Three types of mouse peptidylarginine deiminase: characterization and tissue distribution.

Three types of mouse peptidylarginine deiminase were separated by DEAE-Sephacel ion-exchange column chromatography, and we propose designating them peptidylarginine deiminase type I, II, and III according to the order of elution. The type II enzyme was widely distributed in various tissues including the skeletal muscle, whereas the type I enzyme was localized in the epidermis and uterus, and the type III enzyme was detected in the epidermis and hair follicles. These enzymes were distinguished by their molecular weights and substrate specificity. The molecular weights were estimated to be approximately 54,000 (type I) and 100,000 (type II and III) by Sephacryl S-200 gel filtration column chromatography. On SDS-PAGE the type II and III enzymes gave Mr = 81,000 and Mr = 76,000, respectively. Among the substrates tested, the type I enzyme showed highest activity toward BZ-L-Arg-NH2, type II toward BZ-L-Arg-O-Et, and type III toward protamine. Western blot analysis showed that antibodies against the type II enzyme were immuno-crossreactive to the type III enzyme.     

Immunocytochemical demonstration of skeletal muscle type peptidylarginine deiminase in various rat tissues

We have performed an immunocytochemical study of peptidylarginine deiminase (EC 3.5.3.15) in various rat tissues using an antiserum to the enzyme purified from rat skeletal muscle. Staining was observed in skeletal muscle fibers, glia cells of the central nervous system, serous cells of submandibular gland, demilunar cells (serous cells) of sublingual gland, uterine endometrium and myometrium, and certain cells in the lamina propria of intestinal villi. Possible involvement of the enzyme in multiple cellular processes were discussed.     

Isolation and characterization of cDNA clones encoding rat skeletal muscle peptidylarginine deiminase

Various mammalian tissues contain protein-arginine deiminases (EC 3.5.3.15), which convert the arginine residues in normal peptide bonds to the citrulline residues in calcium ion-dependent manners. Here, we describe the complete primary structure of rat skeletal muscle peptidylarginine deiminase deduced from the sequences of its cDNA clones isolated by recombinant DNA technology. We have isolated three overlapping cDNA clones which constitute a 4,507-base pair cDNA sequence including a 2,452-base pair 3'-untranslated region. The coding region consists of 1,995 base pairs encoding 665 amino acid residues. A potential N-linked glycosylation site is present at asparagine-534. The molecular weight of the enzyme calculated from the deduced amino acid sequence is 75,122. Direct repeat sequences resembling the rodent B2 type repetitive sequences appear in the 3'-untranslated region (nucleotides 3,090-3,198 and 3,270-3,391). Northern hybridization demonstrated the presence of its mRNA in poly(A)+ fractions of spinal cord, cerebrum, cerebellum, and submaxillary gland as well as skeletal muscle. The sizes of peptidylarginine deiminase mRNAs in these tissues were estimated to be 4.5-5.0 kilobases. No positive bands were detected on the blots of the corresponding RNA fractions of liver and kidney. Possible similarity of the amino acid sequence of peptidylarginine deiminase to those of other calcium binding proteins is discussed.     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

Isozymes of fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase in rat and bovine heart, liver, and skeletal muscle

The distribution of Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase in rat and bovine heart, liver, and skeletal muscle tissues was examined. With DEAE-cellulose chromatography, two peaks (I and II) of Fru 6-P,2-kinase activity were detected in all tissue extracts. Peak I was the predominant form both in rat and bovine heart tissue, while peak II was the major form in liver and skeletal muscle. Antibodies to heart enzyme reacted specifically with peak I, and antibodies to liver enzyme reacted with peak II from both liver and skeletal muscle. All the isozymes were bifunctional. All the tissues examined contained other isozymes in minor amounts.     

Estrogen regulates peptidylarginine deiminase levels in a rat pituitary cell line in culture

A Nonidet P-40 extract of growth hormone-producing rat pituitary MtT/S cells was found to contain peptidylarginine deiminase (EC 3.5.3.15), which was indistinguishable from an enzyme preparation from rat muscle in Western immunoblotting and immunoprecipitation. This enzyme was immunocytochemically detected in the cytoplasm but was not secreted into the medium during the cultivation. When the cells were cultured for 2 days with various concentrations of 17 beta-estradiol (E2), the enzyme activity increased in a dose-dependent manner, reaching a maximum level (four- to fivefold higher than control) at about 10(-9) M. This increase in the enzyme activity was evident by 14 hr of culture and became relatively stable after 24 hr. It correlated well with the increase in the amount of the muscle type enzyme per cell as analyzed by Western immunoblotting. Estriol and a synthetic estrogen, diethylstilbestrol, also increased the enzyme activity, whereas testosterone, progesterone, and corticosterone were without effect. An antiestrogen, tamoxifen, which by itself was inactive, partially suppressed the effect of E2. Exposure of MtT/S cells for 14 hr to E2 increased incorporation of 35S-labeled amino acids into the immunoprecipitable peptidylarginine deiminase. This increase was dependent on the concentration of E2, attaining a maximum level (about tenfold higher than the control) at about 10(-9) M. These results indicate that estrogen effects the increase in peptidylarginine deiminase content in the pituitary cells by stimulating enzyme synthesis.     

Affinity chromatography of peptidylarginine deiminase from rabbit skeletal muscle on a column of soybean trypsin inhibitor (Kunitz)-Sepharose

We designed a simple procedure for the purification of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle using substrate affinity chromatography. Of the immobilized substrate ligands tested, i.e. protamine and soybean trypsin inhibitor (Kunitz) (STI), STI-Sepharose was found to be an effective affinity adsorbent for purification of the enzyme. The specific binding of peptidylarginine deiminase to STI-Sepharose was observed in the presence of calcium ion, and the enzyme could be selectively eluted from the affinity adsorbent by washing with chelator. A 1,800-fold purification with a 50% yield was achieved in the three-step procedure, which involved DEAE-Sephacel ion-exchange and STI-Sepharose affinity chromatography. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity and the recovery were considerably higher than have been obtained by any procedures previously reported. The specific interaction of peptidylarginine deiminase with STI immobilized on Sepharose was also investigated quantitatively by frontal affinity chromatography. In this method, a peptidylarginine deiminase solution was applied continuously to an STI-Sepharose column and the retardation of the elution front was measured as a parameter of the strength of the interaction. The dissociation constant for the enzyme with STI was found to be 2.3 X 10(-7)M. This value was in good agreement with that obtained by kinetic analysis in our previous studies. Peptidylarginine deiminase required millimolar Ca2+ for the binding to STI-Sepharose. The Ca2+ dependence of the enzyme binding was quite similar to that of the enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular localization of glycogen synthase with monoclonal antibodies

Two monoclonal antibodies, designated 7H5 and 8E11, were produced against glycogen synthase purified from rabbit skeletal muscle. Both antibodies were of the IgG1 (k) isotype. Western blot analysis of extracts of rat and rabbit tissues showed that antibody 7H5 recognized glycogen synthase from skeletal and cardiac muscles, but not from liver. Antibody 8E11 gave similar results but the responses were weaker. Antibody 7H5 also recognized a 69,000 dalton tryptic fragment of glycogen synthase whereas antibody 8E11 did not bind this fragment. Immunocytochemical staining of rabbit skeletal muscle with antibody 7H5 indicated two major sites of glycogen synthase localization. A granular localization present in the cytoplasm and a band-like staining associated with the Z-disk region of the myofibrils. Rabbit cardiac muscle presented a similar pattern though less cytoplasmic staining was apparent. An assay of subcellular fractions for glycogen synthase indicated that the enzyme in cardiac and skeletal muscles is distributed between the soluble (80-90%) and myofibrilar (10-20%) fractions of the tissues. These results provide direct evidence for the presence of glycogen synthase in subcellular fractions other than the soluble fraction of skeletal and cardiac muscles.     

Sensitive immunoassay for rat parvalbumin: tissue distribution and developmental changes

A sensitive enzyme immunoassay for measurements of rat parvalbumin was established using antibodies raised in rabbits with parvalbumin purified from skeletal muscles. Antibodies in the antiserum were purified with a parvalbumin-coupled Sepharose column. The sandwich-type immunoassay system for parvalbumin was composed of polystyrene balls with immobilized purified antibodies and the same antibodies labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg parvalbumin/tube. The assay did not cross-react with other calcium binding proteins, including human S-100a0 and S-100b proteins, rat 28-kDa calbindin-D, and bovine calmodulin. High concentrations of parvalbumin were observed in the skeletal muscles, especially in those composed of fast-twitch fibers, and in the diaphragm and tongue, but not in heart muscle. A relatively high concentration was estimated in the central nervous tissue. Parvalbumin was detected in the cerebral cortex and cerebellum of gestational 15-day fetuses. However, the levels of parvalbumin in the muscle tissues and central nervous tissue were very low in rats before 1 week of age. Thereafter, they increased sharply, reaching the adult levels by 5 weeks in most of the tissues. Parvalbumin concentrations in adult rat soleus muscle increased less than 20-fold within 10 days after transection of the ipsilateral sciatic nerve, while the concentrations in the extensor digitorum longus muscle did not change in the same period.     

Phylogenetic conservation of epitopes in mammalian aldose reductase: application to immunoquantitation

Rabbit antibodies raised against bovine kidney aldose reductase (ALR2) were shown to be monospecific by Western blot analysis of kidney homogenates. In addition, the antiserum (alpha-BKALR2) reacts with a single electrophoretic species in homogenates from rabbit, porcine, and human kidney. ALR2 has been detected in homogenates of bovine kidney, heart, brain and lens, and estimation of the enzyme level in these tissues was accomplished by densitometric analysis of Western blots. Standard curves using highly purified bovine kidney ALR2 were linear in the range of 5-100 ng; a similar sensitivity was seen in tissue homogenates. The results presented here for the ALR2 level in bovine tissues (kidney greater than heart greater than brain greater than lens) are in agreement with literature values for those tissues from which the enzyme has previously been purified. The interspecies similarity in electrophoretic mobility and the retention of antibody reactivity suggest extensive phylogenetic epitope conservation in mammalian aldose reductase.     

Quantitation of tyrosine hydroxylase, protein levels: spot immunolabeling with an affinity-purified antibody

Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and 125I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background 125I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.     

Location of phosphoglycerate mutase in rat skeletal muscle. An immunocytochemical and biochemical study

The subcellular distribution of phosphoglycerate mutase was studied by immunogold techniques. With the aid of highly affinity-purified anti-phosphoglycerate mutase antibodies, the enzyme was found in both cytosol and nucleus of rat skeletal muscle. No evidence of interaction with contractile proteins was observed in cytosol. Nuclear location was also confirmed biochemically using purified nuclear preparations from rat skeletal muscle. Only one immunoreactive nuclear band was observed by Western blot experiments and corresponded to that of phosphoglycerate mutase mobility. Activity measurements from nuclear extracts showed that 25% of total specific activity is found in the nuclei.     

Immunological characterization of phosphoprotein phosphatases

Phosphoprotein phosphatases regulate the biological activities of proteins through their involvement in cyclic phosphorylation/dephosphorylation cascades. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 and types 2A, 2B, and 2C. To elucidate the relationship between the different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes were tested for immunological antigenic relatedness. Two heterologous antibody preparations were employed for this purpose. One was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (calcineurin, holoenzyme). Specific subunit cross-reactivity was examined by protein blot ("Western") analysis. The antibody to the type 2A phosphatase reacted with the catalytic subunits of every type 2 enzyme tested, including both the catalytic and Ca2+-binding subunits of the Ca2+/calmodulin-dependent type 2B phosphatase (calcineurin), bovine cardiac type 2A phosphatase, and turkey gizzard smooth muscle phosphatase-1 (type 2A1). It did not react with any type 1 phosphatase (catalytic subunit or ATP-Mg-dependent). The antigenic relatedness of calcineurin and the bovine cardiac type 2A phosphatase (Mr 38,000) was demonstrated further by protein blot analysis showing that the anti-calcineurin antibody cross-reacted with both enzymes. The mutual cross-reactivity poses an intriguing problem because these enzymes are so different in their molecular structures and modes of regulation. The degree of evolutionary conservation exhibited by the antigenic cross-reactivity of the type 2 enzymes from a broad range of species and tissues suggests a strong selective pressure on maintaining one or more features of these important regulatory enzymes.     

Antiserum against the catalytic subunit of adenosine 3'':5''-cyclic monophosphate-dependent protein kinase. Reactivity towards various protein kinases.

An antiserum against the catalytic subunit C of cyclic AMP-dependent protein kinase, isolated from bovine heart type II protein kinase, was produced in rabbits. Reaction of the catalytic subunit with antiserum and separation of the immunoglobulin G fraction by Protein A-Sepharose quantitatively removed the enzyme from solutions. Comparative immunotitration of protein kinases showed that the amount of antiserum required to eliminate 50% of the enzymic activity was identical for pure catalytic subunit, and for holoenzymes type I and type II. The reactivity of the holoenzymes with the antiserum was identical in the absence or the presence of dissociating concentrations of cyclic AMP. Most of the holoenzyme (type II) remains intact when bound to the antibodies as shown by quantification of the regulatory subunit in the supernatant of the immunoprecipitate. Titration with the antibodies also revealed the presence of a cyclic AMP-independent histone kinase in bovine heart protein kinase I preparations obtained by DEAE-cellulose chromatography. Cyclic AMP-dependent protein kinase purified from the particulate fraction of bovine heart reacted with the antiserum to the same degree as the soluble enzyme, whereas two cyclic AMP-independent kinases separated from the particle fraction neither reacted with the antiserum nor influenced the reaction of the antibodies with the cyclic AMP-dependent protein kinase. Immunotitration of the protein kinase catalytic subunit C from rat liver revealed that the antibodies had rather similar reactivities towards the rat liver and the bovine heart enzyme. This points to a relatively high degree of homology of the catalytic subunit in mammalian tissues and species. Broad applicability of the antiserum to problems related to cyclic AMP-dependent protein kinases is thus indicated.     

Immunoquantitation of aldose reductase in human tissues

Rabbit antibodies raised against bovine kidney aldose reductase (ALR2) were shown to be monospecific for human ALR2 by Western blot analysis of human muscle homogenates. The human enzyme was detected, by reaction with the antiserum (alpha-BKALR2), in homogenates of adrenal gland, muscle, lens, brain, testes, kidney, and placenta, but not in erythrocytes or leukocytes. The amount of enzyme in each tissue was determined by densitometric analysis of autoradiographs of Western blots probed with alpha-BKALR2 and [125I]protein A. Standard curves of radiographic intensity versus amount of purified human muscle ALR2 were linear in the 20 to 200-ng range; a similar sensitivity was seen in tissue homogenates containing up to 675 micrograms total protein. The results presented here for the ALR2 level in human tissues (adrenal greater than muscle greater than lens approximately brain approximately testes greater than kidney approximately placenta) are in agreement with literature values for those tissues from which the enzyme has previously been purified. A notable exception was the absence of detectable ALR2 in human erythrocytes. A quantitative comparison of immunoradiographic response showed that bovine kidney ALR2 was about sevenfold more reactive with a alpha-BKALR2 compared to the human muscle enzyme.     

Isolation and properties of cytochrome c oxidase from rat liver and quantification of immunological differences between isozymes from various rat tissues with subunit-specific antisera

Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c--Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase-conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.     

Heart cross-reactive antigens of mutans streptococci share epitopes with group A streptococci and myosin

Monoclonal antibodies (mAb) generated by immunization of mice with membranes of Streptococcus pyogenes Manfredo (M type 5) were tested for their reactivity with sodium dodecyl sulfate extracts of Streptococcus mutans GS5, Streptococcus rattus BHT (formerly S. mutans, serotype b), and S. mutans Ingbritt 175 in the Western blot. The reaction of the sodium dodecyl sulfate-extracted proteins of whole S. mutans, serotype b), and S. rattus with the mAb revealed that the shared cross-reactive antigens were near m.w. 62,000 to 67,000. Several higher m.w. proteins in S. mutans Ingbritt 175 reacted with the mAb but were not observed in the other two strains of streptococci. Cytoplasmic membranes of S. rattus BHT reacted with these mAb in the enzyme-linked immunosorbent assay and Western blot. The most reactive BHT membrane component detected by these mAb was a 62-kDa polypeptide that was similar in m.w. to the membrane component recognized by these antibodies in purified S. pyogenes membranes. The reactivity of the mAb with BHT membranes in the enzyme-linked immunosorbent assay was absorbed by rabbit skeletal muscle myosin. The patterns of reactivity observed in the BHT membrane immunoblots in this study closely resemble those previously obtained using polyclonal rabbit anti-human heart sera.     

Localization of mitochondrial 60-kD heat shock chaperonin protein (Hsp60) in pituitary growth hormone secretory granules and pancreatic zymogen granules.

We used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of Hsp60 in rat tissues. Western blot analysis, employing both monoclonal and polyclonal antibodies raised against mammalian Hsp60, shows that only a single 60-kD protein is reactive with the antibodies in brain, heart, kidney, liver, pancreas, pituitary, spleen, skeletal muscle, and adrenal gland. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows strong labeling of mitochondria in all tissues. However, in the anterior pitutary and in pancreatic acinar cells, Hsp60 also localizes in secretory granules. The labeled granules in the pituitary and pancreas were determined to be growth hormone granules and zymogen granules, respectively, using antibodies to growth hormone and carboxypeptidase A. Immunogold labeling of Hsp60 in all compartments was prevented by preadsorption of the antibodies with recombinant Hsp60. Biochemically purified zymogen granules free of mitochondrial contamination are shown by Western blot analysis to contain Hsp60, confirming the morphological localization results in pancreatic acinar cells. In kidney distal tubule cells, low Hsp60 reactivity is associated with infoldings of the basal plasma membrane. In comparison, the plasma membrane in kidney proximal tubule cells and in other tissues examined showed only background labeling. These findings raise interesting questions concerning translocation mechanisms and the cellular roles of Hsp60.     

Purification procedure and monoclonal antibodies: two instruments for research on vertebrate porins.

On Western blots of skeletal muscle preparations of different vertebrate classes, four monoclonal anti-human type 1 porin antibodies recognize one single band of either 30.5 or 31 kDa, respectively. To confirm that it is eukaryotic porin which is labeled by the antibodies, we used a purification procedure developed for human type 1 porin for porins from skeletal muscle of shark, frog, and turkey. Applied to different mammalian species and tissues, this procedure exclusively provides type 1 porin. However, applied to shark skeletal muscle, it provides two porin isotypes in nearly equal amounts. In the case of frog skeletal muscle, the procedure provides mainly type 2 porin and a lower amount of type 1 porin. Applied to turkey skeletal muscle, the method provides exclusively type 2 porin. As demonstrated by two-dimensional Western blots, both shark and frog porin isotypes and the turkey type 2 porin are recognized by our antibodies. Furthermore, we elucidated the entire amino acid sequence of frog type 2 porin.