Specific inhibition by ATP and other properties of an endonuclease of Neurospora crassa

1968 ◽  
Vol 46 (10) ◽  
pp. 1285-1291 ◽  
Author(s):  
E. Z. Rabin ◽  
M. Mustard ◽  
M. J. Fraser
Keyword(s):  
Neurospora Crassa ◽  
Nuclease Activity ◽  
Enzyme Concentration ◽  
Specific Inhibition ◽  
Endonuclease Activity ◽  
Dna And Rna ◽  
Period 2 ◽  
Lag Period ◽  
Ageing Period ◽  
Soluble Material

An endonuclease specific for single-stranded DNA and RNA was purified from Neurospora crassa conidia by the method of Linn and Lehman (J. Biol. Chem. 240, 1287 (1965)). The activity of the enzyme was measured by following the rate of release of acid-soluble material at 37 °C from either denatured (single-stranded) or native DNA. After an initial lag period, the length of which was inversely proportional to enzyme concentration, the release of acid-soluble material occurred at a rate which was directly proportional to enzyme concentration. Freshly purified enzyme catalyzed the release of acid-soluble material from denatured DNA 50–70 times faster than from native DNA. This ratio of activities increased to approximately 200 following storage at 0–4 °C. Throughout this "ageing" period 2 × 10−4 M ATP inhibited the activity of the enzyme toward both denatured and native DNA by 50%. The following nucleoside phosphates, at a concentration of 4 × 10−4 M, had no effect on the activity of the endonuclease toward denatured DNA: ADP, AMP, ITP, XTP, CTP, dTTP, and UTP. GTP at 4 × 10−4 M inhibited this activity by 15%. Both ATP and dATP at 1 × 10−4 M inhibited endonuclease activity against denatured DNA by about 25%. The inhibition by ATP was noncompetitive over the range of concentration 0.11–0.75 mg DNA per milliliter. Specific inhibition of nuclease activity by ATP has not been previously reported.Several other properties of the endonuclease were examined. There is evidence that endonuclease action was inhibited by accumulation of the products of digestion. The enzyme lost activity toward denatured DNA in a medium containing 0.3 M NaCl but activity was partially restored in the presence of 4 × 10−3 M mercaptoethanol. The endonuclease did not digest DNA–RNA hybrid to any appreciable extent.

scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

Effect of Cytokinins on the Expansion and Metabolism of Excised Cucumber Cotyledons

1980 ◽  
Vol 7 (3) ◽  
pp. 227
Author(s):  
C Tsui ◽  
Tao Guo-qing ◽  
Chen Hui-ying ◽  
Son Yan-ru ◽  
Lian Han-ping ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Soluble Sugars ◽  
Dry Weight ◽  
Acid Synthesis ◽  
Cell Number ◽  
Cucumis Sativus L ◽  
Biochemical Processes ◽  
Dna And Rna ◽  
Lag Period ◽  
Hydrolysis Of

Expansion of excised cucumber (Cucumis sativus L.) cotyledons was stimulated by treatment with cytokinin, and commenced after a lag period of about 4 h. Expansion induced by benzyladenine (BA) was due mainly to increase of fresh weight, but cell number increased slightly. Hydrolysis of protein and lipid was stimulated by BA, and soluble sugars increased simultaneously. However, there was no significant change in the dry weight of cotyledons during the period of expansion. It is assumed that the transformation of lipid to sugar in the cotyledon is stimulated by BA. The respiration of cotyledons was evidently stimulated by BA and was entirely inhibited by respiratory inhibitors, e.g. NaN,, malonate and dinitrophenol. Inhibitors of protein and nucleic acid synthesis, such as chloramphenicol and actinomycin D, inhibited only the BA-induced expansion. They had no effect on the expansion of controls. These results suggest that different biochemical processes are involved in the expansion of cotyledons induced by BA and in controls. The former is related not only to respiration but also to the synthesis of protein and nucleic acid. BA increased DNA and RNA content per cotyledon. The increase of total RNA is due mainly to the increase of 25 S and 18 S rRNA.

Regulators of Cell Division in Plant Tissues. XXII* Physiological Aspects of Cytokinin-induced Radish Cotyledon Growth

1975 ◽  
Vol 2 (2) ◽  
pp. 129
Author(s):  
M.E Gordon ◽  
D.S Letham
Keyword(s):  
Translational Control ◽  
Total Protein ◽  
Specific Activity ◽  
Red Light ◽  
Growth Control ◽  
Growth Stimulation ◽  
Protein Levels ◽  
Dna And Rna ◽  
Dna And Rna Synthesis ◽  
Lag Period

The cytokinin 6-benzylaminopurine (BAP) markedly stimulated the lateral expansion of excised immature radish cotyledons after a lag period of about 10 h. This growth occurred principally by cell enlargement, especially in the light which enhanced the response. However, a marked response 'to cytokinin occurred in the complete absence of red light during germination, cotyledon excision and incubation. Contact with BAP for 5 h significantly stimulated growth, but a maximum response required more than 24 h of contact; potassium chloride also promoted cotyledon expansion and acted synergistically with cytokinin. The response to cytokinin did not appear to be mediated by ethylene, gibberellins, polyamines or cyclic nucleotides. Growth induction did not alter the respiration rate and appeared to be inde- pendent of chloroplast function. Inhibitors of DNA and RNA synthesis and of protein synthesis on cytoplasmic ribosomes almost completely abolished BAP-induced growth, control growth being less markedly affected. There were, however, no significant BAP-induced increases in total DNA or RNA levels or specific activity before the initiation of growth stimulation. Similarly, BAP had no effect on any individual RNA species until after the lag period, when there was a small enhancement of uridine incorporation into RNA species with similar electrophoretic mobility to rRNA. Although total protein levels were not affected by BAP, the cytokinin enhanced amino acid incorporation into protein within the lag period, an effect which persisted when transcription was strongly inhibited by actinomycin D. Phosphorylation of total protein was stimulated by BAP only well after the onset of cytokinin-induced growth. Protein methylation, however, was stimulated by BAP during the lag period, and the effect was at least as early as the BAP-enhanced incorporation of methionine into protein. The possible role of translational control in the mechanism of cytokinin action is discussed.

Use of Platelet Aggregometer to monitor the chymosin-initiated coagulation of casein micelles

1986 ◽  
Vol 53 (3) ◽  
pp. 359-370 ◽  
Author(s):  
Neal A. Bringe ◽  
John E. Kinsella
Keyword(s):  
Light Transmission ◽  
Average Rate ◽  
Enzyme Concentration ◽  
Casein Micelle ◽  
Clotting Time ◽  
Casein Micelles ◽  
Catalysed Reaction ◽  
High Enzyme ◽  
Lag Period ◽  
Limiting Value

SUMMARYThe chymosin-initiated coagulation of casein micelles was followed by monitoring light transmission using a Platelet Aggregometer. The release of macropeptide by chymosin was monitored using fluorescamine. The lag period in the clotting reaction was proportional to clotting time and the reciprocal of enzyme concentration. The average rate of coagulation, which was approximately equal to the reciprocal of clotting time (Tc), increased in proportion to enzyme concentration at low enzyme concentrations and reached a limiting value at high enzyme concentrations. The percentage hydrolysis at the Tc was 47 ± 5% in the presence of 20 mM-CaCl2 and it was calculated that a 5-fold decrease in the speed of the enzyme-catalysed reaction would decrease this value at the Tc to 43 ± 5%. The possible uses and limitations of the Platelet Aggregometer for determining the influence of the chemical environment on the velocity of the chymosin-catalysed reaction and para-casein micelle aggregatability are discussed.

Two Forms of a Mitochondrial Endonuclease in Neurospora crassa

1975 ◽  
Vol 53 (7) ◽  
pp. 823-825 ◽  
Author(s):  
Charles E. Martin ◽  
Robert P. Wagner
Keyword(s):  
Molecular Weight ◽  
Neurospora Crassa ◽  
Mitochondrial Membrane ◽  
Nuclease Activity ◽  
Inner Mitochondrial Membrane ◽  
Detergent Treatment ◽  
Approximate Molecular Weight ◽  
Membrane Bound

Mitochondrial nuclease activity in Neurospora crassa occurs in membrane-bound and soluble forms in approximately equal proportions. These activities apparently are due to the same enzyme, which has an approximate molecular weight of 120 000. A portion of the insoluble enzyme appears to be associated with the inner mitochondrial membrane and is resistant to solubilization by detergent treatment as well as by physical disruption methods.

scholarly journals The hydroxylation of tyrosine by an enzyme from third-instar larvae of the blowfly Calliphora erythrocephala.

1975 ◽  
Vol 147 (3) ◽  
pp. 565-573 ◽  
Author(s):  
R N Pau ◽  
C Kelly
Keyword(s):  
Phenol Oxidase ◽  
Enzyme Concentration ◽  
Polyacrylamide Gels ◽  
Dihydroxybenzoic Acid ◽  
Calliphora Erythrocephala ◽  
Glycine Buffer ◽  
Hydrogen Donors ◽  
Lag Period ◽  
Tyrosine Concentration ◽  
Third Instar Larvae

1. Two pro-(phenol oxidase) were distinguished when the blood of late-third-instar larvae of Calliphora erythrocephala was electrophoresed in polyacrylamide gels with Tris-glycine buffer, pH 8.3. One pro-(phenol oxidase), after activation by an enzyme readily catalyses the oxidation of both L-tyrosine and L-3,4-dihydroxyphenylalanine (L-dopa). The second enzyme catalyses the oxidation of L-dopa but not of L-tyrosoine. 2. One of the pro-(phenol oxidases) was purified over 2000-fold from homogenates of whole larvae. This enzyme, after activation, catalyses the oxidation of both dopa and tyrosine. On electrophoresis in polyacrylamide gels with Tris-glycine buffer, pH 8.3, it has the same mobility as the enzyme in the blood which catalyses the oxidation of both tyrosine and dopa. 3. The pro-(phenol oxidase)-activating enzyme was purified over 100-fold from homogenates of whole larvae. 4. The oxidation of L-tyrosine, in the presence of the activated purified phenol oxidase, reached a steady maximum rate after a lag period that was directly related to tyrosine concentration and inversely related to enzyme concentration. 5. The effect of the addition of electron donors on the lag period was studied. Dopa, dopamine (3,4-dihydroxyphenethylamine) and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine are the most effective hydrogen donors. 3,4-Dihydroxybenzoic acid, the oxidation of which was not catalysed by the activated pro-(phenol oxidase), did not affect the lag period.

scholarly journals Nonsense mediated decay and a novel protein Period-2 regulate casein kinase I in an opposing manner to control circadian period in Neurospora crassa

2020 ◽  
Author(s):  
Christina M. Kelliher ◽  
Randy Lambreghts ◽  
Qijun Xiang ◽  
Christopher L. Baker ◽  
Jennifer J. Loros ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Rna Binding ◽  
Casein Kinase ◽  
Circadian Period ◽  
Casein Kinase I ◽  
Nonsense Mediated Decay ◽  
Protein Levels ◽  
Period 2 ◽  
White Collar Complex ◽  
Novel Protein

AbstractCircadian clocks in fungi and animals are driven by a functionally conserved transcription-translation feedback loop. In Neurospora crassa, negative feedback is executed by a complex of Frequency (FRQ), FRQ-interacting RNA helicase (FRH), and Casein Kinase I (CKI), which inhibits the activity of the clock’s positive arm, the White Collar Complex (WCC). Here, we show that the period-2 gene, whose mutation is characterized by recessive inheritance of a long 26-hour period phenotype, encodes an RNA-binding protein that stabilizes the ck-1a transcript, resulting in CKI protein levels sufficient for normal rhythmicity. Moreover, by examining the molecular basis for the short circadian period of period-6 mutants, we uncovered a strong influence of the Nonsense Mediated Decay pathway on CKI levels. The finding that circadian period defects in two classically-derived Neurospora clock mutants each arise from disruption of ck-1a regulation is consistent with circadian period being exquisitely sensitive to levels of casein kinase I.

Neurospora endo-exonuclease in heat-shocked mycelia: evidence for a novel heat shock induced function

1989 ◽  
Vol 67 (9) ◽  
pp. 642-652 ◽  
Author(s):  
Dindial Ramotar ◽  
Murray J. Fraser
Keyword(s):  
Dna Repair ◽  
Heat Shock ◽  
Neurospora Crassa ◽  
Recovery Period ◽  
Nuclease Activity ◽  
Active Enzyme ◽  
Inhibitor Complex ◽  
High Level ◽  
Recombinational Dna Repair

Shifting rapidly growing mycelial cultures of Neurospora crassa from 30 to 45 °C for 2 h resulted in 75% of the active endo-exonuclease, an enzyme implicated previously in recombinational DNA repair, being released from nuclei and vacuoles into the cytosol, where it was inactivated by a heat shock induced inhibitor. Cycloheximide, at a level which inhibited [35S]methionine incorporation into the protein of unshocked cells by 95%, did not block the release of endo-exonuclease from the organelles during heat shock, but rather allowed a 6- to 15-fold accumulation of the nuclease activity in the cytosol. The heat shock also resulted in depletion of about 65% of nuclear, and over 35% of cytosolic, trypsin-activable endo-exonuclease (comprised of both endo-exonuclease–inhibitor complex and endo-exonuclease precursor?). Heat shock also caused the release of about 40% of the total trypsin-activable endo-exonuclease plus active enzyme from mitochondria. During a subsequent 1-h recovery at 30 °C, trypsin-activable endo-exonuclease, but not active enzyme, was restored in full to the nuclei. Some restoration of vacuolar endo-exonuclease was also observed. In the cytosol, neither the level of active enzyme nor the level of trypsin-activable endo-exonuclease was appreciably restored during this recovery period. The accumulation of the new inhibitor in the cytosol of heat-shocked mycelia was estimated to be 20-fold. Although cycloheximide prevented this accumulation, it is not clear whether the inhibitor itself is synthesized in response to heat shock or whether it is generated from a preexisting form by some other heat shock induced function. The high level of inhibitor that accumulated during heat shock persisted throughout the 1-h recovery period. The properties of this inducible inhibitor showed both similarities to and differences from a highly specific constitutive inhibitor of endo-exonuclease purified from unshocked mycelia.Key words: Neurospora crassa, endo-exonuclease, heat shock, inducible, inhibitor.

Specific inhibitors of Neurospora endo-exonuclease

1989 ◽  
Vol 67 (9) ◽  
pp. 632-641 ◽  
Author(s):  
Zafer Hatahet ◽  
Murray J. Fraser
Keyword(s):  
Neurospora Crassa ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Exonuclease Activity ◽  
Endonuclease Activity ◽  
Average Molecular Mass ◽  
Strand Breaks ◽  
Heat Stable ◽  
First Order Kinetics ◽  
Pbr322 Dna

A heat-stable, trypsin-sensitive cytosolic inhibitor of Neurospora crassa endo-exonuclease has been purified 300-fold in 90% yield from crude extracts of mycelia. On electrophoresis in sodium dodecyl sulfate – polyacrylamide gels, it showed a relatively broad band corresponding to polypeptides with an average molecular mass of about 24 kDa. The protein inhibited the single strand specific endonuclease activity of endo-exonuclease noncompetitively, but the inhibition was not complete (maxima of 70–95%) in the presence of excess inhibitor. The inhibition of the double strand exonuclease activity was competitive and complete when inhibitor was present in excess. In addition, the inhibitor completely blocked the formation of site-specific double strand breaks and nicking by endo-exonuclease in linearized pBR322 DNA. The ribonuclease activity of endo-exonuclease was also inhibited by this protein. The inhibition of Neurospora endo-exonuclease was very specific. Although inhibition was seen also for an immunochemically related nuclease derived from mycelia of Aspergillus nidulans, no inhibition was observed for two other nucleases of Neurospora, nor for a wide variety of commercially available nucleases. A bound form of inhibitor was also partially purified from the cytosol of Neurospora in association with inactive, but trypsin-activable endo-exonuclease. Activity gel analysis showed that this was an endo-exonuclease–inhibitor complex. On heating at 80 °C, inhibitor was released from the complex with the same first order kinetics as inactivation of the trypsin-activable endo-exonuclease and in approximately equivalent amounts. The bound inhibitor was also 24 kDa in size, but completely inhibited both the endo- and exonuclease activities of endo-exonuclease. It was found unexpectedly that heat-inactivated endo-exonuclease stimulated the exonuclease activity of the active enzyme without affecting the endonuclease activity. The stimulated exonuclease activity was also completely blocked by inhibitor.Key words: Neurospora crassa endo-exonuclease, inhibitor, constitutive, cytosolic.

The action of calf rennet and other proteolytic enzymes on κ-casein

1969 ◽  
Vol 36 (1) ◽  
pp. 11-20 ◽  
Author(s):  
R. C. Lawrence ◽  
L. K. Creamer
Keyword(s):  
Protein Interactions ◽  
Proteolytic Enzymes ◽  
Enzyme Concentration ◽  
Ion Concentration ◽  
Protein Protein Interactions ◽  
Low Concentrations ◽  
Lag Period ◽  
High Concentrations ◽  
The Stability ◽  
Calcium Ion Concentration

SummaryThe hydrolysis of κ-casein by a number of rennets and other proteolytic enzymes has been followed by measuring the increase in opacity due to the formation of insoluble aggregates of para-κ-caseins. The stability of these precipitates varied markedly, some being solubilized rapidly by the further action of the enzyme. The turbidity obtained with certain enzymes was dependent upon the calcium ion concentration, indicating that the para-κ-caseins produced were not identical for all enzymes.For high concentrations of calf rennet, the rate of aggregation was linear with respect to time. With low concentrations of enzyme, increase in turbidity was preceded by a lag period which was lengthened by decreasing the enzyme concentration or increasing the κ-casein concentration. This increase in lag is favoured by a high κ-casein/para-κ-casein ratio, suggesting that the aggregation of newly formed para-κ-casein is prevented by the unchanged κ-casein. In addition, small amounts of αs1- or β-caseins present in the κ-casein also markedly affected the aggregation of para-κ-casein, indicating that all 3 major casein components can inhibit the aggregation of para-κ-casein in the absence of calcium ions. In the light of these observations the possible role of protein-protein interactions in casein coagulation by calf rennet is discussed.

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