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.     

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 characterization of serum biotinidase in profound biotinidase deficiency.

The biochemical and immunological characterization of biotinidase was performed in sera from 100 normal individuals, 68 children with profound biotinidase deficiency (less than 10% of mean normal activity) who were identified symptomatically and by newborn screening, and 63 of their parents. On isoelectric focusing, serum enzyme from normal individuals exhibits extensive microheterogeneity, consisting of at least four major and five minor isoforms at pH 4.15-4.35. Patients with profound biotinidase deficiency can be classified into at least nine distinct biochemical phenotypes, on the basis of (a) the presence or absence of cross-reacting material (CRM) to biotinidase, (b) the number of isoforms, and (c) the distribution frequency of the isoforms. None of the patients with CRM had an abnormal Km of the substrate for the enzyme. All of the parents had normal isoform patterns. The mean activities, CRM concentrations, and specific activities were not significantly different between parents of CRM-positive children and parents of CRM-negative children. There is no relationship between either the age at onset or the severity of symptoms and the isoform patterns or CRM status of the symptomatic children. The isoform patterns of children identified by newborn screening are not different from those of symptomatic children.     

Biochemical and immunological characterization of the duck hepatitis B virus envelope proteins.

To examine the envelope proteins of duck hepatitis B virus (DHBV), which are encoded by the pre-S/S open reading frame of the viral genome, an antiserum was raised in rabbits against a fusion protein comprising most of the pre-S coding segment. By using this antiserum, viral particles could be precipitated from serum, and two pre-S proteins with molecular sizes of approximately 35 and 37 kilodaltons were detected in the sera and livers of DHBV-infected ducks after Western blotting and after biosynthetic labeling of a primary duck liver cell culture. In serum, the pre-S proteins were shown to exist predominantly in DHBV-DNA-free particles associated with a 17-kilodalton protein which, by N-terminal amino acid sequence analysis, was shown to represent the viral S protein which is encoded by the 3' proximal segment of the DHBV pre-S/S open reading frame. To compare the immunogenic potential of the S and pre-S proteins, serum particles and gel-purified S protein were used to immunize rabbits. In neither case was a significant immune response against the DHBV S protein observed. However, a good antibody titer against DHBV pre-S was obtained even after immunization with small amounts of the pre-S antigen.     

Biochemical and immunological characterization of pea nuclear intermediate filament proteins

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6–12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.Abbreviations IF Intermediate filament - L Lamin fraction - LM Lamina-matrix fraction - MAb JLA20 Anti-chicken actin monoclonal antibody - MAb LN43 Anti-human lamin B2 monoclonal antibody - MAb PL19 Anti-pea lamin #19 monoclonal antibody - MAb TIB 131 Anti-intermediate filament monoclonal antibody - N Nuclei fraction - NEM Nuclear envelope-matrix fraction - NIF Nuclear intermediate filament - PAb PL3 Anti-pea lamin #3 polyclonal antibody     

Biochemical and immunological characterization of rat spleen prostaglandin D synthetase

Rat spleen prostaglandin D synthetase (Christ-Hazelhof, E., and Nugteren, D. H. (1979) Biochim. Biophys. Acta 572, 43-51) is very similar to rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi O. (1985) J. Biol. Chem. 260, 12410-12415) as judged by their pI (4.7-5.2), Mr (26,000-27,000), and self-inactivation during the isomerase reaction from prostaglandin H2 to prostaglandin D2. However, the amino acid compositions of these two enzymes were quite different. Furthermore, the spleen enzyme was associated with the glutathione S-transferase activity, differing from the brain enzyme. The synthetase and transferase activities of the spleen enzyme showed almost identical pH and glutathione dependencies, the optimum pH = 8.0 and Km for glutathione = 300 microM. The Km values for prostaglandin H2 and 1-chloro-2,4-dinitrobenzene (a substrate for the transferase) were about 200 microM and 5 mM, respectively. The synthetase activity was dose-dependently inhibited by 1-chloro-2,4-dinitrobenzene (IC50: approximately 5 mM) and more strongly by nonsubstrate ligands, such as bilirubin and indocyanine green (IC50: 150 and 2 microM, respectively). Both the synthetase and transferase activities of the purified enzyme dose-dependently decreased and showed identical immunotitration curves by incubation with antibody against this enzyme, but remained unchanged when treated with antibody against the brain enzyme. The antibody specific for the spleen enzyme absorbed almost all of the synthetase activity and about 10% of the transferase activity in the spleen, but not the transferase activity in the liver, heart, and testis. These results show that the two types of prostaglandin D synthetase are similar but different enzymes and that the spleen enzyme is a unique glutathione S-transferase differing from other isozymes and their subunits reported previously.     

Biochemical characterization of Yin Yang 1 - RNA complexes.

YY1 (Yin Yang 1) is present in the Xenopus oocyte cytoplasm as a constituent of messenger ribonucleoprotein complexes (mRNPs). Association of YY1 with mRNPs requires direct RNA-binding activity. Previously, we have shown YY1 has a high affinity for U-rich RNA; however, potential interactions with plausible in vivo targets have not been investigated. Here we report a biochemical characterization of the YY1-RNA interaction including an investigation of the stability, potential 5'-methylguanosine affinity, and specificity for target RNAs. The formation of YY1-RNA complexes in vitro was highly resistant to thermal, ionic, and detergent disruption. The endogenous oocyte YY1-mRNA interactions were also found to be highly stable. Specific YY1-RNA interactions were observed with selected mRNA and 5S RNA probes. The affinity of YY1 for these substrates was within an order of magnitude of that for its cognate DNA element. Experiments aimed at determining the potential role of the 7-methylguanosine cap on RNA-binding reveal no significant difference in the affinity of YY1 for capped or uncapped mRNA. Taken together, the results show that the YY1-RNA interaction is highly stable, and that YY1 possesses the ability to interact with structurally divergent RNA substrates. These data are the first to specifically document the interaction between YY1 and potential in vivo targets.     

Light-harvesting pigment-protein complexes of Rhodopseudomonas sphaeroides forma sp. denitrificans

The composition of the light-harvesting system of Rhodopseudomonas sphaeroides forma sp. denitrificans was investigated. When chromatophores were solubilized by sodium dodecyl sulfate (SDS) at 0 degrees C and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), at least two B800-B850 pigment-protein complexes, three B870 pigment-protein complexes, a reaction center (RC) complex and two pigmented bands which contained B800, B850, and B870 were resolved. In the re-electrophoresis, the B870 pigment-protein complexes gave rise to a series of multiple pigmented bands. All of these multiple pigment-protein complexes showed almost the same polypeptide composition and absorption spectrum characteristic of the B870 complex. The apparent molecular weights of these B870 complexes showed a regular interval of about 7,000 indicating that these complexes were oligomers of a subunit. It was also found that a predominant B800-B850 pigment-protein complex could be degraded into a small complex via some intermediates. These results indicate that essentially two kinds of pigment-protein complexes construct the light-harvesting system of this bacteria and, upon treatment with SDS, these complexes are degraded into many classes of subunit aggregates showing a complicated profile of pigmented bands on the gel. Pigmented bands which contained both of B800-B850 and B870 complexes were considered to arise from occasional co-migration of distinct B800-B850 and B870 pigment-protein complexes.     

Biochemical and immunological characterization of the major envelope glycoprotein of bovine leukemia virus.

The major envelope glycoprotein of bovine leukemia virus was isolated by lectin-bound Sepharose and DEAE-cellulose column chromatography. This protein was shown to have a molecular weight of about 41,000 and to lack detectable immunological cross-reactivity with glycoproteins of other oncornaviruses. Sera obtained from 100% of cattle examined with clinically diagnosed lymphosarcoma contained high-titered antibody to 125I-labeled bovine leukemia virus glycoprotein, whereas sera from animals in a disease-free herd were antibody negative.     

Biochemical and immunological relationships among fibronectin-like proteins from different sea urchin species

Fibronectin-like proteins were purified from ovaries of the sea urchin species, Paracentrotus lividus (PI), Sphaerechinus granularis (Sg), Arbacia lixula (Al), Pseudocentrotus depressus (Pd), and Anthocidaris crassispina (Ac), by gelatin-Sepharose affinity chromatography. The major component had a molecular mass of 180 kDa and was eluted by 1 M NaCl or 8 M urea, depending on the species used. By substrate adhesion assay, we tested the biological activity of the 180 kDa protein purified from Paracentrotus lividus (P1-180K) and showed that it promotes the adhesion of homologous embryonic cells to the substrate. An antiserum, developed against Temnopleurus hardwickii fibronectin-like protein (Th-180K), was used in Western blots of the proteins purified from the five species. The antibody cross-reacted with Pl-180K, Pd-180K and Ac-180K. A peptide map of P1-180K, obtained by V8 protease partial digestion, was compared with those obtained from the other four proteins and showed an homology between 40 and 56%. This report confirms that fibronectin-like proteins can be purified from sea urchins on the basis of their binding to gelatin-Sepharose; the proteins differ for their binding affinity to gelatin and share different epitopes, suggesting that they are members of a sea urchin fibronectin super family.     

The electrophoretic isolation and partial characterization of three chlorophyll-protein complexes from blue-green algae.

Three chlorophyll-protein complexes have been resolved from blue-green algae using an improved procedure for membrane solubilization and electrophoretic fractionation. One complex has a red absorbance maximum of 676 nm and a molecular weight equivalency of 255 000 +/- 15 000. A second complex has an absorbance maximum of 676 nm, a molecular weight equivalency of 118 000 +/- 8000, and resembles the previously described P-700-chlorophyll a-protein (CPI) of higher plants and algae. The third chlorophyll-protein has a red absorbance maximum of 671 nm and a molecular weight equivalency of 58 000 +/- 5000. Blue-green algal membrane fractions enriched in Photosystem I and heterocyst cells do not contain this third chlorophyll-protein, whereas Photosystem II-enriched membrane fractions and vegetative cells do. A component of the same spectral characteristics and molecular weight equivalency was also observed in chlorophyll b-deficient mutants of barley and maize. It is hypothesized that this third complex is involved in some manner with Photosystem II.     

Comparative immunological characterization of various photosynthetic cytochromes c from pro- and eukaryotic algae

Photosynthetic c-type cytochromes isolated from various pro- and eukaryotic algae have been compared by an immunochemical method. Thereby the extent of cross-reactivity of several cytochromes with antisera to cytochrome c from Spirulina platensis, Bumilleriopsis filiformis, and Scenedesmus acutus was quantitatively determined by antigen-binding tests. When immunological relationship is taken as a measure of structural relationship, the following conclusions can be drawn: (1) c-type cytochromes from Anabaena variabilis, Nostoc muscorum, Calothrix membranacea, and Spirulina platensis show large differences in cross-reactivity. (2) The acidic Spirulina cytochrome c is fairly closely related to the two eukaryotic cytochromes assayed here.Abbreviations SAUG Sammlung von Algenkulturen am Pflanzenphysiologischen Institut der Universität Göttingen, FRG - PCC Pasteur Culture Collection     

Biochemical and immunological characterization of the cloned crystal toxin of Bacillus thuringiensis var. israelensis

The protein components of the cloned crystal toxin of Bacillus thuringiensis var. israelensis were separated by polyacrylamide gel electrophoresis under denaturing conditions. Using an antiserum to the solubilized B. thuringiensis var. israelensis crystal protein as a probe, immunological homology between the crystal protein components of B. thuringiensis var. israelensis and those of the recombinant B. megaterium strain VB131 was tested. The results from this study indicate that the crystal inclusion of the recombinant strain contains only the 130 kilodalton protein and not the 68 or the 28 kilodalton proteins of the crystal toxin of B. thuringiensis var. israelensis and that the 130 kilodalton protein is primarily responsible for the mosquitocidal activity of this organism.     

Biochemical and immunological relationships of prostatic and leukocytic acid phosphatases and their subcellular localization

Human acid phosphatases from various sources were purified by a new rapid procedure including chromatofocusing and gel permeation chromatography on an FPLC system. Isoenzymes 2a and 4 (Mr 105,000 and 76,000, respectively) are the prevalent acid phosphatases in prostate, leukocytes and bone marrow. Additional forms are proteolytic products formed during isolation procedure. The source of elevated acid phosphatase in blood serum in metastasizing prostatic carcinoma can be either prostate itself, the tumour or, more likely, bone marrow cells destroyed during the invasion by the tumour cells. Distribution of isoenzymes is not organ specific. It was found using an antiserum against isoenzyme 4 that immunoreactivity is confined to the specific secretory granules both of prostatic epithelium and leukocytes.     

Biochemical,immunological and genetic characterization of natural gramine-free variants of Hordeum vulgare L.

Many barley cultivars (e.g. Arimar) contain the indole alkaloid gramine, but some do not. Among seven gramine-free cultivars tested, two phenotypic classes were found: those with a normal level of the N-methyltransferase (NMT) activity that catalyzes the last two steps of gramine synthesis (e.g. Proctor); and those having neither NMT activity nor protein recognized by polyclonal antibodies raised against purified NMT (e.g. Morex).A 3 × 3 diallel cross with reciprocals was made using cultivars Arimar, Proctor and Morex. The pattern of occurrence of gramine and NMT activity among the F₁ hybrids suggested that Proctor and Morex carried defective alleles of the same nuclear gene governing an early step in the indole alkaloid pathway, and that Morex also carried a recessive allele at a nuclear locus encoding NMT activity. However, no non-parental alkaloid phenotypes were found in the F₂ generation from an Arimar × Morex cross and the ratio of progeny with gramine to those with no alkaloids was 3 : 1. One explanation of these results is tight linkage between genes controlling two of the steps in gramine biosynthesis.     

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.     

Light-harvesting systems of brown algae and diatoms. Isolation and characterization of chlorophyll ac and chlorophyll afucoxanthin pigment-protein complexes

The present study examined the protein associations and energy transfer characteristics of chlorophyll c and fucoxanthin which are the major light-harvesting pigments in the brown and diatomaceous algae. It was demonstrated that sodium dodecyl sulfate (SDS)-solubilized photosynthetic membranes of these species when subjected to SDS polyacrylamide gel electrophoresis yielded three spectrally distinct pigment-protein complexes. The slowest migrating zone was identical to complex I, the SDS-altered form of the P-700 chlorophyll a-protein. The zone of intermediate mobility contained chlorophyll c and chlorophyll a in a molar ratio of 2 : 1, possessed no fucoxanthin, and showed efficient energy transfer from chlorophyll c to chlorophyll a. The fastest migrating pigment-protein zone contained fucoxanthin and chlorophyll a, possessed no chlorophyll c, and showed efficient energy transfer from fucoxanthin to chlorophyll a. It is demonstrated that the chlorophyll $\text{a}\text{c-}\text{protein}$ and the chlorophyll $\text{a}\text{fucoxanthin-protein}$ complexes are common to the brown algae and diatoms examined, and likely share similar roles in the photosynthetic units of these species.