Biomedical subjects

J Bär

Publications and source records attributed to J Bär.

At least 19 recordsLinked to original sources

PubMed2005

Serum resistin levels of obese and lean children and adolescents: biochemical analysis and clinical relevance.

OBJECTIVE: It was hypothesized that resistin links obesity with diabetes, but this has not been studied in children and adolescents to date. PATIENTS: We determined serum resistin levels of 135 obese (body mass index, 32.0 +/- 6.2 kg/m2; age, 12.6 +/- 3.4 yr) and 201 lean children (body mass index, 18.7 +/- 2.4 kg/m2; age, 12.5 +/- 2.5 yr) by a newly developed and extensively evaluated in-house immunoassay. These results were controlled for their association with markers of puberty, obesity, and insulin sensitivity. RESULTS: The analytical evaluation of our assay revealed different resistin isoforms with major peaks of higher than 660 and 55 kDa in the size exclusion chromatography. Using this assay system we found no difference in the resistin levels of obese compared with lean subjects (P = 0.48). However, resistin was significantly higher in girls than in boys (6.74 +/- 2.42 vs. 5.79 +/- 2.45; P < 0.001). Interestingly, in both obese and lean children, resistin correlated with age (P < 0.01), Tanner stage, and testosterone and estradiol levels (P < 0.05). In contrast, no significant correlation was found with parameters of insulin resistance such as homeostasis model assessment, insulin sensitivity index, or insulin, proinsulin, and glucose concentrations in obese subjects. CONCLUSIONS: Resistin appears to be not the main link between obesity and insulin resistance in children and adolescents but because of its association with Tanner stage, it may be related to the maturation of children during pubertal development. Additionally, we have demonstrated the presence of different molecular isoforms of resistin in human blood, and this may raise problems in comparing data from diverse assay systems.

Adolescent↗

PubMed2001

Molecular analysis of acid ceramidase deficiency in patients with Farber disease.

Farber disease is a rare, autosomal recessively inherited sphingolipid storage disorder due to the deficient activity of lysosomal acid ceramidase, leading to the accumulation of ceramide in cells and tissues. Here we report the identification of six novel mutations in the acid ceramidase gene causing Farber disease: three point mutations resulting in single amino acid substitutions, one intronic splice site mutation resulting in exon skipping, and two point mutations also leading to occasional or complete exon skipping. Of interest, these latter two mutations occurred in adjacent nucleotides and led to abnormal splicing of the same exon. Expression of the mutated acid ceramidase cDNAs in COS-1 cells and subsequent determination of acid ceramidase residual enzyme activity demonstrated that each of these mutations was the direct cause of the acid ceramidase deficiency in the respective patients. In contrast, two known polymorphisms had no effect on acid ceramidase activity. Metabolic labeling studies in fibroblasts of four patients showed that even though acid ceramidase precursor protein was synthesized in these individuals, rapid proteolysis of the mutated, mature acid ceramidase occurred within the lysosome.

Acid Ceramidase↗

PubMed2000

Denaturation of phosphofructokinase-1 from Saccharomyces cerevisiae by guanidinium chloride and reconstitution of the unfolded subunits to their catalytically active form.

Unfolding and refolding of heterooctameric phosphofructokinase-1 from Saccharomyces cerevisiae were investigated by application of kinetic, hydrodynamic, and spectroscopic methods and by use of guanidinium chloride (GdmCl) as denaturant. Inactivation of the enzyme starts at about 0.3 M GdmCl and undergoes a sharp unfolding transition in a narrow range of the denaturant concentration. The inactivation is accompanied by a dissociation of the enzyme into dimers (at 0.6 M GdmCl), which could be detected by changes of the circular dichroism and intrinsic fluorescence. Protein aggregates were observed from 0.7 to 1.5 M GdmCl that unfold at higher denaturant concentrations. Refolding of chemically denatured phosphofructokinase proceeds as a stepwise process via the generation of elements of secondary structure, the formation of assembly-competent monomers that associate to heterodimers and the assembly of dimers to heterotetramers and heterooctamers. The assembly reactions seem to be rate-limiting. Recovery of the enzyme activity (maximum 65%) competes with an nonproductive aggregation of the subunits. alpha-Cyclodextrin functions as an artificial chaperone by preventing aggregation of the subunits, whereas ATP is suggested to support the generation of heterodimers that are competent to a further assembly.

Chromatography, Gel↗

PubMed2000

Purification, molecular and kinetic characterization of phosphofructokinase-1 from the yeast Schizosaccharomyces pombe: evidence for an unusual subunit composition.

Phosphofructokinase-1 (Pfk-1) from Schizosaccharomyces pombe was purified by 54-fold enrichment to homogeneity elaborating the following steps: (a) Disruption of the cells with glass beads; (b) fractionated precipitation with polyethylene glycol 6000; (c) affinity chromatography on Cibacron-Blue F3G-A-Sephadex G 100; (d) ion exchange chromatography on Resource Q. The native enzyme exhibits a mass of 790+/-30 kDa, as detected by sedimentation equilibrium measurements. The apparent sedimentation coefficient was found to be s(20,c)=20.2+/-0.3 S. No significant dependence of the s-value on the protein concentration was observed in the range 0. 07-0.7 mg/ml. Polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate and MALDI-TOF spectra showed that the enzyme is composed of subunits of identical size of 100+/-5 kDa, forming an octameric structure. The N-terminus of the enzyme was found to be blocked. Sequences of tryptic and chymotryptic peptides of the subunit coincide with the proposed amino acid sequence as deduced from the gene from the EMBL library. The Pfk-1 coding sequence of S. pombe was transformed into a Pfk-1 double deletion mutants of Saccharomyces cerevisiae resulting in glucose-positive cells with enzyme activity in the crude cell extract. The kinetic analysis revealed less cooperativity to fructose 6-phosphate (n(H)=1.6) and less inhibition by ATP as compared to the enzyme from baker's yeast. Fructose 2,6-bisphosphate (in micromolar range) and AMP (in millimolar range) were found to overcome ATP inhibition and to increase the affinity to fructose 6-phosphate.

Adenosine Monophosphate↗

PubMed1998

Prostate-specific antigen forms complexes with human alpha 2-macroglobulin and binds to the alpha 2-macroglobulin receptor/LDL receptor-related protein.

PURPOSE: To investigate the binding of the prostate-specific antigen (PSA) to human alpha 2-macroglobulin (alpha 2-M) and to alpha 1-antichymotrypsin (ACT). MATERIALS AND METHODS: Binding analysis was evaluated by electrophoresis, Western-blotting, enzyme-linked immunosorption assay (ELISA) and size exclusion chromatography. Quantification of PSA and of different forms of alpha 2-M was performed using commercial test kits. The cleavage site of PSA in alpha 2-M was analyzed by SDS-PAGE and microsequencing. RESULTS: Binding of PSA to alpha 2-M is initiated by the cleavage of the peptide bond between amino acids Tyr 686 and Glu 687 of the bait region indicating a chymotrypsin-like activity of the PSA. The PSA's proteolytic cleavage triggers the transformation of alpha 2-M as detected by conformation-specific monoclonal antibodies. Kinetic analysis revealed faster binding of PSA to alpha 2-M than to ACT. The PSA bound to alpha 2-M is caged by the inhibitor and thus escapes detection by antibodies. This results in an incorrect calculation of the level of PSA when released from prostate into the blood. Complexes of PSA-alpha 2-M and PSA-ACT were found to bind to the alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2-M-R/LRP) which may be the clearance receptor for PSA. CONCLUSIONS: Quantifying free PSA and PSA-ACT complexes, as routinely done in managing prostate-associated diseases, does not represent the total secretion capacity of the prostate. The proteinase inhibitor alpha 2-M has to be considered as a main contributor to PSA complex formation in the blood.

Amino Acid Sequence↗

PubMed1998

Ribose-5-phosphate isomerase from Saccharomyces cerevisiae: purification and molecular analysis of the enzyme.

Purification and molecular analysis of ribose-5-phosphate isomerase (EC 5.3.1.6) from Saccharomyces cerevisiae is described first time. The enzyme was enriched from a haploid deletion mutant containing the wild-type gene on a multicopy plasmid elaborating the following steps: ammonium sulphate precipitation, interfacial salting out on Sepharose 6B, high performance liquid chromatography on Fractogel EMD DEAE and on Resource Phenyl. The enzyme activity was found to be rather unstable possibly caused by removal of stabilizing cofactors or proteins during the purification procedure. The purified enzyme showed a hyperbolic dependence on the substrate ribose-5-phosphate with a K(m)-value of 1.6 +/- 0.3 mmol/l. For the native enzyme a molecular mass of 115 +/- 10 kDa was determined as found by saccharose density gradient centrifugation, sedimentation equilibrium analysis, size exclusion chromatography and polyacrylamide gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blotting revealed one band with a molecular mass of 31 +/- 2 kDa. Thus, the native enzyme is composed of four subunits of identical size. The molecular mass of the subunit and the identified N-terminal sequence of 33 amino acids fits well the 258 amino acid protein encoded by the S. cerevisiae RKI open reading frame, which was characterized previously only by increasing specific activities of ribose-5-phosphate isomerase in cells after cloning the gene. On the basis of the conserved amino acids an alignment of the amino acid sequence of ribose-5-phosphate isomerase from yeast with those of the enzyme from mouse, spinach and Escherichia coli is presented.

Aldose-Ketose Isomerases↗

PubMed1997

Purification and characterization of phosphofructokinase from the yeast Kluyveromyces lactis.

Phosphofructokinase from Kluyveromyces lactis was purified by 180-fold enrichment, elaborating the following steps: cell disruption, polyethylene glycol precipitation, affinity chromatography, size exclusion chromatography on Sepharose 6B and on Bio-Sil SEC 400 and ion exchange chromatography. The homogeneous enzyme exhibits a molecular mass of 845 +/- 20 kDa as determined by sedimentation equilibrium measurements and a specific activity of 100 units/mg protein. The apparent sedimentation coefficient was found to be s20,c = 20.7 +/- 0.6 S and no significant dependence on the protein concentration was observed in a range from 0.2 to 8 mg protein/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to molecular masses of 119 +/- 5 kDa and 102 +/- 5 kDa, respectively. Thus, the enzyme assembles as octamer composed of two types of subunits. From Western blot analysis applying subunit-specific monoclonal antibodies raised against Saccharomyces cerevisiae phosphofructokinase and from the determination of the N-terminal amino acid sequence, the conclusion was drawn that the 102 kDa-subunit corresponds to the beta-subunit of the S. cerevisiae enzyme. In contrast to bakers' yeast phosphofructokinase, the K. lactis enzyme exhibits no cooperativity with respect to the substrate fructose 6-phosphate. Both activators AMP and fructose 2,6-bisphosphate decrease the Michaelis constant with respect to this substrate. The enzyme from K. lactis is also inhibited by ATP. Fructose 2,6-bisphosphate or AMP diminish the ATP-inhibition. In contrast to the phosphofructokinase from S. cerevisiae, where fructose 2,6-bisphosphate turned out to be more efficient than AMP, both activators exert similar effects on the K. lactis enzyme.

Enzyme Activation↗

PubMed1996

Improved purification of ribulose 5-phosphate 3-epimerase from Saccharomyces cerevisiae and characterization of the enzyme.

D-Ribulose 5-phosphate 3-epimerase from Saccharomyces cerevisiae was purified to homogeneity by 1970-fold enrichment elaborating the following steps: disruption of fresh cells, polyethylene glycol precipitation, ion exchange chromatography, heat-treatment, size exclusion chromatography on Sephadex G-75 and Bio-Sil SEC 125 and hydrophobic interaction chromatography. A molecular mass of 50 +/- 4 kDa was determined for the native enzyme by sedimentation equilibrium experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single band with 26 kDa which has been characterized as an individual polypeptide chain. Thus, the enzyme is a dimer composed of two identical subunits. The specific activity of the purified enzyme with 7700 units/mg protein was found to be 30-fold higher than described in previous papers. The enzyme shows a hyperbolic dependence of the catalytic activity towards ribulose 5-phosphate with a KM-value of 1.5 mmol/l. The N-terminal amino acid sequence analysis of the native enzyme and of several peptides obtained by chemical and proteolytic fragmentation provided a part of the primary structure which fits to the primary structure deduced from the DNA sequence.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of an adenotin-like adenosine binding protein from human platelets.

A low-affinity adenosine binding protein (adenotin) was purified from human platelet membranes by a four-step procedure. Purification was achieved after extraction from human platelet membranes with 0.3% 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Further purification included Sepharose CL6B gel filtration, DEAE-Sepharose CL6B, and hydroxylapatite chromatography. The protein was purified 884-fold to homogeneity with a 25% yield of binding activity from the membranes. 5'-[8(n)-3H]-N-ethylcarboxamidoadenosine ([3H]NECA) binds to the purified protein with a KD of 155 (144-167) nmol/l and a Bmax of 1.85 +/- 0.10 nmol/mg of protein. Sodium dodecylsulfate polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa. The 2-chloro-substituted adenosine analogs 2-chloro-5'-N-methylcarboxamidoadenosine (CIMECA) and 2-chloro-5'-N-ethylcarboxamidoadenosine (CINECA) were identified as new high affinity ligands of the purified protein showing Ki values of 18 nmol/l and 28 nmol/l, respectively. The low-affinity adenosine binding protein showed a pharmacological profile as follows: CIMECA > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine (CIA) > 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21,680) > R-N6-phenylisopropyl-adenosine (R-PIA). Amino-terminal sequence analysis revealed homologies to endoplasmin, glucose regulated protein (GRP94), tumor rejection antigen precursor (GP96), and some stress related proteins.

Adenosine↗

PubMed1993

Limited proteolysis of yeast phosphofructokinase. Sequence locations of cleavage sites created by the actions of different proteinases.

Purified phosphofructokinase 1 from baker's yeast (Saccharomyces cerevisiae) was subjected to proteolysis by thermolysin, endoproteinase lys-C, trypsin and chymotrypsin under defined solvent conditions. In the absence of substrates and allosteric effectors, the catalytic activity of phosphofructokinase rapidly disappeared in the presence of each proteolytic enzyme. The presence of a saturating concentration of ATP protected phosphofructokinase activity from proteolytic inactivation while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate provided transient activation during proteolysis. Changes in the quaternary structure of phosphofructokinase resulting from proteolysis were estimated by high performance size exclusion chromatography while changes in the primary sequence of the individual alpha and beta polypeptide chains were estimated by polyacrylamide-gel electrophoresis in sodium dodecylsulfate. The site(s) of proteolytic cleavage were identified by N-terminal sequence analysis of resolved electrophoretic components. The presence of ATP protects phosphofructokinase from thermolysin proteolysis, while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate restricts proteolysis to one site in each polypeptide chain involving the peptide bonds preceding Leu199 in the alpha chain and Leu192 in the beta chain. The truncated phosphofructokinase retains its octameric structure. The presence of ATP largely restricts endoproteinase lys-C proteolysis to a single site in the alpha chain involving the peptide bond preceding Val914. This cleavage results in the dissociation of the octameric form of phosphofructokinase into two tetramers. The presence of ATP restricts both trypsin and chymotrypsin proteolysis to the N-terminal and C-terminal regions described above, resulting in the preferential stabilization of the tetrameric form of phosphofructokinase. It would appear that the first 200 and last 80 residues which are unique to the sequence of the yeast phosphofructokinase are not directly involved in catalysis or its allosteric regulation. However, the last 80 residues of the alpha polypeptide chain do appear to stabilize an octameric structure which is unique to yeast phosphofructokinase.

Adenosine Monophosphate↗

PubMed1991

Yeast phosphofructokinase: studies on thiol reactivity of a cross-linked enzyme form.

Cross-linked yeast phosphofructokinase exhibiting the basic regulatory properties of the unmodified enzyme but no cooperativity with respect to the substrate fructose 6-phosphate was subjected to stopped-flow thiol titration in order to study the influence of allosteric effectors on the level of protein conformation. As found for native phosphofructokinase, the cross-linked enzyme revealed at least two classes of cysteinyl residues which can be distinguished by their reactivity towards 5,5'-dithiobis-(2-nitrobenzoic acid). In spite of the restricted conformational flexibility caused by the intramolecular cross-linking, several fructose phosphates and AMP were capable of diminishing the apparent first-order rate constant (k) of modification of the fast reacting thiol groups by Ellman's reagent. In the presence of ATP, a drastic decrease of the k-value by more than one order of magnitude became apparent. The data are appropriate to support the hypothesis of the existence of multiple conformational determinants in octameric yeast phosphofructokinase and contribute to the understanding of the extremely different stability of the enzyme in the presence of either fructose 6-phosphate or ATP with respect to proteolytic degradation.

Adenosine Triphosphate↗

PubMed1990

Regulation of phosphofructokinase at physiological concentration of enzyme studied by stopped-flow measurements.

Stopped-flow measurements have been carried out to study some basic allosteric properties of muscle and yeast phosphofructokinase at physiological concentration of enzyme. An important increase in the affinity for fructose-6-P accompanied by an intense decrease in the ATP inhibition was observed with the muscle enzyme, which also became insensitive to fructose-2,6-P2 under these conditions. Yeast phosphofructokinase exhibited a significant diminution in the inhibition by ATP, although with no apparent change in the affinity for fructose-6-P. These results provide strong support in favor of the dependence of the allosteric regulation of phosphofructokinase on its concentration in vivo.

Adenosine Triphosphate↗

PubMed1990

Yeast phosphofructokinase: pre-steady-state and stationary kinetic studies on a cross-linked enzyme form.

A cross-linked form of yeast phosphofructokinase in which up to four of the subunits of the octameric molecule were found covalently linked by dimethyl suberimidate exhibits no cooperativity with respect to fructose 6-phosphate and is only weakly inhibited by ATP. The modified enzyme is activated by AMP and fructose 2,6-bisphosphate. Both effectors abolish ATP inhibition even at low concentration of fructose 6-phosphate and increase the affinity of modified phosphofructokinase to fructose 6-phosphate but are without significant effect on the respective maximum activity. The corresponding kinetic patterns exhibit similarity to those of the native enzyme at high concentration of fructose 6-phosphate. In comparison to native phosphofructo-kinase the extent of activation by AMP and fructose 2,6-bisphosphate is of minor amount. As shown for native phosphofructokinase, the enzyme which had been cross-linked in the absence of any effector shows a lag phase of product formation. This initial transient phase completely disappears if the enzyme is modified in the presence of fructose 6-phosphate, fructose 1,6-bisphosphate, and fructose 2,6-bisphosphate, respectively. The same result is obtained by preincubation of the enzyme cross-linked in the absence of any effector with each of these fructose phosphates. The kinetic properties of the modified enzyme indicate that cooperativity with respect to fructose 6-phosphate is not a prerequisite for the allosteric modulation of enzyme activity by AMP and fructose 2,6-bisphosphate and support the idea of multiple conformational determinants in yeast phosphofructokinase. The results suggest that moderate intramolecular cross-linking can provide a simple experimental tool to stabilize different conformational states of an enzyme.

Adenosine Monophosphate↗

PubMed1990

Phosphofructokinase from baker's yeast: studies on the initial kinetics and the fast reacting thiol groups of a proteolytically modified active enzyme form.

The initial kinetics as well as the reactivity of the fast reacting thiol groups of a tetrameric form of phosphofructokinase from baker's yeast (called 12 S-enzyme), obtained by limited proteolysis in the presence of ATP were studied by the stopped-flow technique. Before attaining the steady state, the reaction shows a lag phase in the product formation, the duration of which decreases with increasing enzyme concentration. The lag phase disappears after preincubation of the enzyme with either fructose 6-phosphate, fructose 1,6-bisphosphate or fructose 2,6-bisphosphate. The occurrence of an initial transient phase suggests that the enzyme converts from a state of low activity into a highly active one after starting the reaction. The modified enzyme was found to contain two fast reacting cysteinyl residues with respect to their reactivity towards 5,5'-dithiobis(2-nitrobenzoic acid). Fructose 6-phosphate, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate, respectively, decrease the reactivity of this class of thiol groups but not the total number of titrable cysteins. This result supports the hypothesis of a conformational change as a consequence of the effector binding.

Kinetics↗

PubMed1989

Phosphofructokinase from baker's yeast: kinetic properties of a proteolytically modified enzyme.

A tetrameric enzyme form of phosphofructokinase from yeast (called 12 S-enzyme), formed by limited proteolysis of the octameric enzyme in the presence of ATP and by subsequent dissociation in two half-molecules shows sigmoidal kinetics with respect to fructose 6-phosphate and inhibition by ATP. Similar to the native phosphofructokinase, the modified enzyme is also efficiently activated by AMP and fructose 2,6-bisphosphate. Both activators increase the affinity for the substrate fructose 6-phosphate and the respective maximum activity. In contrast to the native phosphofructokinase, however, both AMP and fructose 2,6-bisphosphate change the sigmoidal fructose 6-phosphate velocity curve into a hyperbolic one. AMP and fructose 2,6-bisphosphate decrease the ATP inhibition, probably by modulating the affinity of the allosteric sites to ATP.

Adenosine Monophosphate↗

PubMed1988

Studies on the fast reacting thiol groups in phosphofructokinase from baker's yeast.

The fast reacting thiol groups of yeast phosphofructokinase were studied by means of stopped-flow measurements. The enzyme was found to contain four very fast reacting cysteinyl residues determined by their reactivity towards 5,5'-dithiobis(2-nitrobenzoic acid). A second class of eight thiol groups reveals an apparent first order rate constant which is three orders of magnitude lower than the rate constant of the first one. Due to the extreme high reactivity of the first class of cysteinyl residues partial oxidation was already observed under aerobic conditions. Fructose 6-phosphate, fructose 1.6-bisphosphate, and fructose 2.6-bisphosphate, respectively, decrease the reactivity of the first class of thiol groups but not the total number of the accessible cysteins. This result is discussed with regard to conformational changes of the enzyme after binding of the sugar phosphates.

Dithionitrobenzoic Acid↗

PubMed1986

Interaction of enzymes involved in triosephosphate metabolism. Comparison of yeast and rabbit muscle cytoplasmic systems.

The affinity of baker's yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphate aldolase towards the metabolically related enzymes phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase was tested by using a fluorescence-probe technique with fluorescein isothiocyanate attached covalently to the enzymes. The dissociation constants of the enzyme-enzyme complexes, as well as the rate constants of association and dissociation, were determined. Data were compared with the parameters derived from a mammalian (rabbit muscle) system, known from the literature and determined under the same conditions (pH 7.5 or 8.5 in 0.05 M Tris/HCl buffer at 20 degrees C). The comparison reveals similarities in the supramolecular organization of these cytoplasmic enzymes in phylogenetically distant species. Moreover, the fact that in vitro hybrid complexes are formed of stability comparable to that of non-hybrid complexes indicates that this ancient characteristic is probably conserved during evolution. A possible regulatory mechanism is presented, based on the dynamic competition, with each other, of the enzymes involved in triosephosphate metabolism.

Animals↗

PubMed1986

Study on the initial kinetics of yeast phosphofructokinase by stopped-flow measurements.

The initial kinetics of yeast phosphofructokinase was studied by stopped-flow measurements over an enzyme concentration range from 0.5 mg/ml to 0.01 mg/ml. Before attaining the steady state the reaction showed a lag phase in the product formation, the duration of which was found to decrease with increasing enzyme concentration. The lag phase disappeared after preincubation of the enzyme for at least five minutes with either fructose 6-phosphate, fructose 1,6-bisphosphate or fructose 2,6-bisphosphate. Preincubation of the enzyme with either AMP or ADP resulted in a reduction of this phase, while ATP was without effect. Simultaneous addition of fructose 1,6-bisphosphate to the reaction mixture of the enzyme causes a significant shortening of the transient phase, whereas micromolar concentrations of fructose 2,6-bisphosphate are capable of abolishing the lag phase completely. The occurrence of an initial transient phase suggests that the enzyme after starting the reaction converts from a state of low activity to one of high activity. This conversion mainly depends on the concentration of fructose 1,6-bisphosphate generated in the course of the reaction. In addition an association reaction of the enzyme seems to be involved in the process of conversion of the phosphofructokinase during the initial transient phase.

Adenosine Diphosphate↗