Biochemical and immunological characterization of A1 adenosine receptors purified from human brain membranes.

The A1 adenosine receptor was purified approximately 13,000-fold to apparent homogeneity from human cerebral cortex membranes using a novel affinity-chromatography system developed for the purification of rat brain and rat testis A1 adenosine receptors [Nakata, H. (1989) J. Biol. Chem. 264, 16,545-16,551; Nakata, H. (1990) J. Biol. Chem. 265, 671-677]. The purified human brain receptor showed the ligand-binding specificity expected of the A1 adenosine receptor. The Bmax and Kd for the purified receptor with a specific A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, were approximately 16 nmol/mg protein and 2 nM, respectively. SDS/PAGE of the purified receptor preparation showed one broad protein band of molecular mass of approximately 35 kDa, which is very similar to that of purified A1 adenosine receptor from rat brain membranes. Endoglycosidase F treatment of the purified receptor reduced the molecular mass to approximately 30 kDa, suggesting that the human brain A1 adenosine receptor is a glycoprotein. Comparison of the purified human and rat brain A1 adenosine receptors by peptide mapping after the proteolytic digestion showed minor differences between these receptors. Immunological comparisons of the human brain A1 adenosine receptor with rat brain A1 adenosine receptor using polyclonal antibodies against the purified rat brain A1 adenosine receptor showed that the antibodies react preferentially with the rat brain receptor and weakly with human brain receptor.     

Biochemical and immunological studies of three genetic variants of 3-phosphoglycerate kinase 2 from the mouse

Three electrophoretic variants of 3-phosphoglycerate kinase 2 (PGK-2A, PGK-2B, and PGK-2C) were purified from DBA/2J, C3H/HeJ, and C57L/J mice, respectively. PGK-2C exhibits only 2% of the specific activity of PGK-2A and PGK-2B in the reaction leading to the formation of 1,3-diphosphoglycerate. Compared to PGK-2A and PGK-2B, PGK-2C exhibits broader coenzyme specificity and lower K_ms for substrate and coenzymes. Incubation at 45C revealed that PGK-2B is more heat stable than either PGK-2A or PGK-2C. Enzyme immunoinactivation and double immunodiffusion studies showed that mice carrying any one of these three PGK-2 alleles have similar amounts of proteins for PGK-1 and PGK-2 in testes. The results of these studies suggest that low PGK-2C activity in C57L/J mice is a result of a structural rather than a regulatory gene mutation.     

Biochemical and immunological studies of protein kinase C from Trypanosoma cruzi.

Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.     

Biochemical, immunological, and cell genetic studies in glycogenosis type II.

Fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis type II (Pompe disease) were cultured under standardized conditions, and the activity of acid alpha-glucosidase (E.C.3.2.1.20) towards glycogen, maltose, and 4-methylumbelliferyl-alpha-D-glucopyranoside was measured. Glycogen levels in muscle biopsies and in cultured fibroblasts from patients were determined. Residual enzyme activities varying from 7%-22% were detected in fibroblasts from patients with the adult form but not from patients with the infantile form of glycogenosis II. An inverse correlation was found between the severity of the clinical manifestation and the degree of residual enzyme activity in the fibroblasts. The kinetic and electrophoretic properties of acid alpha-glucosidase in fibroblasts from the adult patients and from control individuals were similar. Immunological studies suggested that the decrease of acid alpha-glucosidase activity is caused by a mutation that affects the production or degradation of the enzyme rather than its catalytic activity. Complementation studies were carried out by fusing fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis II, but neither conventional assays on multikaryons nor enzyme assays on single binuclear heterokaryons gave any evidence for genetic heterogeneity among these forms.     

Biochemical and immunological characterization of genetic variants of phosphoglucose isomerase from mouse

Two electrophoretic variants of phosphoglucose isomerase (PGI) were purified from whole body extracts of DBA/2J and C57BL/6J mice by a substrate-affinity elution from an 8-(6-aminohexyl) amino-ATP-Sepharose column followed by preparative isoelectric focusing. Both PGI variants were shown to be dimers of the same molecular weight, sedimentation coefficient, and K _m for fructose-6-phosphate. The isoelectric points were found to be 8.4 and 8.7 for variants from DBA/2J and C57BL/6J mice, respectively. Differential thermal stability was observed for the two variants in 0.1 m tris-HCl buffer, pH 8.0, at 54 C; the half-lives of the purified PGI from DBA/2J and C57BL/6J mice were shown to be 3.4 and 1.8 min, respectively, under those conditions. Similar differences were observed for the enzyme variants in the crude homogenates. Antisera against PGI from DBA/2J mice were raised in rabbits. The variants from DBA/2J and C57BL/6J mice showed no significant differences in their respective inactivation curves by the antisera. Results of amino acid composition analyses and peptide mappings of the two PGI variants indicate that the genetic variation of this enzyme might result from a single charged amino acid substitution.D. J. C. is a National Institutes of Health Visiting Fellow.     

DNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors

The phage DNA-directed synthesis of beta-galactosidase has been examined in a system containing the following purified Escherichia coli factors: RNA polymerase; cyclic AMP receptor protein; N10-formyltetrahydrofolate Met-tRNAf transformylase; initiation factors 1, 2, and 3; elongation factors Tu, Ts, and G; release factors 1 and 2; 20 aminoacyl-tRNA synthetases; L factor (Kung, H. F., Spears, C., and Weissbach, H. (1974) J. Biol. Chem. 250, 1556-1562); and Lalpha (Kung, H.-F., Spears, C., and Weissbach, H. (1976) Fed. proc. 35, 1537). Under these conditions, beta-galactosidase synthesis occurs at less than 1% of the rate obtained with unfractionated extracts, which suggested that other required components were lacking. The difficulty in obtaining large amounts of the purified aminoacyl-tRNA synthetases for these studies made it necessary to modify the system. It was possible to conserve many of the purified aminoacyl-tRNA synthetases since at least 13 of them could be replaced by an Ehrlich ascites extract. The ascites extract plus other E. coli purified factors was used as a basic system to search for additional components required for beta-galactosidase synthesis. The present report describes the purification from E. coli extracts of three fractions, called Lbeta, Lgamma, and Ldelta, that are needed to restore enzyme synthesis.     

Aggregation-dissociation and stability of acid beta-galactosidase purified from porcine spleen

Sucrose gradient centrifugation of the monomeric form (A1) of porcine spleen beta-galactosidase showed a pH-dependent equilibrium between monomer at neutral pH (pH 7.0) and dimer at acidic pH (pH 5.4-3.0), independent of ionic strength. While the oligomeric form (Ao), which was hardly dissociated under physiological conditions, was dissociated only with some protein denaturing agents into similar catalytic subunit to the A1. Both the A1 and Ao were equally active and stable at acidic pH, in the physiological condition inside lysosome (around pH 4.6).     

Genetic control of cell-type-specific levels of mouse beta-galactosidase.

Tissue analyses of mouse β-galactosidase (BGS) activity indicate previously unobserved genetic complexities. The existing genotypic classification of Bgs^h (high-activity) vs Bgs^d (diminished-activity) alleles has been based exclusively on strain differences in brain [Felton, J., Meisler, M., and Paigen, K. (1974). J. Biol. Chem.249, 3267–3272]. However, it now appears that other tissues in a strain with “high BGS” in brain can be relatively low in activity, or that tissues in a “low-BGS” strain can be relatively high. In addition, different cell types within a tissue (e.g., exocrine vs endocrine pancreas) can vary in BGS activity independently of each other. A given tissue ranges, among strains tested here, between 1.8- and 3.6-fold over the activity of the lowest strain. These new results suggest that complex mechanisms may be involved in tissue- and cell type-specific control of BGS activity. The hypothesis is proposed that there may be “controlling genes” determining specific enzyme levels by causing a limited somatic amplification of the structural gene and by controlling the degree of amplification in a given cell type and strain.     

Heterogeneity of purified mouse interferons.

A procedure for obtaining highly purified stable interferon from mouse L cells is described. Interferon with a specific activity of 2.5 times 10-8 reference units/mg of protein is composed of 10 to 11 polypeptides. They differ in their molecular size as determined by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The molecular weight range is estimated to be from the smallest at 20, 000 to the largest at 32, 000. At least six of the polypeptides are glycoproteins and each of the polypeptides can be obtained as an apparent homogeneous entity and each has interferon activity.     

Biochemical and immunological characterization of a histone variant associated with spermatogenesis in the mouse

A chromosomal histone, H2S, specific to the mouse testis has been purified. Amino acid analysis indicated lack of cysteine and a high basic amino acid content typical of histones. Specific antibodies against histones H2S have been generated in rabbits and partially purified using (NH₄)₂SO₄ precipitation and ion-exchange chromatography. Protein transfer experiments indicate presence of antigenically similar histones in the rat and rabbit testes but not in the guinea pig and dog testes. In addition, histone complement of somatic tissues such as lung, kidney, liver and spleen lacked antigenically similar proteins. Immunocytochemical studies using peroxidase-antiperoxidase complex indicated presence of immunoreactive cells in the seminiferous epithelium which were lacking in the interstitium. These data demonstrate histone H2S to be a unique histone associated with spermatogenesis in the mouse.     

Biochemical and immunological characterization of an H2A variant from the mouse testis

A histone H2A variant, protein 'A', has been isolated and purified from the mouse testis. Amino acid composition analysis and electrophoretic properties indicate it to be apparently similar to H2A X X variant present in the mouse L1210 cells. Specific antibodies against protein 'A' have been generated in rabbits and used to study tissue and species distribution in mammals. Protein transfer experiments indicate the presence of antigenically similar proteins in somatic tissues of the mouse. Immunologically similar proteins were also detected in other mammalian testes. The data further indicate that protein A is antigenically distinct from the other members of the H2A family, H2A X 1, H2A X 2 and H2A X Z.     

Purification of mitochondrial creatine kinase. Biochemical and immunological characterization

Mitochondrial creatine kinase was purified from canine myocardium. The preparation exhibited a positively charged isoenzyme free of other creatine kinase isoenzymes and on sodium dodecyl sulfate gel exhibited a single protein band. Amino acid composition showed mitochondrial creatine kinase to be different from that of MM or BB creatine kinase and did not hybridize with the M or B subunits of the cytosolic forms. Antiserum was developed to mitochondrial creatine kinase which did not cross-react with cytosolic creatine kinases. Antiserum to cytosolic creatine kinase exhibited no reaction to mitochondrial creatine kinase. Utilizing the specific antiserum, a radioimmunoassay was developed for the specific detection of mitochondrial creatine kinase. Thus, mitochondrial creatine kinase was purified and shown to be comprised of a unique subunit which is biochemically and immunologically distinct from the cytosolic creatine kinases.     

Biochemical and immunological analysis of an abundant form of glutathione S-transferase,in mouse testis

One of the major forms of glutathione S-transferase (designated as Ft transferase) has been identified and purified to near homogeneity from mouse testis. The purification was achieved by ammonium sulfate fractionation, DEAE cellulose chromatography, hydroxylapatite chromatography and the preparative isoelectric focusing. Purified Ft transferase has an isoelectric point of 4.9 ± 0.3 and was shown to be a homodimer with a native molecular weight of about 50 000.Immunologically, antisera to Ft transferase do not crossreact with F2 or F3 transferase. However, a weak cross reactivity was observed between the antisera to F3 transferase and Ft transferase. Biochemical properties of purified Ft transferase are similar to those transferases isolated from mouse liver. Tissue distributions of the multiple forms of glutathione S-transferase were examined by column isoelectric focusing of various mouse tissue homogenates. It was found that mouse Ft transferase is present only in testis as a major form and in brain as a minor form, but not in other tissues that were examined.     

Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells

Juxtanuclear birefringent caps (FC) containing 10-nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KCl, 1% Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10-nm filaments that is seen in situ. Up to 90% of the FC protein is resolved as two polypeptides of approximately 54,000 and 55,000 molecular weight on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels, and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10-nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10-nm filaments has been achieved by treatment of FC with 6 mM sodium- potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10-nm filaments upon the addition of 0.171 M sodium chloride.     

Biochemical and immunological characterization of rice albumin

Rice albumin fromOryza sativa (Var. Basmati 370) accounts for about 5% of the total seed proteins. A major fraction of rice albumin has been found to be a glycoprotein which is a monomer of 60 kd having iso-electric point 6.54. When rice albumin is digested with trypsin, it shows the presence of 24 peptides as against 28 peptides which were estimated from its amino acid Composition. This indicates the presence of a few peptides which resemble each other in their charge and R_f values. Antibodies against Con A purified rice albumin were affinity purified and were used to quantitate the rice albumin levels during post-anthesis by RIA and ELISA. The latter experiments reveal that maximum albumin is present between 18 and 20 days post-anthesis.NCL Communication No. 4187.     

Light-harvesting complexes of brown algae. Biochemical characterization and immunological relationships

The pigment composition of the light-harvesting complexes isolated from several brown algae belonging to different orders has been analysed by reverse-phase HPLC. Relative to whole chloroplasts, they were markedly enriched in Chl c, fucoxanthin and violaxanthin and conversely depleted in Chl a. The relative molar proportions of the 4 main pigments (Chl a/Chl c/fucoxanthin/violaxanthin) ranged from 100:18:76:6 to 100:30:107:17. The protein moiety of LH complexes of all the species studied were composed of one or two main polypeptide components in the range of 19-22 kDa. These polypeptide subunits were arranged in polymeric particles about 240 kDa in Laminaria saccharina. A polyclonal antibody raised against the LH polypeptide of Fucus serratus has been tested on LH apoproteins of other Chromophytes and Chlorophytes. Phylogenic implications of these results are discussed.     

Highly purified bile-canalicular vesicles and lateral plasma membranes isolated from rat liver on Nycodenz gradients. Biochemical and immunolocalization studies.

1. A liver canalicular plasma-membrane fraction enriched 115-155-fold in five marker enzymes relative to the tissue homogenate was obtained by sonication of liver plasma membranes followed by fractionation in iso-osmotic Nycodenz gradients. 2. Two lateral-plasma membrane fractions were also collected by this procedure; the lighter-density fraction was still associated with canalicular membranes, as assessed by enzymic and polypeptide analysis. 3. The polypeptide composition of the domain-defined plasma-membrane fractions was evaluated. It was demonstrated by immunoblotting that the 41 kDa alpha-subunit of the inhibitory G-protein, associated in high relative amounts with canalicular plasma-membrane fractions, was partially lost in the last stage of purification; however, this subunit was retained by lateral plasma membranes. 4. Antibodies to the proteins of bile-canalicular vesicles were shown to localize to the hepatocyte surface in thin liver sections examined by immunofluorescent and immuno-gold electron microscopy. Two subsets of antigens were identified, one present on both sinusoidal and canalicular plasma-membrane domains and another, by using antisera pre-absorbed with sinusoidal plasma membranes, that was confined to the bile-canalicular domain.