glycolytic intermediates or hormones, including glucagon, epinephrine, and dopamine, had an effect on ATP-Mg2+-dependent adenylate cyclase activity. However, the enzymatic activity was stimulated not only by GTP but also by 5'-AMP and was inhibited by NADH.

Adenosine Triphosphate↗
PubMed1983

Reconstitution of adenylate cyclase in Neurospora from two components of the enzyme.

The ATP-Mg2+-dependent, guanine nucleotide-stimulated adenylate cyclase of Neurospora crassa was shown to be composed of distinct components which, when mixed, can reconstitute a holoenzyme. After brief heat treatment, the adenylate cyclase activity in crude homogenates of Neurospora was found to have reduced activity and greatly reduced sensitivity to stimulation by guanyl-5'-yl-imidodiphosphate (Gpp(NH)p). Gpp(NH)p sensitivity could be restored by addition of a homogenate from a crisp-1 (cr-1) mutant of Neurospora which was shown to have little or no adenylate cyclase activity. These results show that the adenylate cyclase of Neurospora resembles the enzyme of animal systems in that it is composed of distinct regulatory and catalytic components. The data presented also show that the cr-1 mutant is defective in the adenylate cyclase catalytic component but not in the regulatory component. Therefore, cr-1 is unlike most other such eucaryotic mutants which appear to be defective in the regulatory component.

Adenosine Triphosphate↗
PubMed1983

Rapid isolation of Escherichia coli minicells by glass-fiber filtration: study of plasmid-coded polypeptides.

A filtration technique is described to purify Escherichia coli chi 1488 minicells much more rapidly than the usual method involving sucrose gradient centrifugation, and to produce minicells that have not been subjected to osmotic stress. The minicells so prepared are metabolically active as indicated by the in vivo incorporation of [35S]methionine into plasmid-coded polypeptides.

Cell Separation↗
PubMed1982

Isolation and properties of a cyclic AMP-binding protein from Neurospora. Evidence for its role as the regulatory subunit of cyclic AMP-dependent protein kinase.

A cyclic AMP-binding protein with a native molecular weight calculated to be 82,000 was purified 2,000-fold from Neurospora crassa. The apparent subunit molecular weight was 47,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native protein exists as a dimer of identically sized subunits. The 8-N3-cyclic [32P]AMP-labeled protein appeared as a doublet upon two-dimensional gel electrophoresis, with pI = 5.4 and 5.5. Binding studies with both noncyclic and cyclic nucleotides showed cyclic AMP to have the highest binding affinity. The KD for cyclic AMP was 300 nM at 23 degrees C. The cyclic AMP-binding protein has two distinct cyclic AMP binding sites, a slowly dissociating site and a rapidly dissociating site. The cyclic AMP analog, 8-bromo cyclic AMP, was found to bind preferentially the slowly dissociating cyclic AMP binding site. The cyclic AMP-binding protein is thought to be the regulatory subunit of cyclic AMP-dependent protein kinase based upon the findings that it co-purifies with this protein kinase activity upon DEAE-cellulose chromatography, isoelectric focusing, and cyclic AMP affinity resin chromatography. On purification by the latter two procedures, no other cyclic AMP-binding activity was detected. A number of similarities between the Neurospora cyclic AMP-binding protein and the cyclic AMP-dependent protein kinase regulatory subunits from yeast and mammalian tissue are discussed.

Binding, Competitive↗
PubMed1981

Gene-amplification model of carcinogenesis.

A two-stage model of carcinogenesis is proposed based on recent evidence for the occurrence of proto-oncogenes in the vertebrate genome, evidence for gene amplification during carcinogenesis, and studies of the action of tumor promoters. The model is baed on the view that an increase in the level of gene product from such proto-oncogenes is sufficient to induce neoplastic transformation. It proposes that the initial step in carcinogenesis (initiation) is a mutation producing a tandem duplication of a proto-oncogene. Gene amplification can then occur by successive unequal sister chromatid crossing-over events in several cell cycles until sufficient gene product is produced to transform the cell.

Animals↗
PubMed1981

Deficient cyclic adenosine 3',5'-monophosphate control in mutants of two genes of Neurospora crassa.

Strains of Neurospora crassa mutant in either of two genes, Crisp-1 (cr1) and Frost (fr), showed no increase of cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels when subjected to several treatments which produce large increases of cyclic AMP in wild-type Neurospora. Evidently, the previously reported deficiencies of adenylate cyclase in these mutants were sufficient to block the normal increases. This fact suggests that both mutants could be used to help determine which control phenomena involve cyclic AMP and to interrupt the control of established cyclic AMP-regulated functions. Earlier studies had suggested an interdependence of the cyclic AMP level and the electric potential difference across the plasma membrane of Neurospora. Present experiments, therefore, employed several strains with the cr1 mutation to test for possible roles of cyclic AMP in recovery and oscillatory behavior of the Neurospora membrane potential. The results showed all such phenomena to be normal in the adenylate cyclase-defective strains, which demonstrates that variations of cyclic AMP are not obligatorily involved in the apparent control processes. Evidence is also presented that the induction of both glucose transport system II and the alternative oxidase do not require elevated cyclic AMP levels.

2,4-Dinitrophenol↗
PubMed1979

Properties of two cyclic nucleotide-deficient mutants of Neurospora crassa.

Studies on the crisp-1 (cr-1), cyclic adenosine 3',5'-monophosphate (cAMP)-deficient mutants of Neurospora crassa were undertaken to characterize the response of these mutants to exogenous cyclic nucleotides and cyclic nucleotide analogs. A growth tube bioassay and a radioimmune assay for cyclic nucleotides yielded the following results. (i) 8-Bromo cAMP and N6-monobutyryl cAMP but not dibutyryl cAMP are efficient cAMP analogs in Neurospora, stimulating mycelial elongation of the cr-1 mutants. Exogenous cyclic guanosine 3'5'-monophosphate (cGMP) also stimulates such mycelial elongation. (ii) Both cAMP levels and cGMP levels found in cr-1 mycelia are lower than those in wild type. However, the levels of both cyclic nucleotides are normal in conidia of cr-1. The data on cr-1 mycelia and those reported earlier in Escherichia coli (M. Shibuya, Y. Takebe, and Y. Kaziro (Cell 12:528-528, 1977) show a previously unexpected relationship between cAMP and cGMP metabolism in microorganisms. The semicolonial morphology of another adenylate cyclase-deficient mutant of Neurospora, frost, was not corrected by exogenous cyclic nucleotides or by phosphodiesterase inhibitors indicating that the frost morphology is probably not caused by low endogenous cAMP levels. The low adenylate cyclase activity and the abnormal morphology of frost may be related separately to the linolenate deficiency reported in the mutant.

Adenylyl Cyclases↗
PubMed1979

Control of cyclic adenosine 3',5'-monophosphate levels by depolarizing agents in fungi.

It has been reported that diverse treatments which depolarize the plasma membrane of Neurospora crassa produce rapid increases in cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. In the current study, membrane active antibiotics, which are known or putative depolarizing agents, were found to produce similar cyclic AMP increases, not only in N. crassa, but also in the distantly related fungi Saccharomyces cerevisiae and Mucor racemosus. Uncouplers of oxidative phosphorylation, which have been found to depolarize Neurospora, also produced cyclic AMP increases in all three fungi. The time course of the cyclic AMP response to these various treatments was similar in all three fungi. The fungal studies and studies on depolarized central nervous tissue suggest that cyclic AMP increases may be produced in response to plasma membrane depolarization in diverse eucaryotic cells. A model is proposed for eucaryotic microorganisms in which membrane depolarization serves as a signal of breakdown of the plasma membrane integrity. The subsequent cyclic AMP increase, in turn, may mediate cellular response to help protect the plasma membrane from chemical and mechanical threats to its integrity.

Anti-Bacterial Agents↗
PubMed1978

Cyclic AMP and cyclic GMP in germinating conidia of Neurospora crassa.

A new method for obtaining synchronous germination allowed accurate time-course studies of endogenous levels of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-phosphate in germinating conidia of Neurospora crassa. The levels of both cyclic nucleotides remained constant throughout germination, showing that they neither signal nor respond to any of the biochemical changes which are known to occur in the germination process. Conidal germination was approximately normal in three cr-1 mutants of Neurospora which have been shown to be deficient in adenylate cyclase activity. Cyclic AMP levels in the mycelia of cr-1 mutants were low, but surprisingly, the levels in conidia were normal.

Adenylyl Cyclases↗
PubMed1977

Cyclic AMP and the plasma membrane potential in Neurospora crassa.

Diverse treatments, which have been shown by Slayman, C. L. (1977) in Water Relations in Membrane Transport in Plants and Animals (Jungreis, A., Hodges, T. K., Kleinzeller, A., and Schultz, S. G., eds) pp. 69-86, Academic Press, New York, to depolarize the plasma membrane of Neurospora, increase levels of adenosine 3':5'-monophosphate (cyclic AMP) in the organism. The treatments include those producing large transport fluxes of metabolizable or nonmetabolizable compounds, rapid temperature drops, and addition of agents which uncouple oxidative phosphorylation. Severe mechanical stress, which may also act to depolarize the plasma membrane, leads to increases in cyclic AMP. The maximal depolarization appears to precede the maximal cyclic AMP levels. It is proposed that the membrane depolarization produces the increased cyclic AMP levels by stimulating the plasma membrane-bound adenylate cyclase and that cyclic AMP may be important to the maintenance of membrane integrity.

Arginine↗