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Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa.
Biochim Biophys Acta. 1978 Apr 12; 523(2):297-313.BB

Abstract

Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD.

Authors

No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

26408

Citation

Pan, S S., and A Nason. "Purification and Characterization of Homogeneous Assimilatory Reduced Nicotinamide Adenine Dinucleotide Phosphate-nitrate Reductase From Neurospora Crassa." Biochimica Et Biophysica Acta, vol. 523, no. 2, 1978, pp. 297-313.
Pan SS, Nason A. Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa. Biochim Biophys Acta. 1978;523(2):297-313.
Pan, S. S., & Nason, A. (1978). Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa. Biochimica Et Biophysica Acta, 523(2), 297-313.
Pan SS, Nason A. Purification and Characterization of Homogeneous Assimilatory Reduced Nicotinamide Adenine Dinucleotide Phosphate-nitrate Reductase From Neurospora Crassa. Biochim Biophys Acta. 1978 Apr 12;523(2):297-313. PubMed PMID: 26408.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa. AU - Pan,S S, AU - Nason,A, PY - 1978/4/12/pubmed PY - 1978/4/12/medline PY - 1978/4/12/entrez SP - 297 EP - 313 JF - Biochimica et biophysica acta JO - Biochim Biophys Acta VL - 523 IS - 2 N2 - Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD. SN - 0006-3002 UR - https://www.unboundmedicine.com/medline/citation/26408/Purification_and_characterization_of_homogeneous_assimilatory_reduced_nicotinamide_adenine_dinucleotide_phosphate_nitrate_reductase_from_Neurospora_crassa_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0005-2744(78)90033-5 DB - PRIME DP - Unbound Medicine ER -