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Isolation and characterization of pigeon breast muscle cytosolic 5´-nucleotidase-I (cN-I)

100%

Tkacz-Stachowska K., Lechward K., Skladanowski A. C.

2005

vol. 52

issue 4

789-796

5´-Nucleotidase specific towards dCMP and AMP was isolated from avian breast muscle and characterized. It was found to be similar to a type-I form (cN-I) identified earlier as the AMP-selective 5´-nucleotidase responsible for adenosine formation during ATP breakdown in transfected COS-7 cells. Expression pattern of the cN-I gene in pigeon tissues indicated breast muscle as a rich source of the transcript. We purified the enzyme from this source using two-step chromatography and obtained an active homogenous preparation, free of ecto-5´-nucleotidase activity. The tissue content of the activity was calculated at 0.09 U/g wet weight. The specific activity of the enzyme preparation was 4.33 U/mg protein and it preferred dCMP and AMP to dAMP and IMP as a substrate. Its kinetic properties were very similar to those of the enzyme purified earlier from heart tissue. It was strongly activated by ADP. Inhibition by inorganic phosphate was more pronounced than in heart-isolated cN-I. Despite this difference, a similar physiological function is suggested for cN-I in both types of muscle.

Mass spectrometry identification of membrane-bound respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus)

86%

Polcyn W.

2008

vol. 55

issue 4

753-760

Respiratory nitrate reductase (NR) from Bradyrhizobium sp. (Lupinus) USDA 3045 has biochemical properties of the membrane-bound NR type. However, in the completely sequenced rhizobium genomes only genes for the periplasmic type of dissimilatory NR were found. Therefore purification and identification of the enzyme by tandem mass spectrometry (MS/MS) was undertaken. MS/MS spectra representing 149 unique tryptic peptides derived from purified 137-kDa subunit matched the NCBInr-deposited NarG sequences. MS/MS sequencing of two other SDS/PAGE bands (65- and 59-kDa) identified them as derivatives of the NarH-type protein. Applying additional validation criteria, 73% of the sequence of the NarG subunit (902 aa) and 52% of NarH sequence (266 aa) was assembled (UniProt KB acc. no. P85097 and P85098). This is the first unambiguous identification of an active NarGH-like NR in rhizobia. Moreover, arguments are provided here for the existence of a functional enzyme of this type also among other rhizobial species, basing on immunoblot screening and the presence of membrane-associated NR-active electrophoretic forms.

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2 Acta Biochimica Polonica

1 2008

1 2005

1 Lechward K.

1 Polcyn W.

1 Skladanowski A. C.

1 Tkacz-Stachowska K.

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Isolation and characterization of pigeon breast muscle cytosolic 5´-nucleotidase-I (cN-I)

Mass spectrometry identification of membrane-bound respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus)