Homodimers and heterodimers of Pho1-type phosphorylase isoforms in Solanum tuberosum L. as revealed by sequence-specific antibodies.

Higher plants possess two types of glucan phosphorylase (EC 2.4.1.1). One isozyme type, designated as Pho1, is located in the plastid whereas the other type, Pho2, is restricted to the cytosol. For Solanum tuberosum L. two Pho1 type phosphorylases have been sequenced [Nakano, K. & Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236; Sonnewald, U., Basner, A., Greve, B. & Steup, M. (1995) Plant Mol. Biol. 27, 567-576]. Both proteins (referred to as Pho1a and Pho1b, respectively) are highly similar (81-84% amino acid identity over most parts of the two sequences) with the exception of the N-terminal transit peptide and the large insertion located between the N- and the C-terminal domains. In this communication antibodies that bind specifically to either Pho1a or Pho1b were used to study both isoforms at the protein level. The antibodies were applied to both potato tuber and leaf extracts following either denaturing or non-denaturing electrophoresis. Pho1a but not Pho1b was immunochemically detectable in tuber extracts whereas leaf extracts contained both the Pho1a and Pho1b protein. During denaturing electrophoresis the two antigens comigrated. When the leaf Pho1 isoforms were separated by affinity electrophoresis three bands of activity were resolved; all of them were recognized by the anti-Pho1a antibodies, but only two of these reacted with the anti-Pho1b antibodies. The isoform binding exclusively to the anti-Pho1a antibodies comigrated with the Pho1 isozyme from potato tubers. Immunoprecipitation experiments performed with anti-Pho1a antibodies removed the entire Pho1 phosphorylase activity from both tuber and leaf extracts. Addition of anti-Pho1b antibodies to tuber extracts did not affect the enzyme pattern, whereas in leaf extracts one isoform remained unchanged but the two other bands were strongly retarded. This indicates that the Pho1a protein is present in all three forms and Pho1b is associated with Pho1a. Association of Pho1a and Pho1b was further demonstrated by cross-linking experiments using bis(sulfosuccinimidyl)suberate as linker. Immunoprecipitation experiments were also performed using extracts of transformed Escherichia coli cells that expressed either Pho1a or Pho1b or both simultaneously. Under these conditions a homodimeric Pho1b phosphorylase was observed that had a lower electrophoretic mobility than the heterodimer from leaves. In leaves of transgenic potato plants antisense inhibition of the Pho1a gene affected the formation of (Pho1a)2 more strongly than that of the heterodimer. Thus, in leaves, Pho1a exists both as a homodimer, (Pho1a)2 and as heterodimer, (Pho1a-Pho1b); a part of it appears to be covalently modified. Pho1b, in the homodimeric form, is often below the limit of detection. In tubers the homodimer, (Pho1a)2, is the only detectable Pho1-type enzyme. To our knowledge this is the first report on a heterodimeric structure of plant phosphorylase.

Glycogen phosphorylase activity and immunoreactivity during pre- and postnatal development of rat brain.

Catalytic activity and immunoreactivity of glycogen phosphorylase were studied in pre- and postnatal rat brain. The catalytic activity was assayed in brain homogenates; immunoreactivity was investigated by immunoblot analysis using a monoclonal anti-bovine brain glycogen phosphorylase antibody. The cellular localization and intensity of immunoreactivity were analysed on paraffin-embedded sections utilizing the same monoclonal antibody. The catalytic activity increased 10-fold from embryonic day 16 to adult; immunoreactivity became detectable on embryonic day 16 and increased in intensity as the enzyme activity rose to adult values. The first cellular elements to be stained immunohistochemically were ependymal cells lining the ventricles, ependymal cells of the choroid plexus, meningeal cells and a selected population of neurons in the brain stem. The immunoreactivity of plexus cells and meningeal cells was reduced or absent in the adult rat brain. The earliest appearance of glycogen phosphorylase immunoreactivity in astroglial cells was seen at postnatal day 9 in the hippocampus. The staining pattern of the adult brain was reached at day 22 post partum. The developmental changes in glycogen deposition and in glycogen phophorylase activity and immunoreactivity may indicate a variable physiological role of glycogen metabolism for different cell types in the pre- and postnatal periods.

M4 and M9 autoantigens in primary biliary cirrhosis--a negative study.

We have investigated the potentially important finding that anti-M9 and anti-M4 antibodies in the sera of patients with primary biliary cirrhosis (PBC) recognise epitopes on the enzymes glycogen phosphorylase and sulphite oxidase, respectively. To test this, preparations of these two enzymes were applied in ELISA and immunoblotting, and the reactivities of sera from large groups of well-defined PBC patients (and other chronic liver disease patients) were studied. No specific reactivity with either putative antigen was observed in ELISA nor immunoblotting. However, with both antigens a positive correlation was observed between ELISA optical density readings and serum IgG levels in both the PBC and chronic liver disease groups which suggests some non-specific binding of immunoglobulin to the proteins. We conclude that antimitochondrial antibodies (AMA) do not specifically recognise glycogen phosphorylase and sulphite oxidase and the use of these antigens to determine the 'AMA profile' and indicate prognosis is not valid.

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver.

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35-1.0 micron) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 microns. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.

M4 and M9 antibodies in the overlap syndrome of primary biliary cirrhosis and chronic active hepatitis: epitopes or epiphenomena?

Before the identification of the major mitochondrial antigens of primary biliary cirrhosis as components of the 2-oxo-acid dehydrogenase enzyme family, mitochondrial autoantigens were believed to be extremely heterogeneous and were divided into nine subtypes termed M1 to M9. This classification was based on the data derived from the relatively nonspecific biochemical and immunological techniques that were available. After the cloning and definition of the major autoantigens, more than 95% of the sera of patients with primary biliary cirrhosis were found to react with components of the 2-oxo-dehydrogenase enzymes; these enzymes correspond to the old M2 classification. Two other "M" species, dubbed M4 and M9, have attracted significant attention because they have been postulated to be prognostic indicators and more recently have been tentatively identified respectively as sulfite oxidase (EC 1.8.3.1) and glycogen phosphorylase (EC 2.4.1.1). Indeed, patients with the "overlap syndrome" are reported to have antibodies to M4 and a poor prognosis, whereas patients with antibodies to M9 have a favorable prognosis. To address the significance and definition of M4 and M9, we performed in-depth studies of sera from 11 patients with the overlap syndrome, 75 patients with primary biliary cirrhosis, 19 chronic active hepatitis patients, 13 patients with primary sclerosing cholangitis, 10 patients with cholangiocarcinoma, 20 patients with systemic lupus erythematosus, 20 patients with alcoholic cirrhosis, 17 patients with scleroderma and 30 normal individuals, using techniques of ELISA, complement fixation, immunoblotting and enzyme inhibition. We report herein that we were unable to show any disease-specific reactivity toward the proposed M4 and M9 antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

A Babesia bovis 225-kilodalton protein located on the cytoplasmic side of the erythrocyte membrane has sequence similarity with a region of glycogen phosphorylase.

A Babesia bovis gene sequence is described which encodes a geographically conserved epitope (recognized by monoclonal antibody (mAb) 23.8.34) of a 225-kDa protein located on the surface of merozoites and associated with the infected erythrocyte membrane. The gene sequence, derived from both genomic and cDNA copies, is 2044 bp long and has one long open reading frame encoding about one third of the 225-kDa protein. The open reading frame is expressed in an approximately 6,400 nucleotide RNA transcript. A 73-amino acid sequence occurs as 4 complete and 1 partial tandem repeats at the carboxy terminus of the partial protein sequence. The epitope recognized by mAb 23.8.34 was localized to the repeat region. Based on epitope localization with mAb 23.8.34, the repeat was exposed on the surface of merozoites and located near the cytoplasmic face of the erythrocyte membrane. The amino terminus of the protein was non-repetitive and had 21% identity (60% similarity) to glycogen phosphorylase over a region of 151 amino acids. In addition, the corresponding 5' DNA sequence hybridized to as many as 8 restriction fragments on Southern blots of genomic DNA. In contrast, the DNA sequence of the repeat hybridized to a single fragment. Both the repeat and multiple non-repeat DNA sequences were detected in a different geographic strain of B. bovis. These results indicate that the 5' end of the 225-kDa protein gene is related to a larger gene family, independent of the 3' end of the gene encoding the repeat.

Molecular heterogeneity in McArdle's disease.

Biopsies were taken from a group of eleven patients with McArdle's disease, a congenital deficiency in muscle glycogen phosphorylase. The biopsies were screened by Western and Northern blotting for phosphorylase protein, phosphorylase-bound pyridoxal-5'-phosphate (the cofactor of the enzyme) and for phosphorylase mRNA. Of the eleven patients, three expressed phosphorylase mRNA at near normal levels and at the expected size. One of these patients also expressed low levels of phosphorylase protein that correlated with a small amount of measurable phosphorylase activity. These data support the contention of molecular heterogeneity in the presentation of this phenotype.

Brain isozyme of glycogen phosphorylase: immunohistological localization within the central nervous system.

An antibody specific for the predicted carboxyterminal sequence of the human brain isozyme of glycogen phosphorylase (alpha-1,4-D-glucan:orthophosphate D-glucosyltransferase, EC 2.4.1.1) was generated to verify the carboxyterminal amino acid sequence of this protein. The isozyme-specific antibody was used to examine the localization of this protein in primate and non-primate brain. The highest levels of the brain isozyme in cerebrum and cerebellum were found in fibrous astrocytes, many with glial processes that appear to terminate upon blood vessels.

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis recognize an epitope of glycogen phosphorylase.

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis (PBC) were previously found to recognize two antigenic determinants at 98 and 59 kD, using a purified antigen fraction derived from rat liver mitochondria in the Western blot. Here we show that these antibodies are directed against an epitope of the enzyme glycogen phosphorylase. By Western blotting, a determinant at 98 kD was obtained testing anti-M9 positive sera against phosphorylase from skeletal muscle, and after plasmin treatment a degradation product appeared at 59 kD. Both determinants were identical to the M9-specific determinants 98 and 59 kD as shown by absorption studies. When these antibodies were eluted from the 98 and 59 kD determinants of the M9 antigen after immunoblotting, they again recognized the same epitopes on plasmin-treated phosphorylase. Furthermore, phosphorylase enzyme activity could be also demonstrated in the purified M9 fraction, and anti-M9-positive/anti-M2-negative but not anti-M9-negative/anti-M2-positive sera could be shown to stimulate phosphorylase activity. Testing sera from 1189 patients with different hepatic and non-hepatic disorders against M9 and phosphorylase from skeletal muscle by ELISA, 20% were positive with phosphorylase and only 2% with the M9 fraction. These data indicate that the commercially available phosphorylase from skeletal muscle cannot be recommended as M9 source. It may still contain non-PBC-specific epitopes which are probably recognized by naturally occurring antibodies directed against this highly conserved protein.

Purification of glycogen phosphorylase from bovine brain and immunocytochemical examination of rat glial primary cultures using monoclonal antibodies raised against this enzyme.

The physiological function in brain of glycogen and the enzyme catalyzing the rate-limiting step in glycogenolysis, glycogen phosphorylase (EC 2.4.1.1), is unknown. As a first step toward elucidating such a function, we have purified bovine brain glycogen phosphorylase isozyme BB 1,700-fold to a specific activity of 24 units/mg protein. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining, a single major protein band corresponding to an apparent molecular mass of 97 kDa was observed. Mouse monoclonal antibodies raised against the enzyme were purified and shown to be monospecific as indicated by immunoblotting. Immunocytochemical examination of astroglia-rich primary cultures of rat brain cells revealed a colocalization of glycogen phosphorylase with the astroglial marker glial fibrillary acidic protein in many cells. The staining for the enzyme appeared at two levels of intensity. There were other cells in the culture showing no specific staining under the experimental conditions employed. Neurons in neuron-rich primary cultures did not show positive staining. The data suggest that glycogen phosphorylase may be predominantly an astroglial enzyme and that astroglia cells play an important role in the energy metabolism of the brain.

Phosphorylase-cross-reactive antibodies evoked by streptococcal M protein.

Rabbit antisera evoked by type 5 streptococcal M protein (M5) were screened by enzyme-linked immunosorbent assay (ELISA) for immunological cross-reactivity with purified rabbit muscle phosphorylases a and b. Of 10 pep M5 antisera tested, 3 showed significant cross-reactivity with both forms of the enzyme. ELISA inhibition studies using one of the pep M5 antisera showed that all of the phosphorylase b antibodies were inhibited by pep M5, the immunogen, and phosphorylase b, the ELISA antigen. All of the antibodies were also inhibited by pep M6 and pep M19, but not by pep M24, indicating that the cross-reactive epitopes were shared by multiple serotypes of M protein. Western blot (immunoblot) analyses showed that pep M5 antisera reacted strongly with the subunit of phosphorylase b. In addition, purified phosphorylase partially inhibited the binding of pep M5 antibodies to a 95-kilodalton protein of human myocardium. One of the three cross-reactive pep M5 antisera inhibited the enzymatic activity of phosphorylase a in a dose-related fashion, reaching a maximum inhibition of 75%. The enzymatic activity in the presence of antibody was totally restored when the antiserum was first incubated with pep M5.

Electrophoretic analysis of liver glycogen phosphorylase activation in the freeze-tolerant wood frog.

As an adaptation for overwinter survival, the wood frog, Rana sylvatica is able to tolerate the freezing of extracellular body fluids. Tolerance is made possible by the production of very high amounts of glucose in liver which is then sent to other organs where it acts as a cryoprotectant. Cryoprotectant synthesis is under the control of glycogen phosphorylase which in turn is activated in response to ice formation. To determine the mechanism of phosphorylase activation, a quantitative analysis of phosphorylase protein concentration and enzymatic activity in liver was carried out following separation of the phosphorylated a and nonphosphorylated b forms of the enzyme on native polyacrylamide gels. The results suggest that in gels, the b form is completely inactive, even in the presence of AMP and sodium sulfate, whereas the a form is active and stimulated 3-fold by these substances. Further, phosphorylase activation appears to arise solely from conversion of the b to a form of the enzyme without an increase in phosphorylase concentration or activation of a second isozyme. The quantitative analysis presented here should prove generally useful as a simple and rapid method for examining the physiological and genetic regulation of phosphorylase in animal cells.

The relationship between the two forms of glycogen phosphorylase in Dictyostelium discoideum.

The cellular slime mold, Dictyostelium disoideum, provides an ideal model system to study eukaryotic cell differentiation. In D. discoideum, glycogen degradation provides precursors for the synthesis of developmentally regulated structural products. The enzyme responsible for glycogen degradation, glycogen phosphorylase, exists in active and inactive forms. The active, or 'a' form, is independent of 5'adenosine monophosphate (5'AMP) while the inactive, or 'b' form, is 5'AMP-dependent. The activity of the 'b' form predominates early in development, while the activity of the 'a' form peaks in mid-late development; their combined specific activities remain constant at any point. Polyclonal antibodies raised to the purified forms of this enzyme showed low cross-reactivity. The anti-'a' serum reacted with a 104-kDa protein that was associated with phosphorylase 'a' activity; the anti-'b' serum reacted with a 92-kDa protein that was associated with phosphorylase 'b' activity and weakly cross-reacted with the 104-kDa protein. Immunoblots of peptide maps of the purified enzyme forms showed that each antibody was specific for the proteolytic fragments of its respective antigen. We also demonstrated in vitro phosphorylation of the 'b' form by an endogenous protein kinase. Cyclic AMP perturbation of intact cells caused induction of both phosphorylase-'a' activity and the 104-kDa protein. Immunotitration data suggested that the 'a' form accumulates due to de novo protein synthesis, although this result must be interpreted with caution.

Degradation artefacts during sample preparation for sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Preparation of samples for sodium dodecyl sulphate polyacrylamide gel electrophoresis routinely involves heating the protein in solution containing detergent and reducing agent for at least two minutes. Here we show that this treatment causes fragmentation of the protein glycogen phosphorylase, whether purified or as a component of a skeletal muscle preparation. The fragments are detected as minor bands on western blots and represent the products of discrete breakage point in the peptide sequence. Protease inhibitors cannot suppress the fragmentation. Such small amounts of immunoreactive fragments may be incorrectly identified on western blots as contaminants that were originally present in the antigen preparation. They may also be a source of ambiguity in studies that search for degradation intermediates during proteolysis.

Quantitation of glycogen synthase and phosphorylase protein in mouse liver: correlation between enzymatic protein and enzyme activity.

Phosphorylase and glycogen synthase protein were measured in normal and genetically diabetic (C57BL/KsJ db/db) mice liver extracts using rocket immunoelectrophoresis, and these data correlated with measurements of total phosphorylase and total glycogen synthase activities, respectively. Phosphorylase protein in 5-week-old normal mice was about 5 micrograms/mg protein and reached 8 micrograms/mg protein by 9 weeks. In comparison, the diabetic mice had elevated levels of phosphorylase protein (11-13 micrograms/mg protein) which correlated with an increased total phosphorylase activity compared to normals. The correlation coefficient for the phosphorylase activity vs protein plot was highly significant (r = 0.73, P less than 0.001). The molar concentration of phosphorylase subunit in normal mouse liver was calculated to be 11 microM and up to 23 microM in the diabetic mice. The liver concentration of glycogen synthase was relatively constant in normal mice at 400 ng/mg protein (corresponding to approximately 1.4 microM) but varied from 230 to 441 ng/mg protein (0.9 to 1.8 microM) in diabetic mice. There was little correlation between glycogen synthase activity and enzymatic protein (r = 0.15). These results indicate (1) that phosphorylase is present at concentrations approximately 10 times that of glycogen synthase, and (2) that glycogen synthase activity is relatively more dependent upon factors other than the amount of enzymatic protein.

Quantitation of muscle glycogen phosphorylase mRNA and enzyme amounts in adult rat tissues.

Mammalian glycogen phosphorylases comprise a family of isozymes that are expressed selectively in a variety of cell types. As an initial step towards understanding the molecular processes that regulate the differential expression of the phosphorylase family, we have begun a quantitative examination of isozyme expression in vivo. In this paper, we report quantitative estimates of the amounts of the muscle (M) isozyme and its mRNA in adult rat tissues. Quantitative estimates of the amount of M-phosphorylase were obtained by an analysis involving electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose filters and sequential treatment with M-isozyme specific antibody and radioactively- labeled protein A. M-phosphorylase mRNA amounts were determined by an analysis involving transfer of RNA from agarose gels to nitrocellulose filters and subsequent hybridization with radioactively labelled rat M-phosphorylase cDNA. These studies indicate that M-phosphorylase is present in all tissues tested with the possible exception of liver. These are skeletal muscle, heart, brain, stomach, lung, kidney, spleen and testis. Quantitation of M-phosphorylase amounts indicate that there is a wide spectrum of variation (over 1000-fold range) in the relative amounts of the M-isozymes in these tissues. Relative mRNA levels parallel isozyme levels indicating that the major control of expression of this isozyme is governed by mRNA accumulation.

The diurnal rhythm of liver glycogen phosphorylase: correlating changes in enzyme activity and enzymic protein.

The diurnal rhythm of phosphorylase activity in mouse liver extracts was correlated with the 24 h fluctuations in phosphorylase protein. This last measurement was made using rocket immunoelectrophoresis. The peak activity of phosphorylase appeared coincident (at 18:00 h) with the greatest amount of phosphorylase protein detected. Conversely, the lowest activity measured and lowest enzymic protein content both occurred at 02:00 h. Regression analysis revealed a significant positive correlation between enzyme activity and protein. Thus changes in the cellular concentration of this enzyme are implicated in the diurnal rhythm of liver glycogen.

Association of chicken pectoralis muscle phosphorylase with the Z-line and the M-line of myofibrils: comparison with 'amorphin', the amorphous component of the Z-line.

By immunofluorescence technique, glycogen phosphorylase, one of the soluble glycolytic enzymes, was shown to be localized in the Z-line of chicken pectoralis muscle myofibrils, in addition to the M-line, as previously reported by Heizmann, C.W. and Eppenberger, H.M. (Heizmann, C.W. and Eppenberger, H.M. (1978) J. Biol. Chem. 253, 270-277). After extraction of thick filaments by a solution containing pyrophosphate and high salt (Hasselbach-Schneider solution), or after extraction of thin and thick filaments by a solution containing 0.6 M KI, phosphorylase still remained in the Z-line. Amorphin (Mr 85 000) was reported by Chowrashi, P.K. and Pepe, F.A. (Chowrashi, P.K. and Pepe, F.A. (1982) J. Cell Biol. 94, 565-573) as a new Z-line amorphous component. The amino acid composition of amorphin reported by them was very similar to that of phosphorylase b reported by Heizmann and Eppenberger. The partially purified 85 kDa protein, according to Chowrashi and Pepe, showed cross-reactivity to anti-phosphorylase serum and phosphorylase activity. Although amorphin was reported to be eluted from DEAE-column chromatography around the gradient of 0.5 M KCl, little protein was eluted around 0.5 M KCl in our experiments, and the 85 kDa protein which we identified as phosphorylase b was eluted around 0.2 M KCl. Hence, it should be said that the major 85 kDa protein extracted according to Chowrashi and Pepe was phosphorylase b.

Isolation and comparative studies of glycogen phosphorylase isoenzymes from lymphocytes and polymorphonuclear leucocytes.

Two glycogen phosphorylase isoenzymes have been identified in pig lymphocytes and polymorphonuclear leucocytes by DEAE cellulose chromatography. Both isophosphorylases have been further purified by affinity chromatography on Sepharose-AMP to almost homogeneity. The purified isophosphorylases were composed of subunits of molecular weight similar to the muscle and liver monomers. Isophosphorylase I was more related to the liver enzyme than isophosphorylase II based on immuno-inhibition experiments. Both isoenzymes were markedly different from liver and muscle phosphorylases in their activation by AMP, sodium sulfate and 1,2-dimethoxyethane.

The high autoantibody activity of antibodies to rat skeletal muscle glycogen phosphorylase b produced in rabbits.

Antibodies to rat skeletal muscle glycogen phosphorylase b were prepared in six rabbits by weekly injection of the enzyme emulsified with complete Freund's adjuvant. All the antiserum preparations showed high autoantibody activities to react with the rabbit muscle enzyme in both inhibition of enzyme activity and precipitation. In Ouchterlony double diffusion in agar, the antiserum preparations were precipitable to give a distinct spur between the two precipitin lines formed with rat and rabbit enzymes. When the autoantibody index was taken as per cent cross-reactivity of rabbit enzyme with rat enzyme, the autoantibody indices of inhibition and precipitation of one of the antiserum preparations were as high as 98% and 52.3%, respectively.