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Aims@#Heterologous holoenzyme formation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has been a challenge due to a limited understanding of its biogenesis. Unlike bacterial Rubiscos, eukaryotic Rubiscos are incompatible with the Escherichia coli (E. coli) chaperone system to fold and assemble into the functional hexadecameric conformation (L8S8), which comprises eight large subunits (RbcL) and eight small subunits (RbcS). Our previous study reported three sections (residues 248-297, 348-397 and 398-447) within the RbcL of Synechococcus elongatus PCC6301, which may be important for the formation of L8S8 in E. coli. The present study further examined these three sections separately, dividing them into six sections of 25 residues (i.e., residues 248-272, 273-297, 348-372, 373-397, 398-422 and 423-447).@*Methodology and results@#Six chimeric Rubiscos with each section within the RbcL from Synechococcus replaced by their respective counterpart sequence from Chlamydomonas reinhardtii were constructed and checked for their effect on holoenzyme formation in E. coli. The present study shows that Section 1 (residues 248-272; section of Synechococcus RbcL replaced by corresponding Chlamydomonas sequence), Section 2 (residues 273-297), Section 3 (residues 348-372) and Section 6 (residues 423-447) chimeras failed to fold and assemble despite successful expression of both RbcL and RbcS. Only Section 4 (residues 373-397) and 5 (residues 398-422) chimeras could form L8S8 in E. coli.@*Conclusion, significance and impact of study@#GroEL chaperonin mediates the folding of bacterial RbcL in E. coli. Therefore, residues 248-297, 348-372 and 423-447 of Synechococcus RbcL may be important for interacting with the GroEL chaperonin for successful holoenzyme formation in E. coli.
Subject(s)
Synechococcus , Ribulose-Bisphosphate Carboxylase , Escherichia coli , HoloenzymesABSTRACT
Objective To observe the effect of 7 flavonoids on recombinant human protein kinase CK2 holoenzyme activity and investigate their structure-activity relationship. Methods Recombinant hu-man protein kinase CK2 α' and β subunits were mixed at equal molar ratio to reconstitute CK2 holoen-zyme. The CK2 activity was assayed by detecting incorporation of 32p of [γ-32P] ATP into the substrate for the inhibitory effect by flavonoids and calculation of IC50 was performed according to probability unit (PROBIT) method. Results Myricetin, quercetin, morin, luteolin, kaempferol, apigenin, and chrysin were shown to obviously inhibit recombinant CK2 holoenzyme activity in a concentration-dependent man-ner with IC50 values of 1.18, 0.51, 16.16, 0.86, 1.88, 1.72, and 13.63 umol/L, respectively. Myricetin, quercetin, luteolin, kaempferol, and apigenin were more effective than DRB and A3, which were known as CK2 inhibitors in vitro. Whereas morin and chrysin displayed a similar effect to DRB. Structure-activity study indicated that the major structural requirements for the potent inhibition of CK2 by these flavonoids were hydroxyl group at position 6, 3' and 4'. Different from these requirements, absence of a hydroxyl group at position 3 did not modify their inhibitory potency, while addition of hydroxyl groups at positions 2' or 5' was detrimental to the inhibitory effect on CK2. Conclusion The inhibitory effect of flavonoid on protein kinase CK2 in vitro may be determined by the position of their hydroxyl groups.
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AIM To study the direct effect of tyrphostin AG114 on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS Recombinant human protein kinase CK2 α and β subunits were cloned and expressed by genetic engineering, and purified to homogeneity. The two subunits were mixed at equal molar ratio and reconstituted CK2 holoenzyme, which exerted the maximum biological activity. The CK2 activity was assayed by detecting incorporation of 32P of ［γ-32P］ATP or ［γ-32P］GTP into the substrate in various conditions. RESULTS The recombinant human CK2 was a second messenger (Ca2+, cAMP and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. AG114 strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 20.8 μmol·L-1, which lay between IC50 of 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole(DRB) and N-(2-aminoethyl)-5-chloronaphthalene-1-sulfonamide(A3), known as CK2 special inhibitors. Kinetic studies of AG114 inhibition on recombinant human CK2 showed that the inhibition was mixed competitive with GTP and noncompetitive with casein. CONCLUSION AG114 not only is an effective inhibitor of protein tyrosine kinases, but also is a novel potent inhibitor of protein kinase CK2. The recombinant human protein kinase CK2 might be used as a molecular target for simpler screening method and development of more effective inhibitors of CK2.
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AIM: To study the effects and kinetics of sodium quercetin-7-sulphate (SQMS) on recombinant human protein kinase CK2 holoenzyme. METHODS: The recombinant human CK2 holoenzyme activity was assayed by detecting incorporation of 32p of [γ-32P]ATP into the substrate at various conditions. RESULTS: SQMS was shown to strongly inhibit the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 1.37 μmol·L-1, Kinetic studies of SQMS on recombinant human CK2 showed: the inhibition was competitive with ATP and noncompetitive with casein. CONCLUSION: SQMS is an inhibitor of protein kinase CK2, and the inhibitory action of SQMS on CK2 is competitive with ATP and noncompetitive with casein.
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Object To study the direct effect of quercetin on recombinant human protein kinase CK2 holoenzyme and its kinetics. Methods Recombinant human protein kinase CK2? and ? subunits were cloned and expressed by gene engineering, and were purified. The two subunits were mixed at the same molar ratio, thus reconstituting CK2 holoenzyme, which displayed the maximum bioactivity. The CK2 activity was assayed by detecting incorporation of 32 P of [? 32 P] ATP into the substrate in the various conditions. Results The recombinant human protein kinase CK2 was the second messenger (Ca 2+ , cAMP and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. It was found that quercetin strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 522 nmol/L, which was much more effective than DRB and A3, known as CK2 special inhibitors. Kinetic studies of quercetin on recombinant human protein kinase CK2 showed: the inhibition was competitive with ATP and noncompetitive with casein. Conclusion Quercetin is a potent inhibitor of recombinant human protein kinase CK2. The inhibition may be another molecular mechanism of antitumor effect of quercetin. This study provides a simple and rapid screening method for the development of more effective inhibitors of recombinant human protein kinase CK2.
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AIM To study the effects and kinetics of sodium quercetin 7 sulphate (SQMS) on recombinant human protein kinase CK2 holoenzyme. METHODS The recombinant human CK2 holoenzyme activity was assayed by detecting incorporation of 32 P of [? 32 P]ATP into the substrate at various conditions. RESULTS SQMS was shown to strongly inhibit the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 1 37 ?mol?L -1 , Kinetic studies of SQMS on recombinant human CK2 showed: the inhibition was competitive with ATP and noncompetitive with casein. CONCLUSIONSQMS is an inhibitor of protein kinase CK2, and the inhibitory action of SQMS on CK2 is competitive with ATP and noncompetitive with casein.
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AIM To study the direct effect of amiloride on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS Recombinant human protein kinase CK2 ? and ? subunits were cloned,expressed and purified by gene engineering. The two subunits were mixed at the same molar ratio to form CK2 holoenzyme,which displayed the maximum biological activity. CK2 activity was assayed by detecting incorporation of 32 P of [? 32 P]GTP into the substrate in various conditions. RESULTS Amiloride inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 257 57 ?mol?L -1 . Kinetic studies of amiloride on recombinant human CK2 showed that the inhibition was competitive with GTP with the K i of 236 30 ?mol?L -1 ,and noncompetitive with casein with the K i of 163 63 ?mol?L -1 . CONCLUSION Amiloride inhibits the activity of cAMP or ATP dependent enzymes as well as the kinase with GTP as phosphate donor,and the recombinant human protein kinase CK2 may be used as a molecular target for simple screening and development of effective inhibitors of CK2.