Hans Werner Hofer

Phosphorylation of phosphofructokinase from skeletal muscle: Correlations between phosphorylation and muscle function

Abstract The specific radioactivity of [ 32 P]-phosphate incorporated into muscle phosphofructokinase was in equilibrium with the specific radioactivity of the γ-phosphate group of ATP. The incorporation was independent of the presence of cycloheximide. The total content of covalently bound phosphate in phosphofructokinase was correlated with the functional state of the muscle from which the enzyme was purified. Muscle dissected post mortem led to phosphofructokinase containing less than 2 phosphate groups per tetramer. Muscle dissected in vivo gave phosphofructokinase with 4 phosphates per tetramer when kept at rest and 8 phosphates per tetramer when stimulated to contract.

Phosphorylation of phosphofructokinase by protein kinase C changes the allosteric properties of the enzyme

The Ca2+- and phospholipid-dependent protein kinase C from rat brain phosphorylates rabbit muscle phosphofructokinase at the same trypsin-labile site as cyclic AMP-dependent protein kinase. However, protein kinase C also effectively phosphorylates one or more separate sites. Incubation of phosphofructokinase in the presence of protein kinase C, phospholipids, Ca2+, and ATP appears to affect the allosteric properties of phosphofructokinase by shifting the fructose 6-phosphate saturation curve to lower substrate concentrations in a time-dependent manner and decreasing cooperativity of the enzyme.

Epidermal Growth Factor-, Transforming Growth Factor-β-, Retinoic Acid- and 1,25-Dihydroxyvitamin D3-Regulated Expression of the Novel Protein PTPIP51 in Keratinocytes

The novel protein PTPIP51 (protein tyrosine phosphatase-interacting protein 51), which has been found to interact with protein tyrosine phosphatases of the PTP1B/TcPTP subfamily, is expressed in all suprabasal layers of human epidermis. Hence, a human keratinocyte cell line (HaCaT) grown on culture slides was used as a simplified model system to study the influence of hormonal agents on the regulation of PTPIP51 expression. Results were obtained by immunocytochemistry and subsequent statistical analysis. Additionally, immunoblotting was performed to detect the possible occurrence of distinct molecular weight forms as described previously. Subcellular localization of PTPIP51 protein was analyzed by specific staining of cellular organelles. HaCaT cells were subjected to treatment with factors that are crucial for the regulation of proliferation and differentiation of keratinocytes in human epidermis: epidermal growth factor (EGF), transforming growth factor-β(TGF-β), retinoic acid (RA) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Epidermal growth factor receptor (EGFR) expressed in HaCaT cells was inhibited by PD153035. Only about 35% of untreated HaCaT cells were immunoreactive for the PTPIP51 protein. Whereas cells treated with increasing concentrations of 1,25 (OH)2D3 showed a stepwise numerical increase of PTPIP51-positive cells, treatment with RA did not influence the number of PTPIP51-positive cells except when supraphysiological concentrations were applied. Concentration-dependent increase of cells stained positive for PTPIP51 was also observed when HaCaT cells were subjected to EGF treatment. Additional treatment of these cells with PD153035 led to a slight decrease in the fraction of PTPIP51-positive cells, which was not statistically significant. Immunoblotting results suggest a specific pattern of different molecular weight forms of PTPIP51 being expressed in HaCaT cells. Subcellular analysis revealed an association of the protein with mitochondria in nonconfluent cells, whereas confluent cells lack such correlation. The intracellular distribution of PTPIP51 resembled the localization of its interacting partner TcPTP. Furthermore, PTPIP51 was found to be present in both the nucleus and cytoplasm of HaCaT cells. In summary, the results indicate a possible association of PTPIP51 expression with differentiation as well as with apoptosis of keratinocytes.

Allosteric and non-allosteric phosphofructokinases from Lactobacilli purification and properties of phosphofructokinases from L. Plantarum and L. Acidophilus

Phosphofructokinase (ATP : D-fructose-6-phosphate 1 phosphotransferase, EC 2.7.1.11) from two different lactobacilli, Lactobacillus plantarum and Lactobacillus acidophilus were isolated and purified. Both enzymes have a molecular weight of 154 000 and consist of four subunits of identical size. Antisera from sheep immunized against the purified phosphofructokinase from L. plantarum showed immunologic cross reaction with the enzyme from L. acidophilus. In spite of the close molecular relationship indicated by the immunologic cross reaction, the kinetic behaviour of the two enzymes was strikingly different. Phosphofructokinase from L. plantarum showed pure Michaelis-Menten behaviour. Phosphofructokinase from L. acidophilus, however, showed sigmoidal substrate saturation curves for fructose 6-phosphate in the presence of slightly alkaline pH and high ATP concentrations; it was activated by fructose 1,6-biphosphate and inhibited by ADP. The results indicate that even enzymes which are structurally very similar may differ greatly with respect to their kinetic and regulatory properties and suggest that allosteric and non-allosteric phosphofructokinases have the same origin in evolution.

Identification of the phosphorylated sites of phosphofructokinase from skeletal muscle after in vivo and in vitro phosphorylation

Phosphofructokinase from mice muscle was radioactively labelled either in vivo by the injection of [32P]-phosphate or in vitro by the incubation with cAMP-dependent protein kinase and [γ-32P]-ATP. Two labelled peptides were obtained after tryptic digestion in either case showing that at least two sites were phosphorylated. Independent of the labelling method, the labelled peptides showed an analogous pattern on the peptide maps, indicating that both methods led to the phosphorylation of the same sites.

Interaction of immobilized phosphofructokinase with soluble muscle proteins

Selected glycolytic enzymes (including phosphoglucose isomerase, aldolase, glyceraldehyde phosphate dehydrogenase, enolase, pyruvate kinase and lactate dehydrogenase), as well as glycogen phosphorylase, creatine kinase, and adenylate kinase, bound to phosphofructokinase immobilized on an agarose gel. The affinity of phosphofructokinase to these various proteins differed, with phosphorylase exhibiting the strongest binding. Binding was reversed either by: (1) elution with high-ionic-strength buffer (0.4 M KCl); (2) the addition of a 5-10 mM concentration of ATP; or (3) high concentrations of fructose 6-phosphate (5 mM).

Protein phosphotyrosine phosphatases in Ascaris suum muscle

Two forms of protein tyrosine phosphatases were partially purified from the musculo-cutaneous layer of Ascaris suum. A 50-55-kDa soluble form of the phosphatase cross-reacted with antisera raised against human PTP-1B and TC-PTP. Like the enzyme of human origin the phosphatase from Ascaris exhibited a preference for anionic substrates (tyrosine-phosphorylated carboxymethylated and maleylated lysozyme) and was inhibited by micromolar concentrations of vanadate, molybdate, Zn2+, heparin, and poly(Glu4Tyr). As revealed by immuno-cytochemistry, the phosphatase was mainly localized and appeared equally distributed in the cytoplasm, apart from the myofibrils, possibly in loose association with cytoskeletal elements. A second tyrosine phosphatase of 180 kDa molecular mass was mainly found in detergent extracts from a microsomal fraction. It showed no cross-reactivity with antisera raised against soluble mammalian phosphatases and dephosphorylated a basic substrate (Tyr-phosphorylated myelin basic protein). It was resistant to common inhibitors of mammalian tyrosine phosphatases except Zn2+ and thiol reagents.

Perfusion of the psoas muscle of the rabbit. Metabolism of a homogeneous muscle composed of "fast glycolytic" fibres.

A method was developed for the hemoglobin-free perfusion of the rabbit psoas muscle in situ. This muscle consists almost exclusively of fast-twitch (type IIb) glycolytic fibres and was therefore used as a model of a homogeneous muscle of the glycolytic, metabolic type.

Interaction of phosphofructokinase with antibodies: Kinetic properties of phosphofructokinase in complexes with antibodies

The allosteric properties of phosphofructokinase (EC 2.7.1.11) from rabbit muscle are influenced by enzyme concentration, most probably due to changes in the association state of the enzyme. In this study, the behaviour of dispersed pre-cipitates of phosphofructolinase as produced by treatment with antibodies has been investigated. The enzyme is not capable of rapid dissociation in the precipitated state as is confirmed by the lack of inactivation upon dilution and by the absence of shifts in substrate saturation curves as measured in the presence of different concentrations of the enzyme. The Hill coefficient of phosphofructokinase is decreased from 1.96 to 1.04 by antibody treatment. The V at neutral pH is increased 3-fold while the K0.5 for fructose 6-phosphate is reduced significantly. On the other hand, antibody-treated phosphofructokinase retains its sensitivity to allosteric activation by glucose 1,6-bisphosphate in the rpesence of high ATP concentrations.

Phorbol ester-dependent regulation of nuclear protein tyrosine phosphatase in situ

Incubation of splenal lymphocytes with phorbol ester (50 nM PMA) influenced nuclear protein tyrosine phosphatase activity in a time-dependent manner. The activity was elevated after a short incubation (90 s) but was decreased in comparison to untreated cells after 30 and 120 min of incubation. The presence of H7 suppressed the changes. Okadaic acid, an inhibitor of protein phosphatases 2A and 1, led to a similar increase in the activity of nuclear protein tyrosine phosphatase during short-term incubations as phorbol ester but eliminated the subsequent activity decrease. Immunoblots revealed that the same amounts of two forms (49,000 and 60,000 M(r)) of protein tyrosine phosphatases were present in the nuclei from phorbol ester-stimulated and non-stimulated cells. The 60,000 M(r) form co-migrated with a phosphotyrosine-containing protein. The amount of phosphotyrosine was increased in comparison to control cells after 30 min of phorbol ester treatment.