Cloning and expression analysis of the chloroplast fructose-1,6-bisphosphatase gene from Pyropia haitanensis

Fructose-1,6-bisphosphatase（FBPase） is one of the key enzymes in Calvin circle and starch biosynthesis. In this study, the full-length of cpFBPase gene from Pyropia haitanensis was cloned by using rapid amplification of cDNA ends（RACE） technology. The nucleotide sequence of PhcpFBPase consists of 1 400 bp, including a 5′ untranslated region（UTR） of 92 bp, a 3′？UTR of 69 bp, and an open reading frame（ORF） of 1 236 bp, which can be translated into a 412-amino-acid putative peptides with a molecular weight of 44.3 kDa and a theoretical pI of 5.23. Multiple sequence alignment indicated that the protein belonged to the chloroplast FBPase enzyme. Phylogenetic analysis showed that the protein assembled with the cpFBPase of a thermal tolerant unicellular red micro-algae Galdieria sulphuraria. Expression patterns analyzed by qRT-PCR revealed that the expression of PhcpFBPase gene in the thallus phage was 7-fold higher than in the conchocelis phage, which suggested the different mechanisms of inorganic carbon utilization among the different life phages of P. haitanensis. And the different response modes of PhcpFBPase mRNA levels to high temperature and desiccation stress indicated that PhcpFBPase played an important role in responsing to abiotic stress.

Clinical study of applying fructose-1,6-diphosphate and captopril to enhance the protective effects of cardioplegia solution on ischemic myocardium

In the present experiment,fructose-1,6-diphosphate（FDP）and captopril（Cap）wereadded to the cold potassium cardioplegia solution and the levels of malondialdehyde（MDA）,cre-atine phosphokinase MB（CPK-MB）,thromboxane B（TXB2）and 6-keto-PGF1α in plasma weremeasured during open-heart surgery.Quantitative study of myocardial ultrastructure and obser-vation of cardiac resuscitation were also undertaken.The findings suggested that FDP,especiallywhen combined with Cap could significantly strengthen the protective effects of cold potassiumcardioplegia solution on ischemic myocardium.

Status epilepticus due to fructose-1,6-bisphosphatase deficiency caused by FBP1 gene mutation

Introduction:Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare inherited disorder in gluconeogenesis,characterized by hypoglycemia,ketonuria,metabolic acidosis and convulsions.Case presentation:We describe two brothers with FBPase deficiency.The proband developed severe hypoglycemia and progressed to status epilepticus,and the brother showed slightly hypoglycemia with a good prognosis.Whole exome sequencing (WES) identified compound heterozygous variants [c.333+1333+2delinsTC and c.490G>A (p.Gly164Ser)] in fructose-1,6-bisphosphatase 1 gene in the two brothers,which were inherited from the father and the mother,respectively.Conclusion:Genetic analysis provided a solid basis for a definite diagnosis and the determination of precision therapies for the patient.

AMP makes native snake muscle fructose-1,6-bisphosphatase to an alkaline enzyme

A substance in the crude preparation of NADP+ has been found, which activates snake muscle fructose-1,6-bisphosphatase at pH 9.2 and inhibits the enzyme at pH 7.5. After isolation and extensive characterization, the substance has been determined to be AMP. The activation depends on the concentrations of Mg2+ and could be observed only at concentrations above 1 mmol/L. In the presence of AMP, snake muscle fructose-1,6-bisphosphatase resembles an alkaline enzyme. Kinetic studies indicate that AMP and Mg2+ competitively regulate the activity of the enzyme. AMP releases the inhibition of Mg2+ at high concentration at alkaline pH. It has been reported that fructose-1,6-bisphosphatase with a pH optimum in the alkaline region is caused by limited proteolysis. AMP is also able to make fructose-1,6-bisphosphatase to be an alkaline enzyme. This finding indicates that proteolysis may not be the only reason for shift of the optimum pH of fructose-1,6-bisphosphatase to alkaline side and it may imply some

Cloning of a NaCl-induced fructose-1,6-diphosphate aldolase cDNA from Dunaliella salina and its expression in tobacco

Using Rapid Amplification of cDNA ends (RACE) technique, the full-length cDNA en-coding a NaCl-induced fructose-1, 6- diphosphate aldolase (DsALDP) was obtained. It was shown that the DsALDP had a relatively high homology (66%—73%) to chloroplast fructose-1, 6-diphos- phate aldolase (AldP) in many plants according to their amino acid sequences. The phylogenetic analysis further confirmed that AldP in alga is the nearest to DsALDP. As to its expression pattern, DsALDP was de novo synthesized by NaCl induction. Its expression level was significantly changed with inducing time. After the selected DsALDP cDNA subcloned into a binary vector pBI121, the new construct was introduced into tobacco by Agrobacterium tumefaciens. The results of Southern blot and RT-PCR analysis of four transgenic T1 plants indicated that DsALDP was integrated into genome of these transgenic plants and effectively expressed. Aldolase activities have been detected in T1-1, T1-2 and T1-3 plants by bioassay under 100—200 mmol/L NaCl. It was also observed that proline contents in them were differentially increased.

PRODUCTION OF FRUCTOSE-l,6-DIPHOSPHATE(FDP) BY PERMEABILIZED SACCHAROMYCES CEREVISIAE AND A KINETIC MODEL FOR FDP ACCUMULATION

Permeable yeast cells were used in the batch production of fructose-1,6-diphosphate(FDP).The optimum reaction conditions were reported to be:reaction temperature 30℃,tolueneconcentration 8%(V/V),and initial ratio of glucose to inorganic phosphorus(Pi)10:1.Addition ofAMP was found to be very beneficial to the FDP production.A multienzyme system model for FDPaccumulation was developed,in which FDP was regarded as a substrate of phosphor-fructokinase(PFK),to simulate the activation effect of FDP on PFK.The model simulations were in good agree-ment with the experimental data.

Preliminary crystallographic analysis of recombinant chicken liver fructose-2,6-bisphosphatase

The bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphos-phosphatase was expressed in high yield Escherichia coli and purified to homogeneity. Single crystals of chicken liver bisphophatase domain suitable for X-ray diffraction were obtained by the hanging drop vapor diffusion method. The crystals belong to tetragonal space group P41212 or P43213 with two molecules per asymmetric unit. The determined cell dimensions are: a=b= 10.02 nm, c= 13.98 nm, α=β=г=90°. EHffraction data were collected on Weissenberg camera with synchrotron radiation at 0.32nm resolution.

Conformation of 60-residue peptide fragment from N-terminal of porcine kidney fructose 1,6-bisphosphatase

Limited digestion of fructose 1,6-bisphosphatase with subtilisin produces an S-peptide with an about 60-residue peptide fragment that is non-covalently associated with the enzyme. The 60-residue peptide fragment con-sists of the most part of allosteric site for AMP binding. It could be separated from S-protein by gel filtration with a Sephadex G-75 column equilibrated with 9% formic acid. According to X-ray diffraction results the S-peptide consists of two α-helices without β-strand and the α-helix content is about 60% in the 60-residue-peptide fragment. When the enzyme is subjected to limited proteolysis with subtilisin, the secondary structure of the enzyme does not show a de-tectable change in CD spectrum. The CD spectra of the isolated S-peptide were measured under different concentra-tions. In the absence of GuHCl, S-peptide had 30% a-helix and 38.5% turn-like structure but had no β-strand, sug-gesting that the N-terminal 60-residue fragment, which is synthesized initially by ribosome, would

Preparation of monoclonal antibodies against chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and their effects on enzyme activities

The effects of the monoclonal antibodies (McAbs) directed against chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2, 6-P2ase) on the structure and function of the enzyme were studied. Using chicken liver 6PF-2-K/Fru-2,5-P2asc as antigen, 7 clones of monoclonal antibodies specifically binding with the antigen were obtained. The epitopes of the antigen recognized by the 6 McAbs localized on the fructose-2,6-bisphosphatase domain of chicken liver 6PF-2-K/Fru-2, 6-P2ase, and the other (H2) are on the 6-phosphofructo-2-kinase domain. All of the 7 McAbs could activate the kinase activity of the bifunctional enzyme by twofold and had a similar effect on the bisphosphatase activity of the bifunctional enzyme which resulted in a fourfold increase of the bisphosphatase activity of the bifunctional enzyme. However, the McAbs did not affect the activity of the separated fructose-2, 6-bisphosphatase domain. The results suggested that the Fru-2, 6-P2ases in the bifunctional enzyme

Vacuole import and degradation pathway: Insights into a specialized autophagy pathway

Glucose deprivation induces the synthesis of pivotagluconeogenic enzymes such as fructose-1,6-bisphos-phatase, malate dehydrogenase, phosphoenolpyruvatecarboxykinase and isocitrate lyase in Saccharomycescerevisiae. However, following glucose replenishment,these gluconeogenic enzymes are inactivated and de-graded. Studies have characterized the mechanismsby which these enzymes are inactivated in response toglucose. The site of degradation of these proteins hasalso been ascertained to be dependent on the dura-tion of starvation. Glucose replenishment of short-termstarved cells results in these proteins being degradedin the proteasome. In contrast, addition of glucose tocells starved for a prolonged period results in theseproteins being degraded in the vacuole. In the vacuoledependent pathway, these proteins are sequestered inspecialized vesicles termed vacuole import and degra-dation (Vid). These vesicles converge with the endo-cytic pathway and deliver their cargo to the vacuolefor degradation. Recent studies have identified thatinternalization, as mediated by actin polymerization, isessential for delivery of cargo proteins to the vacuolefor degradation. In addition, components of the targetof rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissoci-ates from cargo proteins following glucose replenish-ment. Future studies will be needed to elaborate on the importance of internalization at the plasma membrane and the subsequent import of cargo proteins into Vid vesicles in the vacuole dependent degradation pathway.