Hello, see attached info may be useful for you. Good luck!

Thank you so much for the lead Karen! I haven't referenced hopped enough to confirm yet if this is exactly what I need, but it looks very promising. I appreciate it!

Amended on 24 April 2019

I have searched for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase/PFKFB in my human protein database in vain (please see files; HepG2 fucoidin, JMBT alopecia, and The Fascio effect). Further, Insulin and Insulin receptor/IR, and Glucagon and Glucagon receptor/GL-R are not present in Humans. It is interesting that a bacterium of Agrobacterium tumefaciens has no PFKFB (please see file; Dr. C-K. Chern Agrobacterium).

This may due to the famous Species differences and/or the Species differences in the Fructose and/or Mannose metabolism.

By the way, enzyme assay should be performed by using HPLC-photometric method. which is quantitative, reliable, and sensitive (please see file; Guinea pig Carboxylase assay). In the case of carboxylic acids and sugars, which weakly absorb UV or visible light, substrate and produced product should be derivatized by suitable derivatizing reagents, which are easily available commercially.

Direct utilization of photometer without separation/purification is not quantitative at all due to violence against the Lambert-Beer law. HPLC-MS method is also not quantitative at all, since MS is a non-quantitative detector (kindly informed via the telephone by a courageous engineer employed to Shimadzu Co., Kyoto, Japan). MS and Electron microscope can not become to be quantitative due to detection of very small areas.

Furthermore, RIA and ELISA are not quantitative due to low specificity (please see file; The Fascio effect). Avidin is recently found to be the lipoic acid-biotin binding protein by us (please see file; Lipoic acid Avidin).

PCR is also not the quantitative method, since affinity or reversed Km values (kcat/Km = Amo) may decrease at high temperature region using procaryotic non-glycosylated enzymes (please see file; J Chrom B Rat BIN LIP Km). Eucaryotic glycoprotein enzyme shows heat resistance due to the effect of glycochains (Please see file; Thermo BIN).

About the absence of AKT in humans, I have recently answered the question as follows; i.e.,

Amended on 29 September 2018 I have searched for Akt/Protein Kinase B/RAC-alpha serine/threonine-protein kinase/Proto-oncogene c-Akt and SDF-1/Stromal cell-derived factor 1/C-X-C motif chemokine 12/CXCL12/Intercrine reduced in hepatomas/Pre-B cell growth-stimulating factor among our human protein database in vain (please see file; HepG2 Fucoidan). Therefore, Akt Pathway or PI3K-Akt Pathway seems to be not working in humans. It seems that signal pathways show representative species differences. Biotinidase shows species differences very much (please see file; J Chrom B. rat BIN LIP Km). It is also noteworthy that Serine/threonine-protein kinase mTOR/mammalian target of rapamycin mTOR/FK506-binding protein 12-rapamycin complex-associated protein 1, nerve growth factor (NGF), and Apoptosis-related proteins are not found in my human protein database. We have previously shown that urinary excretion of biotinidase is different between European and Asian (please see file; Racial difference Urine BIN). Only the liver (with pseudo liver cancer) exceptionally seemed to have RAC-alpha serine/threonine-protein kinase/RAC-PK-alpha/Proto-oncogene c-Akt/Protein kinase B/Akt (153-480; LKLLGK-) at 0.0 μg/mg of tissue protein. But, this tissue uniquely has AKT kinase-transforming protein/v-Akt (AKT8 Murine leukemia virus/AKT8 MuLV; 174-501; LKLLGK-) at 2.0 μg/mg of tissue protein. PDMD method identifies protein from amino acid sequence. Then, we may have only detected this viral AKT kinase-transforming protein/v-Akt which has protein serine/threonine kinase activity, and human RAC-alpha serine/threonine-protein kinase/RAC-PK-alpha/Proto-oncogene c-Akt/Protein kinase B/Akt gene may not have been expressed in this tissue. By the way, I have been very sorry for the publish of "Nagamine T, Hayakawa K, Kusakabe T, Takada H, Nakazato K, Doku MH, Iha K. Inhibitory effect of fucoidan on HuH-7 hepatoma cells through down regulation of CXCL12. Nutrition & Cancer 61; 340-347, 2009". This paper seems to have used non-quantitative Western blot, Immuno assay, and Reverse Transcription Polymerase Chain Reaction (RT-PCR) methods. Then, this paper can not use the term "down regulation" at all. I was forced to become to be a co-author by a medical doctor (MD). However, I am happy to be published the quantitative PDMD method in 2014 from The Greece (please see files again; "HepG2 fucoidan" and "JMBT Alopecia"). Serum of 1y11mo girl (with excess biotin and anorexia) has C-C motif chemokine 17/CCL17/CC chemokine TARC/Small-inducible cytokine A17/Thymus and activation-regulated chemokine at 6.2 μg/mg of serum protein. This up regulation seems to be due to Tacaribe virus/TCRV, since this serum has uniquely RNA-directed RNA polymerase (TCRV) at 2.0 μg/mg of serum protein. LC tissue (named as No.6) has Phosphatidylinositol 3-kinase regulatory alpha subunit/PI3K regulatory subunit alpha at 6.8 μg/mg of tissue protein. This up regulation seems to be due to unique infection by Fusion glycoprotein Fo (Bovine respiratory syncytial virus/BRSV) at 5.2 μg/mg of tissue protein. This regulatory protein binds to the activated (phosphorylated) protein-Tyr kinases, and can not binds to PI3K/RAC-alpha serine/threonine-protein kinase, which is absent in humans. By the way, Famous Late Prof. Dr. Kunio Yamauchi (Department of Agricultural Chemistry, The University of Tokyo, Yayoi-cho, Bunko-ku, Tokyo, Japan) has found that α- S2 Casein and β- Lactoglobulin genes are absent in humans. Italian Pediatricians Bertino E et al. have shown it in "Acta Biomed Ateneo Parmense. 1997;68 Suppl 1:15-9. Absence in human milk of bovine beta-lactoglobulin ingested by the mother. Unreliability of ELISA measurements. Bertino E, Coscia A, Costa S, Farinasso D, Prandi G, Fabris C, Cavaletto M, Giuffrida MG, Conti A., Cattedra di Neonatologia, Università di Torino, Italy". This paper also indicated the unreliable or non-quantitative nature of ELISA method. I have quantitatively proven the presence of α- S2 Casein at 50.3 μg/mg of milk protein and β- Lactoglobulin at 27.5 μg/mg of milk protein only in the bovine milk by PDMD method (my unpublished observation). Further, milk biotinidase is present at 5.5 μg/mg of milk protein in the human breast milk, but absent in the bovine milk due to species differences (please see file; The Fascio effect). Furthermore, major component proteins of human breast milk and bovine milk are shown as a reference (our unpublished result). Species differences between humans and bovine seem to be very big.

Bovine milk from Japanes cows 1st V-type proton ATPase 116 kDa subunit a isoform 1/Vacuolar proton pump subunit 1/Vacuolar adenosine triphosphatase subunit Ac116 at 126.4 μg/mg of milk protein 2nd Solute carrier family 43 member 3/SLC43A3 at 124.9 3rd α-Lactalbumin/Lactose synthase B protein at 110.0 4th Negative regulator of reactive oxygen species/Leucine-rich repeat-containing protein 33 at 104.3 5th α s1 Casein at 101.1 6th Aldehyde oxidase/Azaheterocycle hydroxylase 1 at 81.9 7th β- Casein at 78.8 8th κ- Casein at 74.3 9th Sialidase/Neuraminidase (Salmonella typhimurium) at 58.2 10th α- S2 Casein at 50.3 11th β- Lactoglobulin at 27.5 12th Fibrillin-1/MP340 at 15.3

Human breast milk from a healthy Japanese female 1st α-Lactalbumin/Lactose synthase B protein/Lysozyme-like protein 7 at 156.0 2nd β-Casein at 127.5 3rd Lactoferrin at 83.0 4th RING finger protein 86/Tripartite motif-containing protein 2/UBE2D1-dependent E3 ubiquitin-protein ligase at 55.0 4th Vacuolar protein sorting-associated protein 13B/Cohen syndrome protein 1 at 55.0 6th Major capsid protein/MCP (Saimiriine herpesvirus 2/SaHV-2) at 51.0 7th Protein Smp (Escherichia coli) at 40.3 8th Tetraspanin-14/Transmembrane 4 superfamily member 14 at 33.8 9th Lysozyme at 30.9 10th α s1 Casein at 30.0 11th MARCKS-related protein/Macrophage myristoylated alanine-rich C kinase substrate at 25.8 12th Human breast milk lipoamidase at 23.0

You could do a spectrophotometric assay for Fru-2,6-BPase by coupling the reaction to two other reactions. You would need a cocktail which included (a) an appropriate buffer for your enzyme (probably Tris pH 8.0 and definitely not phosphate, which will inhibit the reaction); (b) an abundance of Glucose-6-phosphate isomerase; (c) an abundance of Glucose-6-phosphate dehydrogenase; (d) some NADP+. You would also include your desired concentration of Fru-2,6-BP. Monitor the gain of NADPH at 340nm. I have not determined the necessary concentrations of the enzymes or the NADP+ to perform this assay, nor have I looked in the literature to see if there is any metal requirement (e.g. magnesium), but I have used an assay very like this before. In this proposed assay, since you have an abundance of the coupling enzymes, any produced Fru-6-P would immediately be reacted to form Glc-6-P which in turn is immediately reacted to form 6-phosphogluconolactone, accompanied by the reduction of NADP+ to NADPH. It is a 1:1 stoichiometric relationship between loss of Fru-2,6-BP and gain of NADPH, with the reaction effectively being:

Fru-2,6-BP + H2O + NADP+ --> 6-phosphogluconolactone + Pi + NADPH+ + H+

I hope you can make an assay that works based off of this idea. I will add, however, that spectrophotometry isn't the best possible method to use, but it's a good place to start for studying the enzyme in vitro.

Thank you for your answer Benjamin. The principles of your assay are definitely sound and are very similar to the assay Karen originally cited. Of course any type of enzymatic assay is not the best approach when trying to figure out kinetics. However, we intend to do more in-depth enzymatic and metabolite analysis via mass spec, but first confirm a change in the PFKFB activity before investing in such a large experiment. I think I have all of the information I need as of right now and I will continue to work through the solution. I will update you all if I am with whether or not I was successful.

This may be helpful....