Rat Brain Protein Phosphatase 2A: An Enzyme that May Regulate Autophosphorylated Protein Kinases

Abstract: Protein phosphatase 2A (PP2A) isolated from whole rat brain homogenate supernatants has been compared with that extracted from rat synaptosomal membranes. Both purified enzymes are comprised of the three known PP2A polypeptide chains of 65 (A subunit), 55 (B/B' subunit), and 38 (C subunit) kDa and have okadaic acid inhibition curves ( K _i = 0.05 n M ) nearly identical to that reported for skeletal muscle PP2A. The isolated 38-kDa subunit of rat brain PP2A appears to contain phosphotyrosine based on cross-reactivity with a specific monoclonal antibody (PY-20). Amino acid compositions and sequences of peptides isolated from the 65- and 38-kDa species correspond to regions of the cDNA-deduced sequences of the regulatory and catalytic subunits of protein phosphatase 2A from several sources. Studies reported here also demonstrate that autophosphorylated protein kinases, particularly Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II), are excellent substrates for brain PP2A. Furthermore, Ca²⁺-dependent K⁺-depolarization of hippocampal synaptosomes was accompanied by a sequential increase, then decrease, in CaM kinase II phosphorylation level over a 45-s time course. The decrease was blocked by 1 n M okadaic acid. These data demonstrate that the type 2A protein phosphatase is present at the synapses of CNS neurons where its localization could alter the functions of phosphoproteins involved in synaptic plasticity.     

Antibodies to a Segment of Tyrosine Hydroxylase Phosphorylated at Serine 40

Abstract: A synthetic peptide corresponding to residues 32–47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser⁴⁰ and used to generate antibodies in rabbits. Reactivity of the anti-pTH_32–47 antibodies with phospho- and dephospho-Ser⁴⁰ forms of TH protein and peptide TH_32–47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody-capture ELISAs, anti-pTH_32–47 was more reactive with the phospho-TH than with the dephospho-TH forms. Conversely, antibodies against the nonphosphorylated peptide reacted preferentially with the dephospho-TH forms. In western blots, labeling of the ∼60-kDa TH band by anti-pTH_32–47 was readily detectable in lanes containing protein kinase A-phosphorylated native TH at 10–100 ng/lane. In blots of supernatants prepared from striatal synaptosomes, addition of a phosphatase inhibitor was necessary to discern labeling of the TH band with anti-pTH_32–47. Similarly, anti-pTH_32–47 failed to immunoprecipitate TH activity from supernatants prepared from untreated tissues, whereas prior treatment with either 8-bromoadenosine 3',5'-cyclic monophosphate or forskolin enabled removal of TH activity by anti-pTH_32–47. Lastly, in immunohistochemical studies, anti-pTH_32–47 selectively labeled catecholaminergic cells in tissue sections from perfusion-fixed rat brain.     

Molecular assembly of proteins and conjugated polymers: Toward development of biosensors

A molecular assembly in which a conjugated polymer is interfaced with a photodynamic protein is described. The conjugated polymer, functionalized with biotion, is designed such that it can be physisorbed on or chemically grown off a glass surface. The streptavidin-derivatized protein is immobilized on the biotinylated polymer matrix through the strong biotin-streptavidin interactions. The assembly, built on the surface of an optical fiber or on the inside walls of a glass capillary, forms an integral part of a biosensor for the detection of environmental pollutants such as organophosphorus-based insecticides. The Protein in the system can be replaced by any biological macromolecule of interest. We study one specific case, the enzyme alkaline phosphatase. The enzyme catalyzes a reaction producing an intermediate compound that chemiluminesces, and the chemiluminescence singnal is monitored to detect and quantify insecticides such as paraoxon and methyl parathion. Preliminary results indicate ppb level detection with response time less than 1 minute. (c) 1995 John Wiley & Sons, Inc.     

Quinate:NAP(P)+-oxidoreductase from Larix sibirica: purification,characterization and function

Quinate:NAP(P)⁺-oxidoreductase (QORase, EC 1.1.1.24), which catalyzes the interconversion of quinic and 3-dehydroquinic acids, was purified from the needles and developing xylem cells of Larix sibirica. The enzymes from these two tissues were partially characterized and compared. QORase from needles had optimum pH at 9.0 and apparent K_m values of 1.84 mM for quinic acid and 0.19 mM for NADP⁺. The enzyme was activated by phosphoenolpyruvate. Gallic and protocatechuic acids were formed in a reaction mixture of purified enzyme from needles as final products of quinic acid transformation. QORase from developing xylem cells showed pH optimum at 10.0 and had apparent Km values of 0.70 mM for quinic acid and 0.05 mM for NADP⁺. The enzyme was not affected by PEP. The divalent cations Co²⁺ and Mn²⁺ at least doubled activity of QORase from both sources but Mg²⁺ affected the enzyme from needles only. The spatial organization and regulation of quinic acid metabolism in the autotrophic and heterotrophic cells of conifers and the role of QORase in this process are discussed.     

Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

A circularly permuted (cp) variant of the phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been constructed with N- and C-termini created within the coenzyme binding domain. The cp variant has a kcat value equal to 40% of the wild-type value, whereas Km and KD values for NAD show a threefold decrease compared to wild type. These results indicate that the folding process and the conformational changes that accompany NAD binding during the catalytic event occur efficiently in the permuted variant and that NAD binding is tighter. Reversible denaturation experiments show that the stability of the variant is only reduced by 0.7 kcal/mol compared to the wild-type enzyme. These experiments confirm and extend results obtained recently on other permuted proteins. For multimeric proteins, such as GAPDH, which harbor subunits with two structural domains, the natural location of the N- and C-termini is not a prerequisite for optimal folding and biological activity.     

High-sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry.

We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied.     

Spin-Trapping and Direct Epr Investigations on the Hepatotoxic and Hepatocarcinogenic Actions of Luteoskyrin, An Anthraquinoid Mycotoxin Produced by Penicillium Islandicum Sopp. Generations of Superoxide Anion and Luteoskyrin Semiquinone Radical in the Redox Systems Consisted of Luteoskyrin and Liver Nadph- Or Nadh-Dependent Reductases

Luteoskyrin is a hepatotoxic and hepatocarcinogenic bisdihydroanthraquinone produced by Penicillium islandicum Sopp. By observing the EPR spectra of DMPO-spin adducts and luteoskyrin semiquinone radical, we investigated in vitro whether luteoskyrin is reduced to its semiquinone radical leading to the generation of active oxygen species in redox systems catalyzed by NADPH-dependent cytochrome reductases of the liver. We found (1) the formation of luteoskyrin semiquinone radical in the NADPH-cytochrome P-450 reductase system under anaerobic conditions, (2) the generation of O- in the systems composed of luteoskyrin, NAD(P)H, and either rat liver microsomal NADPH-cytochrome P-450 reductase or submitochondrial particles and (3) dicoumarol showed no effect on the O- generation in the case of submitochondrial particles. From these results we proposed that luteoskyrin liver injuries are induced by the active oxygen species generated in the process of autoxidation of luteoskyrin semiquinone radical which is produced in the one-electron redox systems catalyzed by the liver NAD(P)H-dependent cytochrome reductases.     

Involvement of protein phsophatase-1 in cytoskeletal organization of cultured endothelial cells

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effect of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vien endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton. © 1995 Wiley-Liss, Inc.     

Decrease in protein tyrosine phosphatase activities in vanadate-treated obese Zucker (fa/fa) rat liver

The inhibitory action of vanadate towards protein tyrosine phosphatase (PTPase) has been considered as a probable mechanism by which it exerts insulin-like effects. In this study, we have examined thein vivo effects of vanadate on PTPases in the liver of obese Zucker rats, a genetic animal model for obesity and type II diabetes. These animals were characterized by hyperinsulinemia and mild hyperglycemia. The number of insulin receptors were significantly (p<0.01) decreased in liver. After chronic administration of vanadate in obese rats, 80% decrease in the plasma levels of insulin was observed. The insulin receptor numbers were significantly (p<0.01) higher in vanadate-treated obese rats as compared to the untreated ones. The hepatic PTPase activities in cytosolic and particulate fractions, with phosphorylated poly glu:tyr (41) and the insulin receptor peptide (residues 1142–1153) as substrates, increased in obese rats. In vanadate-treated obese rat livers, the PTPase activities in both subcellular fractions with these substrates decreased significantly (p<0.001). The decreases in PTPase activities from these groups of rats were further supported by chromatography on a Mono Q column. These data support the view that inhibition of PTPases plays a role in the insulin-mimetic action of vanadate.     

Autophagy and acid phosphatase activity in the corpora allata of adult mated females of Diploptera punctata

The corpora allata exbibit cycles of synchronous cell growth and atrophy during ovarian cycles in adult females of the cockroach Diploptera punctata. In the present report, the process of synchronous autophagy of organelles which results in cellular atrophy was investigated. In general, unwanted organelles were sequentially sequestered by several different mechanisms and then targeted for destruction. Autophagy was initiated on day 4 when corpus allatum cells were largest and most actively synthesizing juvenile hormone. The first sign of the initiation of autophagy was aggregation of ribosomes in an isolation membrane. By day 5, many organelles were isolated in the autophagic vacuoles. The ribosomecontaining vacuoles were wrapped by flattened stacks of Golgi cisternae to form conspicuous whorl-like autophagosomes. This is a previously undescribed type of autophagic vacuole with the entire complex of Golgi cisternae forming part of the autophagic membranes. Smooth endoplasmic reticulum was wrapped into membranous autophagic vacuoles with concentric arrays of doubel membranes. Plasma membrane was invaginated and then isolated in a multivesicular body. These three different types of isolated vacuoles did not show acid phosphatase activity as indicated by histochemical staining with -glycerophosphate as substrate. Subsequently, these autophagosomes fused with each other and with 1° or 2° lysosomes to form giant autophagolysosomes. Some mitochondria appeared to have coalesced directly into autophagolysosomes. Golgi complexes were evident during this period; they actively participated in making lysosomal enzymes. Cytoskeletons were frequently observed in the vicinity of autophagic vacuoles and were presumably involved in the transport of the vacuoles. As a result of lysosomal degradation lipofuscins and dense bodies were frequently observed by days 9–12 indicating atrophy of corpus allatum cells. Structural parameters, especially those present early in autophagy, such as the isolation membrane, ribosome-containing vacuoles and whorl-like autophagosomes, can be used to search for potential growth regulators responsible for the induction of autophagy, of the corpora allata, and the subsequent termination in juvenile hormone synthesis.