Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [¹⁴C]-α-methyl glucopyranoside ([¹⁴C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [¹⁴C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

Desensitization of the δ-Opioid Receptor Correlates with Its Phosphorylation in SK-N-BE Cells: Involvement of a G Protein-Coupled Receptor Kinase

Abstract: Phosphorylation of G protein-coupled receptors is considered an important step during their desensitization. In SK-N-BE cells, recently presented as a pertinent model for the studies of the human δ-opioid receptor, pretreatment with the opioid agonist etorphine increased time-dependently the rate of phosphorylation of a 51-kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino-terminal region of the cloned human δ-opioid receptor, revealed that it corresponded to the δ-opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the δ-opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the δ-opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn²⁺, known to act as G protein-coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human δ-opioid receptor, that a close link exists between receptor phosphorylation and agonist-promoted desensitization and that desensitization involves a GRK.     

NAD+ kinase: molecular weight determination by low-angle laser-light scattering

Low-angle laser-light scattering (LALLS) was employed to measure the absolute molecular weight of chicken liver NAD+ kinase (NADK). The weight-average molecular weight (Mw) was found to be 275 000 +/- 15 000. The corresponding value for the second virial coefficient was -1.65 X 10(-3) ml X mol X g2. The value for Mw is in close accord with estimates reported for pigeon liver (270 000) and C. utilis (260 000) NADK. If the active enzyme is a dimer, the weight difference between pigeon/chicken liver and rabbit liver (136 000) NADK would indicate that the latter enzyme is an active monomer unit.     

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

Flavin-dependent thymidylate synthase: A novel pathway towards thymine

For several decades only one chemical pathway was known for the de novo biosynthesis of the essential DNA nucleotide, thymidylate. This reaction catalyzed by thyA or TYMS encoded thymidylate synthases is the last committed step in the biosynthesis of thymidylate and proceeds via the reductive methylation of uridylate. However, many microorganisms have recently been shown to produce a novel, flavin-dependent thymidylate synthase encoded by the thyX gene. Preliminary structural and mechanistic studies have shown substantial differences between these deoxyuridylate-methylating enzymes. Recently, both the chemical and kinetic mechanisms of FDTS have provided further insight into the distinctions between thyA and thyX encoded thymidylate synthases. Since FDTSs are found in several severe human pathogens their unusual mechanism offers a promising future for the development of antibiotic and antiviral drugs with little effect on human thymidylate biosynthesis.     

Free radical mechanisms in enzyme reactions

Free radicals are formed in prosthetic groups or amino acid residues of certain enzymes. These free radicals are closely related to the activation process in enzyme catalysis, but their formation does not always result in the formation of substrate free radicals as a product of the enzyme reactions. The role of free radicals in enzyme catalysis is discussed.     

Mitogen-Activated Protein Kinase Kinase 3 Is Required for Regulation during Dark-Light Transition

Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition.     

Neurofilament Protein Phosphorylation in Spinal Cord of Experimentally Diabetic Rats

This study was designed to determine if the known decrease in slow axonal transport of proteins in the sciatic nerve of experimentally diabetic rats is related to altered phosphorylation of neurofilament proteins (NFPs). Rats were rendered diabetic with 50 mg/kg of streptozotocin, i.p. At 3 and 6 weeks later, NFPs were prepared from spinal cord. The in vivo phosphorylation state of NFPs was examined by using phosphate-dependent (RT97) and -independent (RMd09) antibodies against high-molecular-mass NFPs on Western blots. Neurofilament-associated kinase activity was also measured in vitro by incubation of NFPs with [32P]ATP. Phosphorylation of all three NFPs (high, medium, and low molecular mass) occurred, as confirmed by gel electrophoresis and autoradiography. At 30 min of incubation, protein-bound radioactivity in NFPs from diabetic animals was reduced to 86.7 +/- 3.4 and 54.3 +/- 19.6% of that in nondiabetic animals at 3 and 6 weeks of diabetes, respectively (p less than 0.001 and p less than 0.05, respectively). NFPs were also incubated with acid phosphatase and rephosphorylated. Results showed that the increased in vivo phosphorylation contributed to the decreased in vitro phosphorylation. Extraction of protein kinases and addition back to the NFPs revealed, in addition, a reduced activity in the diabetic animals of the protein kinases measured in vitro.     

Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.     

High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver

Saturation and competitive binding analyses demonstrated the presence of a high affinity (K_D = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver.