Binding of murine monoclonal antibodies to the active and inactive configurations of aequorin

Murine monoclonal IgG1 antibodies (MAb), designated Aq-11 and Aq-12, were prepared against the photoprotein aequorin from jelly fish. Aequorin is a calcium-sensitive photoprotein which consists of a single polypeptide chain, apoaequorin, and a functional chromophore, coelenterazine. Native aequorin consists of two species with molecular masses of 25 and 23.5 kDa. MAb Aq-12 was found by immunoblot analysis to bind specifically to the 25 kDa species, while MAb Aq-11 reacted with the 23.5 kDa protein. Activation of apoaequorin with coelenterazine was associated with a shift of the 23.5 kDa molecule to the 25 kDa species. In contrast, treatment with calcium ions induced a shift back to the 23.5 kDa form. These changes between the active and inactive forms were identified by reactivity with MAbs Aq-11 and Aq-12. The results thus indicate that these MAbs should be useful in monitoring activation of this photoprotein.     

GSK-3beta regulation in skeletal muscles by adrenaline and insulin: evidence that PKA and PKB regulate different pools of GSK-3

We have recently shown that while adrenaline alone has no effect on the activation of Protein Kinase B (PKB) in rat soleus muscle, it greatly potentiates the effects of insulin (Brennesvik et al., Cellular Signalling 17: 1551-1559, 2005). In the current study we went on to investigate whether this was paralleled by a similar effect on GSK-3, which is a major PKB target. Surprisingly adrenaline alone increased phosphorylation of GSK-3beta Ser9 and GSK-3alpha Ser21 and adrenaline's effects were additive with those of insulin but did not synergistically potentiate insulin action. Dibutyryl-cAMP (5 mM) and the PKA specific cAMP analogue N6-Benzoyl-cAMP (2 mM) increased GSK-3beta Ser9 phosphorylation, whereas the Epac specific cAMP analogue 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (1 mM) did not. Wortmannin (PI 3-kinase inhibitor; 1 microM) blocked insulin-stimulated GSK-3 phosphorylation completely, but adrenaline increased GSK-3beta Ser9 phosphorylation in the presence of wortmannin. The PKA inhibitor H89 (50 microM) reduced adrenaline-stimulated GSK-3beta Ser9 phosphorylation but did not influence the effects of insulin. Insulin-stimulated GSK-3 Ser9 phosphorylation was paralleled by decreased glycogen synthase phosphorylation at the sites phosphorylated by GSK-3 as expected. However, adrenaline-stimulated GSK-3 Ser9 phosphorylation was paralleled by increased glycogen synthase phosphorylation indicating this pool of GSK-3 may not be directly involved in phosphorylation of glycogen synthase. Our results indicate the existence of at least two distinct pools of GSK-3beta in soleus muscle, one phosphorylated by PKA and another by PKB. Further, we hypothesise that each of these pools is involved in the control of different cellular processes.     

Differences in reactivity of antibodies to active versus inactive PLTP significantly impacts PLTP measurement

Due to conflicting reports concerning the relationship between phospholipid transfer protein (PLTP) activity and mass in plasma, the protein concentration and activity of PLTP were assessed in fractions isolated by fast protein liquid chromatography from the plasma of healthy normolipidemic individuals. Using both polyclonal and monoclonal antibodies, PLTP was identified by Western blot analysis after both SDS and non-denaturing gradient gel electrophoresis, and quantitated by dot blot. PLTP activity was determined using a labeled vesicle/HDL assay. PLTP mass corresponded substantially with the activity distribution using the polyclonal antibody on dot blot with some inactive PLTP being present. However, the monoclonal antibody preferentially reacted with inactive PLTP, primarily associated with LDL and large HDL, overestimating inactive PLTP. Western blot analysis of non-denaturing gradient gels, using the polyclonal antibody, indicated that active PLTP was associated with numerous discrete HDL subpopulations (7.6-12.0 nm) with the major portion being 9-12 nm. Inactive PLTP was associated with particles of 12 to >17 nm. The monoclonal antibody demonstrated a different pattern of reactivity on gradient gels, showing strong reactivity with the inactive PLTP in particles of 12 to >17 nm, but less reactivity with particles of 7.6-12 nm. The differences in reactivities of antibodies for active versus inactive PLTP can account for some of the discrepancies reported in the literature regarding the relationship between PLTP mass and activity.     

Protein structural changes associated with active and inactive states of hydrogenase from Thiocapsa roseopersicina

Electrophoretic and isoelectrofocusing behavior of the hydrogenase from Thiocapsa roseopersicina under various conditions revealed remarkable properties of this enzyme: there are two active forms which differ in their molecular masses as well as in oxygen sensitivity; the apparent molecular masses of the active hydrogenase forms (90 and 49 kDa) differ considerably from those in the inactive state (64, 34, and 15 kDa); the active forms and some of the inactivated ones can be transformed into each other reversibly; urea can unfold the 64 and 34 kDa proteins but not the 49 kDa form at room temperature; the pI of these proteins are different in the presence of urea. The results suggest large rearrangements in the hydrogenase protein structure which are associated with the enzymatically active and inactive states. It is concluded that reversible formation of disulfide bonds cannot be the major cause for maintaining the enzyme conformation. Strong hydrophobic interactions are suggested to be primarily responsible for the structural stability and for the rearrangements.     

Monoclonal antibodies can discriminate between some active and inactive forms of leukocyte interferon

Antiviral activity of recombinant human leukocyte A interferon was inactivated by heating at 65 degrees C or by reduction of disulfide bonds. The specific immunoreactivity, as measured by radioimmunoassays measuring binding to monoclonal antibodies, decreased concomitantly with the antiviral activity. Although the monoclonal antibodies did bind to inactivated interferon, their binding affinity to inactivated interferon was in general very much lower than their binding affinity to active interferon. Therefore, this immunoassay could replace the antiviral assay for detection of biologically active interferon. In addition, most of these antibodies should be especially useful for purification of the interferons since they discriminate between the native active and inactive denatured species. Screening for such antibodies is convenient and simple. The general use of antibodies that preferentially interact with native molecules provides a powerful new principle for choosing monoclonal antibodies with extraordinary potential in assay and purification.     

An inactive pool of GSK-3 at the leading edge of growth cones is implicated in Semaphorin 3A signaling

Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase that has been implicated in several aspects in embryonic development and several growth factor signaling cascades. We now report that an inactive phosphorylated pool of the enzyme colocalizes with F-actin in both neuronal and nonneuronal cells. Semaphorin 3A (Sema 3A), a molecule that inhibits axonal growth, activates GSK-3 at the leading edge of neuronal growth cones and in Sema 3A-responsive human breast cancer cells, suggesting that GSK-3 activity might play a role in coupling Sema 3A signaling to changes in cell motility. We show that three different GSK-3 antagonists (LiCl, SB-216763, and SB-415286) can inhibit the growth cone collapse response induced by Sema 3A. These studies reveal a novel compartmentalization of inactive GSK-3 in cells and demonstrate for the first time a requirement for GSK-3 activity in the Sema 3A signal transduction pathway.     

GSK-3-associated signaling is crucial to virus infection of cells

Signal transduction pathways play important roles in virus infection, replication, and associated pathogenesis. Some of the best understood cell signaling networks are crucial to virus infections such the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and the WNT/β-catenin pathways. Glycogen synthase kinase-3 (GSK-3) is a lesser known signaling molecule in the field of virus research. Interestingly, GSK-3 forms the crux of multiple cell signaling pathways. However, recent studies indicate that GSK-3 may perform key roles in the response to viral infection, replication and pathogenesis. The effects of activated or inactivated forms of GSK-3 on virus infection are still not yet clearly understood phenomenon. The comprehension of the molecular mechanisms underlying the regulation of GSK-3-associated signaling pathways in terms of different stages of virus replication could be important not only to understand the pathogenesis of virus, but also possibly leading to new therapeutic targets. This review will focus on recent advances in understanding the roles of GSK-3 on viral replication, pathogenesis and the immune responses.     

Structural characterization of the GSK-3beta active site using selective and non-selective ATP-mimetic inhibitors

GSK-3beta is a regulatory serine/threonine kinase with a plethora of cellular targets. Consequently, selective small molecule inhibitors of GSK-3beta may have a variety of therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes and cancer. In order to characterize the active site of GSK-3beta, we determined crystal structures of unphosphorylated GSK-3beta in complex with selective and non-selective ATP-mimetic inhibitors. Analysis of the inhibitors' interactions with GSK-3beta in the structures reveals how the enzyme can accommodate a number of diverse molecular scaffolds. In addition, a conserved water molecule near Thr138 is identified that can serve a functional role in inhibitor binding. Finally, a comparison of the interactions made by selective and non-selective inhibitors highlights residues on the edge of the ATP binding-site that can be used to obtain inhibitor selectivity. Information gained from these structures provides a promising route for the design of second-generation GSK-3beta inhibitors.     

Binding of active and inactive hemolytic factor of sea anemone nematocyst venom to red blood cells

Sea anemone nematocyst venom, in the presence of Ca²⁺, induced the lysis of red blood cells after an induction period. In the absence of Ca²⁺, however, no lysis occurred, but the hemolytic factor was shown to bind to the cells. This binding was shown to be requisite for the Ca²⁺ dependent lysis to ensue. After freeze thawing, the venom proteins responsible for lysis lost their hemolytic activity, yet still bound to the cells. The freezethawed inactivated venom competitively blocked hemolysis by active venom.     

Differential stability of proteolytically active and inactive recombinant metalloproteinase in Chinese hamster ovary cells

Stability of heterologous protein expression during production is critical for regulatory approval of vaccine and therapeutic products. Leishmania GP63, a zinc metalloproteinase that is a potential vaccine candidate, has been expressed on the surface of Chinese hamster ovary (CHO) cells. Flow cytometry was used to follow the stability of GP63 expression. Expression of proteolytically active GP63 (GP63WT) was unstable whether or not methotrexate (MTX) selection was maintained. In contrast, expression of an active site mutant (GP63E265D) was stable under MTX selection. In the absence of selection, the decline in GP63E265D expression was more gradual than the loss of GP63WT expression. Different molecular mechanisms accounted for these losses and resulted in higher growth rate nonproducer populations. A dynamic population model was used to calculate the conversion rates of GP63WT producers to nonproducers. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 594-600, 1997.     

Intracellular distribution of active and inactive transglutaminase in stimulated cultured C6 glioma cells

The cellular distribution of active and inactive transglutaminase (TGase) was studied in C6 glioma cells before and during stimulation by a serum-containing medium. The activity of the enzyme was determined in the soluble and insoluble fractions obtained by freezing and thawing the cells, followed by centrifugation at 12,000g for 5 min. In the soluble fractions, the activity of TGase decreased 2.5 h post-stimulation and increased after 5 and 8 h. In the corresponding insoluble fractions, no significant changes in the activity of the enzyme were noted up to 8 h after stimulating the cells with fresh medium. An immunological approach was next used to determine the quantity of TGase antigen during the stimulation of the cultured glioma cells. In the soluble fraction, the quantity of the antigen decreases significantly at 2.5, 5, and 8 h. In contrast, in the insoluble fraction, a significant increase in TGase antigen was detected 8 h after the addition of fresh medium. Cycloheximide completely inhibited the increase in the quantity of TGase antigen in the insoluble fraction, 8 h post-stimulation, while actinomycin D caused a partial inhibition. Trypsin, neuraminidase, or Sendai viruses increased the activity of TGase significantly, when added to nonstimulated cells. Trypsin had no effect on TGase activity when added to the cells 2 h after stimulation with a serum-containing medium. These findings suggest that an inactive form of the enzyme is present in the insoluble cellular fraction. A model has been proposed to explain the variations in TGase activity, its distribution and translocation during cellular stimulation.     

Cooperative regulation of GSK-3 by muscarinic and PDGF receptors is associated with airway myocyte proliferation

Muscarinic receptors and platelet-derived growth factor (PDGF) receptors synergistically induce proliferation of airway smooth muscle (ASM), but the pathways that regulate these effects are not yet completely identified. We hypothesized that glycogen synthase kinase-3 (GSK-3), a kinase that represses several promitogenic signaling pathways in its unphosphorylated form, is cooperatively inhibited by PDGF and muscarinic receptors in immortalized human ASM cell lines. PDGF or methacholine alone induced rapid GSK-3 phosphorylation. This phosphorylation was sustained only for PDGF; however, methacholine potentiated PDGF-induced sustained GSK-3 phosphorylation. Synergistic effects of methacholine also were observed on PDGF-induced retinoblastoma protein (Rb) phosphorylation and cell proliferation. Suppression of GSK-3 inhibitory function using SB 216763 also augmented PDGF-induced Rb phosphorylation and cell cycle progression; this synergy was similar in magnitude to that seen for methacholine with PDGF. GSK-3 phosphorylation induced by methacholine required PKC, since it was abolished by GF 109203X and G? 6976; however, inhibition of PKC had no effect on cell responses to PDGF. PKC inhibition also specifically abolished the synergistic effect of methacholine on PDGF-induced GSK-3 phosphorylation and cell proliferation. Collectively, these results show that GSK-3 plays a key repressive role in ASM cell proliferation. Moreover, muscarinic receptors mediate PKC-dependent GSK-3 inhibition, and this appears to be a primary mechanism underpinning augmentation of PDGF-induced cell growth.     

Monoclonal antibodies that distinguish between active and inactive forms of human postheparin plasma hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified from human postheparin plasma. Specific monoclonal antibodies (MAbs) were produced that discriminate between active (native) and inactive (denatured) forms of the enzyme. Mice immunized with native H-TGL resulted in MAbs that recognized only the native protein. The antibodies did not react with H-TGL treated with 1% sodium dodecyl sulfate or heated at 60 degrees C. The loss of immunoreactivity with heating correlated directly with the loss of enzyme activity and there was a corresponding increase in immunoreactivity with the MAbs prepared against the denatured enzyme. Western blot analysis of postheparin plasma with the MAbs against denatured H-TGL gave a single protein band of 65 kD; preheparin plasma showed no detectable immunoreactivity with either MAb. These immunochemical studies suggest that there are no circulating active or inactive forms of H-TGL in man. Furthermore, the MAbs provide the necessary reagents for development of immunoassays for H-TGL.