Hog Brain Research Articles

Glycogen- and PP1c-targeting Subunit GM Is Phosphorylated at Ser48 by Sarcoplasmic Reticulum-bound Ca2+-Calmodulin Protein Kinase in Rabbit Fast Twitch Skeletal Muscle

Multifunctional Ca(2+)-calmodulin-dependent protein kinase (CaMKII) is a Ser/Thr protein kinase uniformly distributed within the sarcoplasmic reticulum (SR) of skeletal muscle. In fast twitch muscle, no specific substrates of CaMKII have yet been identified in nonjunctional SR. Previous electron microscopy data showed that glycogen particles containing glycogen synthase (GS) associate with SR at the I band level. Furthermore, recent evidence implicates CaMKII in regulation of glucose and glycogen metabolism. Here, we demonstrate that the glycogen- and protein phosphatase 1-targeting subunit, also known as G(M), selectively localizes to the SR membranes of rabbit skeletal muscle and that G(M) and GS co-localize at the level of the I band. We further show that G(M), GS, and PP1c assemble in a structural complex that selectively localizes to nonjunctional SR and that G(M) is phosphorylated by SR-bound CaMKII and dephosphorylated by PP1c. On the other hand, no evidence for a structural interaction between G(M) and CaMKII was obtained. Using His-tagged G(M) recombinant fragments and site-directed mutagenesis, we demonstrate that the target of CaMKII is Ser(48). Taken together, these data suggest that SR-bound CaMKII participates in the regulation of GS activity through changes in the phosphorylation state of G(M). Based on these findings, we propose that SR-bound CaMKII participates in the regulation of glycogen metabolism, under physiological conditions involving repetitive raises elevations of [Ca(2+)](i).

Rpg1p, the subunit of the Saccharomyces cerevisiae eIF3 core complex, is a microtubule-interacting protein.

The essential gene RPG1/TIF32 of Saccharomyces cerevisiae encodes the 110-kDa subunit of the translation initiation factor 3 (eIF3) core complex. In this study, the Rpg1p-specific monoclonal antibody PK1/1 was used to analyse the cellular distribution of Rpg1p by epifluorescence and confocal laser scanning microscopy (CLSM). In budded cells, a portion of Rpg1p was obviously co-localised with microtubules. In addition, CLSM revealed an accumulation of Rpg1p in a patch at the very end of cytoplasmic microtubules reaching the bud tip. A punctate fluorescence pattern was typical for separated unbudded cells. Distribution of Rpg1p was confirmed using a strain expressing exclusively a hemaglutinin-tagged version of Rpg1p. In nocodazole-treated cells, the pattern of the PK1/1 staining was disturbed. No staining was observed in Rpg1p-depleted cells. In vitro experiments revealed that Rpg1p was specifically co-immunoprecipitated with alpha-tubulin from the yeast cell free extract and this observation was further supported by showing that Rpg1p co-sedimented with hog brain microtubules. We conclude that Rpg1p is a microtubule-interacting protein that indicates an interesting connection between the translation initiation machinery and cytoskeleton in yeast Saccharomyces cerevisiae.

ブタの脳と骨の比較電率-温度特性

ブタの脳と骨の比較電率-温度特性

A membrane form of brain L-glutamate decarboxylase: identification, isolation, and its relation to insulin-dependent mellitus.

A membrane form of L-glutamate decarboxylase (GAD) was identified and purified to apparent homogeneity from hog brain. The purified GAD was established as an integral membrane protein by phase-partitioning assay, charge-shift electrophoresis, and chromatography on a hydrophobic interaction column. This membrane GAD has a native molecular mass of 96 +/- 5 kDa and is a homodimer of 48 +/- 3-kDa subunits. Immunoprecipitation and immunoblotting tests revealed the presence of antibodies against this membrane GAD in sera from patients with insulin-dependent diabetes mellitus. Since this form of GAD appears to be an integral membrane protein and is presumed to have extracellular domains exposed, it seems reasonable to suggest that membrane GAD is more likely than soluble GAD to be involved in the pathogenesis of insulin-dependent diabetes and related autoimmune disorders such as stiff-man syndrome.

P-83 - Effects of several kinds of salts on monoamine oxidase in sonicated hog brain mitochondrial membrane.

P-83 - Effects of several kinds of salts on monoamine oxidase in sonicated hog brain mitochondrial membrane.

Effects of Several Cl- and NO3-salts on the Monoamine Oxidase in Hog Brain Mitochondrial Membrane

Effects of Several Cl- and NO3-salts on the Monoamine Oxidase in Hog Brain Mitochondrial Membrane

Polyamine-linked sepharoses: Preparation and application to mammalian spermine synthase

Seven different polyamine-linked Sepharose derivatives were prepared for the affinity chromatography of spermidine and spermine binding macromolecules: Spermine synthase from rat and hog brain was used as a model protein with a spermidine binding side. Comparative studies of the affinities of the enzymes for the seven matrixes suggested that two negative charges, three to four methylene groups apart, should be present at the decarboxylated S-adenosylmethionine binding site and should improve the binding of the enzyme to the Sepharose derivative. Two negative charges at the spermidine binding site would be expected to do the same. Three affinity matrixes linked with 1,17-diamino-4,9,14-triazaheptadecane, 1,21-diamino-4,9,13,18-tetraazaheneicosane, and 5-spermine carboxylic acid, respectively, had an affinity for spermine synthases higher than that of spermine-Sepharose, which has been used for the purification of spermine synthase. The first of these matrixes was used and proved to be effective for the purification.

Structure and function ofl-glutamate decarboxylase

Membrane bound L-glutamate decarboxylase (GAD) has been solubilized and partially purified from hog brain. The solubilized GAD appears to exist in two forms, alpha and beta, differing in their size and electrophoretic mobility. The alpha form has similar mobility as that of the soluble GAD in 7.5% and 5-25% gradient polyacrylamide gel electrophoresis suggesting that they are similar in size and charge. In addition, gene encoding for mouse brain GAD has been cloned and characterized. Mouse brain GAD cDNA consists of two DNA fragments with 1.6 and 1.0 Kb. The 1.6 and 1.0 Kb fragments contain 1657 and 974 bP, respectively. The significance of multiple forms of GAD is also discussed.

Immunolocalization and molecular properties of a high molecular weight microtubule-bundling protein (syncolin) from chicken erythrocytes.

A protein of apparent molecular weight 280,000 (syncolin), which is immunoreactive with antibodies to hog brain microtubule-associated protein (MAP) 2, was purified from chicken erythrocytes. Immunofluorescence microscopy of bone marrow cells revealed the presence of syncolin in cells at all stages of erythrocyte differentiation. In early erythroblasts syncolin was diffusely distributed throughout the cytoplasm. At later stages it was found along microtubules of the marginal band, as confirmed by immunoelectron microscopy. The association of syncolin with the marginal band was dependent on the integrity of microtubules, as demonstrated by temperature-dependent de- and repolymerization or marginal band microtubules. Syncolin cosedimented in a saturable manner with microtubules assembled in vitro, and it was displaced from the polymer by salt. Brain as well as erythrocyte microtubules, reconstituted with taxol from MAP-free tubulin and purified syncolin, were aggregated into dense bundles containing up to 15 microtubules, as determined by electron microscopy. On the ultrastructural level, syncolin molecules were visualized as globular or ringlike structures, in contrast to the thin, threadlike appearance of filamentous MAPs, such as brain MAP 2. According to ultrastructural measurements and gel permeation chromatography, syncolin's molecular weight was approximately 1 x 10(6). It is suggested that syncolin's specific function is the cross-linking of microtubules in the marginal band and, by implication, the stabilization of this structure typical for nucleated (chicken) erythrocytes.

P-312 - The effects of salts on monoamine oxidase activity in hog brain mitochondrial membrane.

P-312 - The effects of salts on monoamine oxidase activity in hog brain mitochondrial membrane.

Lipids as endogenous Na, K-ATPase inhibitors in plasma of healthy individuals and in dialysis dependent patients

We assayed plasma Na,K-ATPase inhibitory activity due to total lipids and lipid fractions. The effect of dialysis on the Na,K-ATPase inhibitory activity was also studied. Plasma lipid extracts from 11 healthy volunteers and 9 dialysis-dependent patients (pre and post dialysis) were separated into neutral lipids and phospholipids. Further fractionation was by thin layer chromatography. These lipid fractions were analyzed for Na,K-ATPase inhibitory activity by displacement of [3H]-ouabain from hog brain Na,K-ATPase. Total inhibitory activity was significantly increased (p less than 0.001) in the post-dialysis plasma compared to pre-dialysis plasma of the same patient group and to controls (482, 85 and 78 nmol/L respectively; means of the groups in digoxin equivalents). The major inhibitory activity was associated with non-esterified fatty acids with modest contributions from four other lipid fractions. Our results show that endogenous lipids are major plasma Na,K-ATPase inhibitors in vitro under these assay conditions.

Inhibition of brain cation pump enzyme by in vitro lead ion: Effects of low level [Pb] and modulation by homogenate

Rat brain homogenates were preincubated with lead chloride for 20 min at 0°C. Inhibition of K- pnitrophenylphosphatase (K-pNPPase), which measures the dephosphorylation step of Na,K-ATPase, reached steady state within 10 min and was readily reversible. The activity in brain homogenates prepared from 60-day-old rats, expressed (mean ± SE) as a percentage of control (100.0 ± 0.48%), fell to 89.4 ± 0.52% at 0.25 μ m [Pb] to 64.2 ± 1.65% at 5 μ m [Pb]. However, between 5 and 25 μ m [Pb] K-pNPPase activity significantly increased (to 85.4 ± 3.14% at 25 μ m) before it again fell above 30 μ m. Similar results were obtained with brain homogenates prepared from 10-day-old rats. The multiphasic nature of this dose-response curve did not change with changes in buffer, substrate, anion, test tube order, or test tube composition (glass vs plastic), and appeared to be intrinsic to homogenate-Pb interactions. Microsomal preparations also exhibited multiphasicity, but with a shift in the inflection points. However, there was no multiphasicity with commercial hog brain Na,K-ATPase, suggesting an interaction between a component in the rat brain preparations and lead ion. Addition of boiled rat brain homogenate to commercial Na,K-ATPase resulted in partial protection from lead inhibition but did not produce multiphasicity over the ranges tested. Bovine serum albumin provided less protection. We couclude that there is a complex interaction among K-pNPPase activity, some factor in the rat brain homogenate, and low levels of lead ion.

Deuterium isotope effect in gamma-aminobutyric acid transamination: determination of rate-limiting step.

The rate of transamination of gamma-aminobutryic acid (GABA) catalyzed by hog brain gamma-aminobutyrate aminotransferase was substantially reduced when the hydrogen at the gamma-carbon position was replaced by deuterium. The deuterium isotope effect of this reaction has been substantiated by fluorometric, radiometric, and mass spectrometric procedures and assessed kinetically. The ratios of Vmax of the nonlabeled substrate/Vmax of the deuterated substrate obtained under different conditions ranged from 6 to 7. This indicates that the cleavage of the hydrogen from the gamma-carbon is the rate-determining step in GABA transamination. Similar isotope effects have also been shown to occur in the peripheral system in vivo.

Hypotaurine aminotransferase assay: [2-3H]hypotaurine

This chapter discusses the assay of hypotaurine aminotransferase as is the synthesis of the substrate of the enzyme, [2- 3 H]hypotaurine. Hypotaurine aminotransferase catalyzes the α-ketoglutarate-dependent transamination of hypotaurine to form sulfinoacetaldehyde, an unstable intermediate, which rapidly degenerates to acetaldehyde and SO 2 . The rate of the enzyme reaction can be monitored by determining the rate of formation of glutamate, SO 2 , or acetaldehyde, the rate is most easily and specifically quantitated by following 3 H 2 O release when [2- 3 H]hypotaurine is the substrate. Use of [ 3 H]hypotaurine is particularly convenient when assaying enzyme activity in crude tissue fractions including the heavy mitochondrial fraction with which the mammalian enzyme is associated. Hypotaurine aminotransferase activity appears to be restricted to the “heavy” mitochondrial fraction in liver and brain preparations; the activity from hog brain mitochondria copurifies with γ-amino-butyrate aminotransferase and has been purified by the procedure of Bloch-Tardy. Hypotaurine aminotransferase activity is optimal at a pH near 8.2 and is stimulated 20% by the inclusion of l0 μM pyridoxal phosphate in the assay solution. The activity is inhibited by reagents that bind to pyridoxal phosphate (for example, aminooxyacetic acid and hydroxylamine) and by established inhibitors of γ-aminobutyrate aminotransferase (for example, 4-amino-5-fluoropentanoic acid and vinyl-GABA). Both selenotaurine and selenohypotaurine are inhibitors of hypotaurine aminotransferase.

Dehydroepiandrosterone Sulfate as a Digitalis Like Factor in Plasma of Healthy Human Adults

Plasma values for digitalis like factors (DLF) and dehydroepiandrosterone sulfate (DHEA-S) in 11 healthy adults were (mean +/- SD) 44 +/- 6 pmol digoxin equivalents/L and 11 +/- 3 mumol/L respectively. DHEA-S accounted for 62-100% of the total plasma DLF. DHEA-S and plasma DLF displaced [125I]digoxin from digoxin antibody, inhibited hog brain Na,K-ATPase and displaced [3H]ouabain from ATPase in a concentration dependent manner similar to digoxin. Plasma extracts (11 subjects) inhibited Na,K-ATPase and displaced [3H]ouabain from Na,K-ATPase with mean values +/- SD of 1.7 +/- 0.22 and 1.8 +/- 0.25 nmol digoxin equivalents/L plasma respectively. HPLC fractionation of plasma DLF showed several peaks. The major peak was due to DHEA-S, identified by its retention time, radioimmunoassay of DHEA-S and mass spectrometry.

Mr 205,000 sulfoglycoprotein in extracellular matrix of mouse fibroblast cells is immunologically related to high molecular weight microtubule-associated proteins.

Rabbit antibodies raised against microtubule-associated protein 1 (MAP-1) from hog brain were found to crossreact with extracellular matrix components of mouse BALB/c 3T3 cell cultures. As shown by immunofluorescence microscopy of confluent cell cultures, the extracellular MAP-related antigen was located on dense fibrillar network arrays underlying and surrounding the cells. The immunoreactive material was sensitive to trypsin but resistant to collagenase. The microtubule-disrupting drug colcemid had no visible effect on the morphology of the anti-MAP-stained network, whereas treatment with cytochalasin B provoked its abolishment. Simian virus 40-transformed BALB/c 3T3 cells expressed considerably less extracellular antigen than did the nontransformed cells. After in vivo radiolabeling of BALB/c 3T3 cells, a secreted polypeptide of Mr 205,000 was isolated by immuno-precipitation from culture media as well as from cell-free extracellular matrices. This antigen was identified as a sulfoglycoprotein, noncollageneous in nature, that undergoes intermolecular disulfide bonding. Anti-MAP-1 antibodies affinity-purified on the extracellular Mr 205,000 protein were immunoreactive with MAP-1 and MAP-2 from brain and decorated cytoplasmic microtubules as demonstrated by immunoblotting and immunofluorescence microscopy. Thus, a structural relationship between cytoskeletal and extracellular polypeptides is demonstrated.

Characteristics of a (Na+-K+)-ATPase inhibitor in extracts of tea

Extracts of tea were examined for inhibitors of the sodium-potassium pump by investigating the effect of the extracts on 1) isolated preparations of (Na+-K+)-ATPase from hog brain and human blood cells; 2) the displacement of radioactive ouabain from its specific receptor on red blood cells, and 3) the uptake of radioactive rubidium in intact red blood cells. It has been found that extracts of tea were potent inhibitors of the purified hog brain (Na+-K+)-ATPase. However, the inhibition was not specific for the (Na+-K+)-ATPase and the extract of tea did not displace 3H-ouabain in a specific ouabain-receptor assay. Additionally, the tea extracts displayed only a small inhibitory effect on the uptake of 86Rb in intact red blood cells. These observations suggest that the material is not like digitalis and that, unlike cardiac glycosides, it may inhibit the activity of the (Na+-K+)-ATPase by interacting with the enzyme at intracellular sites.

Structural homology of microtubule-associated proteins 1 and 2 demonstrated by peptide mapping and immunoreactivity.

The major high molecular weight microtubule-associated polypeptides from hog brain (MAP-1 and MAP-2) were compared by one- and two-dimensional peptide mapping under varied conditions and by immunological techniques. Partial digestion of MAP-1 and MAP-2 with Staphylococcus aureus V8 protease and analysis in one dimension gave rise to very similar peptide maps independent of whether 125I-, 3H-, or 32P-labeled proteins were used. One-dimensional cleavage patterns of significant similarity were also obtained by partial digestion of MAP-1 and MAP-2 using trypsin or chymotrypsin. Furthermore, a pronounced similarity, although clear nonidentity, of MAP-1 and MAP-2 was also revealed after exhaustive digestion of 125I-labeled proteins with S. aureus V8 protease or trypsin followed by analysis of peptides in two dimensions. For immunological comparison, antisera were used that had been raised in rabbits using electrophoretically purified MAP-1 and MAP-2 components as immunogens. As determined by immunoprecipitation, the antiserum raised to MAP-1 was equally reactive with MAP-1 and MAP-2 components, whereas the antiserum to MAP-2 reacted primarily with MAP-2. Indicating the presence of common as well as unique antigenic determinants on MAP-1 and MAP-2, these results, therefore, were in agreement with the peptide mapping data. Implications of these results for biosynthetic mechanisms as well as differential distribution and functions of MAPs in cells are discussed.

The pattern of MAP-2 binding on microtubules: visual enhancement of MAP attachment sites by antibody labeling and electron microscopy

We used affinity-purified rabbit antibody to hog brain microtubule-associated protein 2 (MAP-2) to examine the pattern of attachment of MAPs to microtubules purified by cycles of in vitro assembly and disassembly. Microtubules were fixed, deposited on EM grids, and labeled with antibody and protein A-gold colloid followed by negative staining. We observed that: The sites of MAP attachment were greatly enhanced by antibody binding in negatively stained preparations. The axial repeat revealed by antibody (100 +/- 5 nm) was greater than the previously reported value of 32 nm based on thin sectioning and negative staining procedures. The antibody was arranged in a broad band and revealed a helical pattern of binding. Microtubules with and without treatment with alpha-chymotrypsin to remove the projection portion of MAP-2 looked similar, suggesting that the antibody-enhanced pattern may reflect the sites of MAP attachment on microtubules. Microtubules with an increased MAP:tubulin ratio exhibited the same 100-nm periodicity.

Phosphorylation of microtubule-associated proteins regulates their interaction with actin filaments.

We have determined the absolute phosphate content of microtubule-associated proteins (MAPs) and established that phosphorylation inhibits the actin filament cross-linking activity of MAPs and both of the major MAP components, MAP-2 and tau. Similar results were obtained with actin from rabbit muscle, hog brain, and Acanthamoeba castellanii. We used the endogenous phosphatases and kinases in hog brain microtubule protein to modulate MAP phosphate level before isolating heat-stable MAPs. MAPs isolated directly from twice-cycled microtubule protein contain 7.1 +/- 0.1 (S.E.) mol of phosphate/300,000 g protein. After incubating microtubule protein without ATP, MAPs, had 4.9 +/- 0.6 phosphates. After incubating microtubule protein with 1 mM ATP and 5 microM cAMP in 2 mM EGTA, MAPs had 8.6 +/- 0.5 phosphates but there was also exchange of three more [32P]phosphates from gamma-labeled ATP for preexisting MAP phosphate. Incubation of microtubule protein with ATP and cAMP in 5 mM CaCl2 resulted in exchange but no net addition of phosphate to MAPs. We fractionated the MAP preparations by gel filtration and obtained MAP-2 with 4.3 to 7.5 and tau with 1.5 to 2.2 mol of phosphate/mol of protein depending on how we treated the microtubule protein prior to MAP isolation. The actin filament cross-linking activity of whole MAPs, MAP-2, and tau depended on the MAP-phosphate content. In all cases, phosphorylation of MAPs inhibited actin filament cross-linking activity. The concentration of high phosphate MAPs required to form a high viscosity solution with actin filaments was 2 to 4 times more than that of low phosphate. MAPs. During incubation of microtubule protein with [gamma-32P]ATP, only MAP peptides are labeled. Treatment of these MAPs with either acid or alkaline phosphatase removes phosphate mainly from MAP-2, with an increase in actin filament cross-linking activity. Thus, both MAP phosphorylation and the effect of phosphorylation on actin cross-linking activity of MAPs are reversible.