Fluorometric determination of adenine and its derivatives by reaction with glyoxal hydrate trimer

Yuki, Hidetaka; Sempuku, Chikao; Park, Manki; Takiura, Kiyoshi

doi:10.1016/0003-2697(72)90403-4

Cited by 35 publications

(10 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…strain PCC 6803 was regulated by the nitrogen source in the medium, with a rapid decline in activity following the addition of NH 4 Cl to nitrate-grown cells. They were not able, however, to demonstrate the presence of any covalent modification of GS in in vivo 32 P labelling experiments. Furthermore, the inactivated Synechocystis GS was reactivated upon the addition of alkaline phosphatase and also by treatment at pH values between 8 and 10 or by the addition of organic solvents but not by the addition of phosphodiesterase (17).…”

mentioning

confidence: 85%

“…32 P labelling of GS was carried out by using a high-speed supernatant. Forty microliters of the high-speed supernatant was mixed with 1 l of NH 4…”

Section: Sds-page Autoradiography and Western Blotting (Immunoblottmentioning

confidence: 99%

“…Theoretical yields of ribose were obtained by similar treatment of both ADP and NAD ϩ (1, 2, 10, and 20 nmol). (iii) Adenine content was determined by the fluorescence assay described by Yuki et al (32). Four to eight nanomoles of inactive GS in a volume of 1 ml was mixed with 1 ml of glyoxal reagent (40% [wt/vol] aqueous solution of glyoxal hydrate trimer) and 3 ml of glacial acetic acid.…”

mentioning

confidence: 99%

See 2 more Smart Citations

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803

1995

View full text Add to dashboard Cite

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP ؉ and, to a lesser extent, NAD ؉ , suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [ P]NAD؉ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.In cyanobacteria, as in most prokaryotes, glutamine synthetase (GS; EC 6.3.1.2) plays a key role in nitrogen assimilation. The enzyme catalyzes the ATP-dependent biosynthesis of glutamine from NH 4 ϩ and glutamate and thus represents a key point for the interaction of carbon and nitrogen metabolism. Given this importance, one would expect that both the synthesis and the activity of the enzyme would be tightly regulated. Three types of GS, termed GSI to GSIII, are found in prokaryotes; GSI is the typical form found in many organisms (31). In the enterobacteriaceae, GS activity is regulated by an adenylylation/deadenylylation system, the activity of the adenyltransferase itself being regulated by the uridylylation state of the protein P II , which also exterts an effect on the transcription of the glnALG operon through its interaction with the NR II protein (14, 23). In gram-positive organisms, GS is not subject to adenylation and activity appears to be regulated by allosteric mechanisms (2).GSs have been purified from a number of cyanobacteria (18) and exhibit the typical size and subunit composition of GSI, although recently the presence of a GSIII has been reported in Synechocystis sp. strain PCC 6803 (24). The regulation of GS activity in cyanobacteria, however, is not well understood. Fisher et al. (4) demonstrated that a cloned cyanobacterial GS was functionally expressed in Escherichia coli but was not subject to adenylation, although cyanobacteria have been shown to possess a P II homolog (28), the activity of which in Synechococcus sp. strain PCC 7942 is regulated by phosphorylation (6). Evidence from several sources (21,25,27,29) suggests that cyanobacterial GS activity is regulated by allosteric processes. In contrast, Joseph and Meeks (9) suggested that GS activity in a symbiotic Nostoc strain might be regulated by a posttranslational mechanism. GS activity has also been shown to be regulated by environmental factors in two unicellular cyanobacteria. Marqués et al. (15) demonstrated a short-term regulation of activity in Synechoc...

show abstract

mentioning

confidence: 85%

“…32 P labelling of GS was carried out by using a high-speed supernatant. Forty microliters of the high-speed supernatant was mixed with 1 l of NH 4…”

Section: Sds-page Autoradiography and Western Blotting (Immunoblottmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803

1995

View full text Add to dashboard Cite

show abstract

“…For the Fe protein, activesite iron was determined by Mg-ATP-induced complexation (14). Phosphate was determined, after digestion, through formation of phosphate-molybdomalachite green complex (10), ribose was determined by reaction with orcinol (5), and adenine was determined by formation of a fluorescent product with glyoxal hydrate trimer (31). Tris was found to quench the fluorescence, and all proteins used in the fluorescent analysis were prepared in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer.…”

mentioning

confidence: 99%

Nitrogenase from the photosynthetic bacterium Rhodopseudomonas capsulata: purification and molecular properties.

Hallenbeck¹,

Meyer²,

Vignais³

1982

Journal of Bacteriology

View full text Add to dashboard Cite

Nitrogenase proteins were isolated from cultures of the photosynthetic bacterium Rhodopseudomonas capsulata grown on a limiting amount of ammonia. Under these conditions, the nitrogenase N2ase A was active in vivo, and nitrogenase activity in vitro was not dependent upon manganese and the activating factor. The nitrogenase proteins were also isolated from nitrogen-limited cultures in which the in vivo nitrogenase activity had been stopped by an ammonia shock. This nitrogenase activity, N2ase R, showed an in vitro requirement for manganese and the activating factor for maximal activity. The Mo-Fe protein (dinitrogenase) was composed of two dissimilar subunits with molecular weights of 55,000 and 59,500; the Fe protein (dinitrogenase reductase), from either type of culture, was composed of a single subunit (molecular weight, 33,500). The metal and acid labile sulfur contents of both nitrogenase proteins were similar to those found for previously isolated nitrogenases. The Fe proteins from both N2ase A and N2ase R contained phosphate and ribose, 2 mol of each per mol of N2ase R Fe protein and about 1 mol of each per mol of N2ase A Fe protein. The greatest difference between the two types of Fe protein was that the N2ase R Fe protein contained about 1 mol per mol of an adenine-like molecule, whereas the N2ase A Fe protein content of this compound was insignificant. These results are compared with various models previously presented for the short-term regulation of nitrogenase activity in the photosynthetic bacteria. 708 on July 31, 2020 by guest

show abstract

“…The orcinol assay (7) was used for pentose; phosphate was measured with Chen's modification of the molybdate assay (8). Adenine was measured by its fluorescence after treatment with glyoxal hydrate (9). Assays for these compounds have been described in detail (2).…”

mentioning

confidence: 99%

Removal of an adenine-like molecule during activation of dinitrogenase reductase from Rhodospirillum rubrum.

Ludden¹,

Burris²

1979

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

During the activation of the inactive dinitro- Dinitrogenase reductase (Rr2) from Rhodospirillum rubrum is inactive as isolated (1) and has been shown to have two pentose, two phosphate, and two adenine-like molecules (Ad) attached per 60,000 molecular weight dimer (2). The role of these groups in activation is discussed in this paper.Other enzymes have been shown to have ribose, phosphate, and adenine covalently attached. The first of these was glutamine synthetase (GS) from Escherichia coli; it has up to 12 AMP molecules per dodecamer (3). The adenylylated form of the enzyme is much less active in the biosynthesis of glutamine than is the unadenylylated form. Cleavage of the bond between a tyrosine residue on the protein and the phosphate of AMP by a specific enzyme (ATase) isolated from E. coli or by snake venom diesterase (SVD) activates the enzyme. The E. coli enzyme can also adenylylate GS with ATP as an AMP donor.The RNA polymerase of E. coli has its template specificity altered by ADP-ribosylation (4). The ADP-ribose donor is NAD+, and linkage to the protein is through arginine-ribose. ADP-ribose is susceptible to hydrolysis either by SVD or by a glycohydrolase from chromatin material (5), but the two enzymes cleave the ADP-ribose in different places. The adenine/ribose/phosphate ratio is 1:2:2 for RNA polymerase and 1:1:1 for the adenylylated GS.The dinitrogenase reductases from other organisms do not appear to have the pentose-, phosphate-, and Ad-containing group (2), and they do not require activation. In this paper, evidence is presented to show that the Ad, but not the phosphate group, is removed upon activation. The role of ATP and metals in activation also is established.MATERIALS AND METHODS Enzymes. The Rrl and Rr2 of R. rubrum and the activating factor (AF) were purified as described (2, 6). Alkaline phosphatase from E. coli was obtained from Sigma. This preparation (type III-R) was essentially free from DNase and RNase activities and had a specific activity of 31 ,umol of p-nitrophenyl phosphate hydrolyzed per min per mg of protein. The stock solution of phosphatase was diluted 1:60 before use, and 50 MIl of the diluted solution was used per assay (0.07 unit per assay). SVD type II also was obtained from Sigma; 1.6 units of the enzyme was dissolved in 2 ml of buffer and 50 ,ul was used per assay (0.04 units per assay). Because of the lability of this preparation, its activity was checked before it was used on R. rubrum Rr2 by measuring its rate of hydrolysis of bis-p-nitrophenyl phosphate. The SVD used was low in 5'-nucleotidase activity, inorganic pyrophosphatase activity, and phosphatase activity but had considerable nucleotide pyrophosphatase activity. Spleen diesterase (SpD) from Sigma was dissolved in buffer so that 50 ,ul contained 0.3 unit; 50 ,ul was used in each assay. It had considerable phosphatase activity.Enzymatic Treatment of Rr2. Ten to 20 nmol of Rr2 was treated with enzymes (SVD, SpD, alkaline phosphatase) and AF in the presence of ATP, metal ions, and other compound...

show abstract

Fluorometric determination of adenine and its derivatives by reaction with glyoxal hydrate trimer

Cited by 35 publications

References 4 publications

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803

Nitrogenase from the photosynthetic bacterium Rhodopseudomonas capsulata: purification and molecular properties.

Removal of an adenine-like molecule during activation of dinitrogenase reductase from Rhodospirillum rubrum.

Contact Info

Product

Resources

About