Purification and differential expression of enolase from maize

Lal, Sadhna; Kelley, Philip M.; Elthon, Thomas E.

doi:10.1034/j.1399-3054.1994.910406.x

Cited by 5 publications

(7 citation statements)

References 3 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A cyanobacterial enolase has been purified for the first time, which allows structural and kinetic comparison with other enolases. Enolase from chemostat cultures of Synechococcus PCC 6301 was purified to a specific activity (68 U·mg −1 ), comparable with that of homogeneous enolases isolated from various prokaryotic and eukaryotic sources (Singh and Setlow 1978, Dannelly and Reeves 1988, Lal et al 1994, Kaufmann and Bartholmes 1992, Mujer et al 1995, Schurig et al 1995). Homogeneity of the final preparation was confirmed by SDS‐ and nondenaturing‐PAGE that each generated a single protein‐staining polypeptide.…”

Section: Discussionmentioning

confidence: 97%

“…Annotation of several cyanobacterial genomes, including that of Synechococcus PCC 6301, indicates the presence of a single highly conserved enolase gene encoding a polypeptide having a predicted M r of about 46 kDa (Knowles and Plaxton 2003, http:/cyano/genome/jp/), which is 10‐kDa lower than the subunit M r estimated for the purified Synechococcus enolase via SDS‐PAGE. Although the reason for this discrepancy is unknown, it is interesting to note that the 56‐kDa subunit M r estimated via SDS‐PAGE for purified Zea mays enolase (Lal et al 1994) is also significantly greater than the M r predicted for the polypeptide (48 kDa) encoded by the corresponding cDNA sequence (Lal et al 1998). Similar discrepancies were also noted for Arabidopsis and tomato enolases (Van Der Straeten et al 1991).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)¹

Weese

Plaxton

2005

Journal of Phycology

View full text Add to dashboard Cite

Enolase from Synechococcus PCC 6301 was purified 1450-fold to electrophoretic homogeneity and a final specific activity of 68 lmol of phosphoenolpyruvate produced . min À 1 . mg protein À 1 . Analytical gel filtration and nondenaturing and SDS-gel electrophoresis demonstrated that this enolase exists as a 118-kDa homodimer composed of 56-kDa subunits. The purified enzyme displayed 1) a broad pH-activity profile with maximal activity occurring at pH 8.0 and 7.5 for the forward and reverse reactions, respectively, 2) a forward-to-reverse maximal activity ratio of about 1.6, 3) a K m (2-phosphoglycerate) of 0.28 mM, and 4) an absolute requirement for a divalent metal cation cofactor that was best satisfied by Mg 2 þ (K m 5 0.62 mM). Enolase activity increased by about 200% after the first purification step (601 C heat treatment), whereas addition of increasing amounts of a clarified extract led to a progressive 70% inhibition in the activity of the purified enzyme. This was reflected by a reduction in enolase's V max from 73 to 22 U . mg À 1 and forward-to-reverse activity ratio from 1.6 to 1.3. This inhibition was negated when the clarified extract was either preincubated with trypsin or warmed to approximately 401 for 5 min. Results are indicative of a heat-labile enolase inhibitor protein in Synechococcus PCC 6301. By contrast, the purified enolase lost no activity when incubated at 701 C for up to 5 min. This study represents the first purification of enolase from the Cyanophyceae. Characterization of the purified enzyme's physical and kinetic features has provided insights into the structural and functional properties of cyanobacterial enolase.

show abstract

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)¹

Weese

Plaxton

2005

Journal of Phycology

View full text Add to dashboard Cite

show abstract

“…3) may suggest that a single protein undergoes differential phosphorylation. There are indications that maize enolase of apparent molecular masses of 55 and 56 kDa can be resolved into three isoforms upon two-dimensional electrophoresis (Lal et al, 1994). The phosphorylation at tyrosine residues of two enolase isoenzyms from chicken fibroblast has revealed different forms of pI 5.2-6.7 (Eigenbrodt et al, 1983).…”

Section: Discussionmentioning

confidence: 99%

Characterization of dual specificity protein kinase from maize seedlings.

Trojanek

Klimecka²,

Fraser³

et al. 2004

Acta Biochim Pol

View full text Add to dashboard Cite

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.

show abstract

“…1988 ;Lal et al . 1991 ), but protein levels were similar under both aerobic and low‐oxygen conditions ( Kelley & Freeling 1984;Lal et al . 1994 ).…”

Section: Introductionmentioning

confidence: 99%

“… 1 Sachs et al . 1980 ; 2 ANP31.5 is a protein of unknown function ( Vogel & Freeling 1992); 3 Lal et al . 1994 ; 4 Probe hybridizes to a single copy gene under conditions used; 5 Probe hybridizes to more than one related gene under the conditions used: anp31.5 , small multigene family (J. Vogel, personal communication); act , multigene family; ant1 , two genes ( Winning et al .…”

Section: Introductionmentioning

confidence: 99%

Transcriptional and post‐transcriptional processes regulate gene expression in oxygen‐deprived roots of maize

1998

View full text Add to dashboard Cite

SummaryTo investigate the regulation of gene expression in maize (Zea mays L.) in response to oxygen deprivation (flooding), we quantitated run-on transcription in isolated nuclei, steady-state mRNA accumulation and mRNA loading with ribosomes for seven genes that encode proteins synthesized predominantly in oxygen-deprived roots (anaerobic polypeptides) and seven genes that encode proteins synthesized in aerobic roots (aerobic polypeptides). Run-on transcription of anaerobic polypeptide genes was induced in response to oxygen deprivation and run-on transcription of the aerobic polypeptide genes continued during the stress treatment. The increased accumulation of mRNAs that encode anaerobic polypeptides occurred concomitant with the induction of gene transcription and efficient association of these mRNAs with large polysomes. The steady-state accumulation of aerobic polypeptide mRNAs was within twofold of aerobic levels and in a number of cases fewer ribosomes were loaded per transcript. These results demonstrate that selected synthesis of anaerobic polypeptides involves transcriptional as well as significant post-transcriptional regulation of gene expression. The repression of synthesis of many aerobic polypeptides occurs without interruption of gene transcription and is due to translational regulation and possibly the sequestration of mRNAs on mRNPs. Ribosome loading patterns indicated that this translational control occurs at both initiation and post-initiation phases in a messagespecific manner.

show abstract

Purification and differential expression of enolase from maize

Cited by 5 publications

References 3 publications

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)¹

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)¹

Characterization of dual specificity protein kinase from maize seedlings.

Transcriptional and post‐transcriptional processes regulate gene expression in oxygen‐deprived roots of maize

Contact Info

Product

Resources

About

Purification and differential expression of enolase from maize

Cited by 5 publications

References 3 publications

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)1

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)1

Characterization of dual specificity protein kinase from maize seedlings.

Transcriptional and post‐transcriptional processes regulate gene expression in oxygen‐deprived roots of maize

Contact Info

Product

Resources

About

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)¹

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)¹