Cyclic nucleotide phosphodiesterase activities in Neurospora crassa.

Cyclic nucleotide phosphodiesterase activities in soluble Neurospora crassa mycelial extracts were resolved into two peaks, phosphodiesterase I and II, by chromatography on DEAE-cellulose columns. Phosphodiesterase I hydrolysed cyclic AMP and cyclic GMP equally well. Phosphodiesterase II was active on cyclic GMP but scarcely active on cyclic AMP. Phosphodiesterase I was resolved by gel filtration and sucrose-density-gradient centrifugation into three peaks having molecular weights of about 57 000, 125 000 and 225 000. This suggests that this enzyme activity has at least three aggregation forms, tentatively defined as monomeric, dimeric and tetrameric. Similarly, phosphodiesterase II was resolved into two forms, having molecular weights of about 170 000 and 320 000. Evidence on the interconversion between phosphodiesterase I forms was obtained.     

Neurospora crassa glutamine synthetase. Purification by affinity chromatography and characterization of subunit structure.

Neurospora crassa glutamine synthetase was purified to homogeneity by a procedure based on affinity chromatography. The enzyme is adsorbed to a matrix of anthranilic acid bound to Sepharose and eluted with AMP. Different experimental approaches indicate that the enzyme has an octameric structure formed by subunits of identical molecular weight.     

Cyclic 3',5'-AMP phosphodiesterase of Neurospora crassa

Cyclic 3′,5′-AMP (cAMP) phosphodiesterase activity can be demonstrated in extracts of $\text{Neurospora}\text{crassa}$. The activity is particulate, has a pH optimum of 7.4, and consists of two forms that have different cAMP binding constants. Methylxanthines, inorganic phosphate, and EDTA are inhibitors of the diesterase as are ATP, ADP, and 8-bromo-cAMP. The enzymatic activity is stimulated by histamine and imidazole. These properties suggest that the $\text{Neurospora}$ enzyme is more closely related to the mammalian than to bacterial cAMP phosphodiesterases.     

Purification and characterization of flavokinase from Neurospora crassa

The ATP-dependent phosphorylation of riboflavin to FMN by flavokinase is the key step in flavin biosynthesis. Flavokinase has been purified from a fungal source for the first time. The enzyme purified from a cell wall lacking mutant of Neurospora crassa, slime, is a monomer of M(r) 35.5 kDa with maximal activity at alkaline pH and high temperature (55 degrees C). The K(m) for both substrates is the lowest reported for flavokinase from any source so far (120 nM for riboflavin and 210 nM for MgATP2-). The enzyme exhibits preference for Mg2+ over Zn2+ as the essential activator and is also significantly activated by several cations. Activation by orthophosphate may be physiologically relevant for the intracellular regulation of flavokinase.     

Purification and characterization of arginase from Neurospora crassa

We have purified an enzymatically active form of arginase from a wild-type strain of Neurospora crassa to homogeneity. The enzyme has a subunit molecular weight of 38,300 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein migrated as a hexamer during gel-filtration chromatography with an apparent molecular weight of 266,000. The enzyme exhibited hyperbolic kinetics at pH 9.5 with an apparent Km for arginine of 131 mM. Antiserum was prepared against the purified enzyme and used to demonstrate the existence of three cross-reactive proteins in crude extracts of wild-type N. crassa. One of these proteins corresponded to the purified protein, whereas the other two were of molecular weights 41,700 and 26,800, respectively. Using the same antiserum, we found that rat liver, but not rat kidney, contains immunoreactive material. We also detected two proteins in extracts of Saccharomyces cerevisiae that were weakly cross-reactive with the antiserum. These data provide evidence for the existence of multiple forms of arginase in fungi as well as in mammals.     

Cyclic nucleotide phosphodiesterase activity in barley seeds

Barley seeds (Hordeum vulgare L. cv. Himalaya) contain an enzymatic activity which catalyzes the hydrolysis of adenosine cyclic 3′: 5′-monophosphate and adenosine cyclic 2′: 3′-monophosphate. A large portion of the enzymatic activity is present in the dry seed, existing in both soluble and particulate form. Secretion of the soluble phosphodiesterase from embryoless seeds is enhanced by gibberellic acid and inhibited by abscisic acid, dinitrophenol, and cycloheximide. Attempts to isolate or detect a phosphodiesterase which specifically hydrolyzes adenosine cyclic 3′: 5′-monophosphate were unsuccessful. Inhibition experiments indicate that probably one enzyme is involved in the hydrolysis of both of these substrates.     

Purification and characterization of epsilon-N-trimethyllysine L-amino oxidase from Neurospora crassa.

epsilon-N-Trimethyllysine L-amino oxidase from Neurospora crassa has been purified to electrophoretic homogeneity. A 1500-fold purification was obtained by centrifugation and successive column chromatography on ion-exchange and gel filtration supports. The enzyme has an estimated molecular weight of 160 000. It transforms epsilon-N-trimethyllysine into alpha-keto, epsilon-N-trimethylhexanoic acid by oxidative deamination. Kinetic studies of this new enzyme are reported and its probable physiological role is discussed.     

Purification and characterization of glutamate decarboxylase from Neurospora crassa conidia.

L-Glutamate decarboxylase, an enzyme under the control of the asexual developmental cycle of Neurospora crassa, was purified to homogeneity from conidia. The purification procedure included ammonium sulfate fractionation and DEAE-Sephadex and cellulose phosphate column chromatography. The final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gels with a molecular weight of 33,200 +/- 200. A single band coincident with enzyme activity was found on native 7.5% polyacrylamide gels. The molecular weight of glutamate decarboxylase was 30,500 as determined by gel permeation column chromatography at pH 6.0. The enzyme had an acidic pH optimum and showed hyperbolic kinetics at pH 5.5 with a Km for glutamic acid of 2.2 mM and a Km for pyridoxal-5'-phosphate of 0.04 microM.     

Ornithine decarboxylase from Neurospora crassa. Purification, characterization, and regulation by inactivation

Ornithine decarboxylase, a highly regulated enzyme of the polyamine pathway, was purified 670-fold from mycelia of Neurospora crassa that were highly augmented for enzyme activity. The enzyme is significantly different from those reported from three other lower eucaryotic organisms: Saccharomyces cerevisiae, Physarum polycephalum, and Tetrahymena pyriformis. Instead, the enzyme closely resembles the enzymes from mammals. The Mr = 110,000 enzyme is a dimer of 53,000 Da subunits, with a specific activity of 2,610 mumol per h per mg of protein. Antisera were raised to the purified enzyme and were rendered highly specific by cross-absorption with extracts of a mutant strain lacking ornithine decarboxylase protein. With the antisera, we show that the inactivation of the enzyme in response to polyamines is proportional to the loss of ornithine decarboxylase protein over almost 2 orders of magnitude. This is similar to the inactivation process in certain mammalian tissues, and different from the process in S. cerevisiae and P. polycephalum, in which enzyme modification, without proportional loss of antigen, accompanies enzyme inactivation. The N. crassa enzyme is therefore suitable as a microbial model for studies of the molecular regulation of the mammalian enzyme.     

Human tumor necrosis factor-alpha receptor. Purification by immunoaffinity chromatography and initial characterization

The receptor for human tumor necrosis factor-alpha (TNF-alpha) was isolated from a subclone of the human histiocytic lymphoma cell line U937. These cells exhibit a single class of high affinity receptors (Kd = 0.51 +/- 0.25 nM) with an average density of 55,000 +/- 5,000 binding sites/cell. After solubilization with detergent, the receptor retained its ability to bind free TNF-alpha but failed to bind to TNF-alpha immobilized on various solid supports. For receptor purification, 125I-TNF-alpha was covalently attached to the receptor on intact cells by the bifunctional cross-linking reagents ethylene glycolbis(succinimidylsuccinate) or 3,3-dithiobis(sulfosuccinimidylpropionate). The cells were then solubilized with the nonionic detergent Triton X-100, and the supernatants, clarified by centrifugation, were passed over an IgG-Sepharose column prepared from TNF-alpha antiserum. The receptor-rich fraction from the antibody column was further purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These two steps together provided approximately 165,000-fold purification of the TNF-alpha receptor. The TNF-alpha receptor-ligand complex obtained by this method had a subunit molecular weight of 100,000 +/- 5,000 when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis but on gel filtration the complex migrated with an apparent molecular weight of 480,000 +/- 32,000. However, the receptor showed a molecular weight of 65,000 +/- 32,000 when gel filtration was performed in the absence of ligand. Additional characteristics of the receptor are discussed.     

Characterization of Neurospora crassa cyclic AMP phosphodiesterase activated by calmodulin.

Activation of cyclic AMP phosphodiesterase I by brain or Neurospora calmodulin was studied. The stimulation required micromolar concentrations of Ca2+, and it was observed at cyclic AMP concentrations between 0.1 and 500 microM. Activation was blocked by EDTA and some neuroleptic drugs such as chlorpromazine and fluphenazine. These drugs inhibit the elongation of N. crassa wild-type aerial hyphae. These results reinforce the evidence towards the recognition of Ca2+-calmodulin as one of the systems controlling cyclic nucleotide concentrations in Neurospora.     

Purification and characterization of recombinant FAD synthetase from Neurospora crassa

FAD Synthetase (FADS) [EC 2.7.7.2], the second enzyme in flavin cofactor biosynthetic pathway converts FMN to FAD, plays an important role in many redox reactions. Neurospora crassa FADS (NcFADS) was cloned and overexpressed in E. coli cells. Recombinant NcFADS was purified in high yields of ～8 mg per liter of bacterial culture using a single step glutathione sepharose affinity chromatography. SDS-PAGE and MALDI-MS revealed that NcFADS has a molecular mass of ～31 kDa. Enzyme kinetic analysis monitored by reverse phase HPLC demonstrate a specific activity and k_cat of 1356 nmol/min/mg and 0.69sec^?1 respectively. Steady state kinetic analysis of NcFADS exhibited a K_m of NcFADS for FMN is 2.7 μM and for MgATP^?2 is 88.7 μM. Isothermal titration calorimetry experiments showed that the recombinant protein binds to the substrates with apparent K_d of 20.8 μM for FMN and 16.6 μM for MgATP^?2. Biophysical characterization using intrinsic fluorescence suggests that the enzyme is in folded conformation. Far-UV CD data suggest that the backbone of the enzyme is predominantly in a helical conformation. Differential scanning calorimetry data shows that the T_m is 53 °C ± 1. This is the first report on cloning, purification and characterization of FADS from N. crassa. The specific activity of NcFADS is the highest than any of the reported FADS from any other source. The results obtained in this study is expected to pave way for intensive research aimed to understand the molecular basis for the extraordinarily high turnover rate of NcFADS.     

Cyclic nucleotide phosphodiesterase activity in human peripheral blood lymphocytes and monocytes.

cAMP and cGMP phosphodiesterase (PDE) activity was assayed in human peripheral blood lymphocytes purified by isopycnic centrifugation as well as in lymphocyte preparations further purified to remove contaminating platelets and monocytes. The 16,000 X G supernatant from sonicates of each of these cell preparations contained two hydrolytic activities for cAMP with apparent Km of 1.1 to 2.5 microM and 33 to 66 microM, and a single hydrolytic activity for cGMP with an apparent Km of 6 to 25 microM. When lymphocytes were disrupted by Dounce homogenization, there was only a single, low Km cAMP PDE activity in the homogenate; however, the 16,000 X G supernatant demonstrated 2 Km similar to that seen in sonicated lymphocytes. Treatment of the Dounce preparations with 0.5% Triton X-100 or 1.0% NP-40 converted these preparations to activities similar to those seen in sonicated preparations. cGMP hydrolytic activity was low or absent in the Dounce preparations and was not altered by centrifugation; however, it was markedly enhanced by detergent extraction. These data indicate that human peripheral blood lymphocytes and monocytes have PDE activities similar to those seen in other tissues.     

Purification of nucleotide-requiring enzymes by immunoaffinity chromatography.

Monospecific (affinity-purified) anti-(yeast glucose-6-phosphate dehydrogenase) IgG inhibits three different NADPH-requiring enzymes, chicken liver dihydrofolate reductase, pigeon liver fatty acid synthetase and chicken liver malic enzyme. The inhibition of all three enzymes was approx. 50% in a 2h incubation with 100 micrograms of IgG. Similarly, with several different NADH-requiring enzymes, an immunocrossreactivity was observed. Monospecific anti-(rabbit muscle glyceraldehyde-3-phosphate dehydrogenase) IgG inhibited yeast alcohol dehydrogenase and pig heart malate dehydrogenase by 39% and 55% respectively. The cross-reactivity observed was tested by affinity chromatography. Immunoaffinity columns made with each monospecific IgG were able to bind each of the enzymes it immunotitrated. Enzymes were eluted with a nondenaturing solvent with little loss of activity. The immunoaffinity column with monospecific anti-(glucose-6-phosphate dehydrogenase) IgG as the bound ligand was also used to purify partially (over 150-fold) both isocitrate dehydrogenase and dihydrofolate reductase from crude rat liver homogenate.     

Purification of vacuoles from Neurospora crassa.

The Neurospora crassa vacuole, defined by its content of basic amino acids, polyphosphate, protease, phosphatases, and alpha-mannosidase, was purified to near homogeneity. The procedure depends upon homogenization of snail gut enzyme-digested cells in a buffer osmotically stabilized with 1 M sorbitol, differential centrifugation of the extract, and sucrose density gradient centrifugation of the organellar pellet. Isopycnic centrifugation of vacuoles in 2.25 M sorbitol-Metrizamide density gradients yielded a peak (density, 1.31 g/cm3) of vacuolar markers coincident with 32P-phospholipids, trichloroacetate-insoluble 14C, and trichloroacetate-soluble 14C. A trail of macromolecular markers in the lighter portions of the gradient reflected, at least in part, heterogeneity of the vacuoles. Almost no contamination by mitochondria or glyoxysomes was detected. Vacuoles were very heterogeneous in size as estimated by velocity sedimentation, but most were larger than mitochondria. Variations of the osmotic strength of the medium were found to alter the equilibrium density of vacuole preparations from 1.06 g/cm3 to over 1.3 g/cm3. This explains the great variation in density reported previously for the "vacuole," the "vesicle," and the "protease particle" of N. crassa, all of which appear to be the same entity.     

Cyclic nucleotide phosphodiesterase activity in rat adrenal gland zones

The distribution of cyclic 3′, 5′ -nucleotide phosphodiesterase activity in the rat adrenal gland has been studied. Phosphodiesterase activity was 10-fold higher in the zona glomerulosa than in the zona fasciculata-reticularis. Kinetic studies carried out at low substrate concentrations suggest the possible presence of multiple forms of phosphodiesterase activity in both zones of the adrenal; however, these forms appear to have similar apparent Km's for cAMP. Thus, the well known differences in the steroidogenic response of the two zones to ACTH stimulation may be partially explained by large differences in total activities of the various forms of phosphodiesterase.     

Cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Centrifugal fractionation showed that 70% of the cyclic nucleotide phosphodiesterase activity of Phaseolus vulgaris seedlings is recovered in the 1     

Purification and chemical characterization of the rodlet layer of Neurospora crassa conidia.

The rodlet layer of Neurospora crassa macroconidia has been purified and chemically characterized. Sheets of rodlets were released from the conidial surface by vigorously shaking conidia in water. Conidia were removed by filtration and low-speed centrifugation, and the rodlets were recovered from the supernatant by high-speed centrifugation. The rodlet pellet comprised 1.9% of the initial dry weight. Chemical analysis was hampered by the insolubility of the rodlets. They were not solubilized by heating in various protein-denaturing buffers and were only partially dissolved by heating in 1 M NaOH at 100 degrees C for 5 min. Nevertheless, they were found to be largely composed of protein (91%, based on total nitrogen). The major amino acids in acid hydrolysates were aspartic acid, glycine, serine, alanine, half-cystine, and valine. Glucosamine was not detected in acid hydrolysates. The sulfur content was 2.5%, and this could be accounted for in half-cystine and methionine. Carbohydrate comprised just over 2%. The phosphorus content was 0.21%, of which less than one-third was accounted for in phospholipid. The total fatty acid content was 1.0%, most of which could be accounted for by the fatty acids of the phospholipids.     

Cyclic nucleotide phosphodiesterase in vertebrates

The cellular concentration of cyclic nucleotides is largely dependent upon the activity of the enzymatic system responsible for their degradation: cyclic nucleotide phosphodiesterase. This enzymatic system thus plays a crucial role in the regulation of the multiple functions which are modulated by cyclic nucleotides in the organism. Many methodological problems, as well as the complexity of the phosphodiesterase system have long maintained a confusion in this field. Recent progresses (purification to homogeneity of some enzymatic forms, discovery of regulatory mechanisms, particularly) have brought a considerable evolution in the knowledge of the system. It is now well established that cyclic nucleotide phosphodiesterase exists under several isoenzymatic forms, the properties and distribution of which largely differ from a tissue to another. Some of these forms are relatively well characterized, while the representativity of others is still discussed. The significance of this multiplicity of isoenzymes, and their interrelationships are presently under study. A very interesting aspect in the study of this enzymatic system is that it is submitted to several physiological regulatory processes. Recent studies on this point suggest that phosphodiesterase might play a major role in the response of the organism to several hormones. These fundamental studies of phosphodiesterase system find a most interesting application in the pharmacological field. Indeed, numerous synthetic compounds which inhibit the enzyme present a strong pharmacological interest.