Comparison of biotin binding protein of pregnant rat serum with rat serum albumin

The purified biotin binding protein of pregnant rat serum was shown to be immunologically similar to rat serum albumin as assessed by a sensitive radioimmunoassay. In radioimmunoassay for rat biotin binding protein, the binding of [125I] rat biotin binding protein to anti-chicken egg yolk biotin binding protein antibodies was displaced by both rat serum (10-100 nl) and purified rat serum albumin (0.1-10 ng). Similarly, in radioimmunoassay for rat serum albumin the binding of [125I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin binding protein antibodies was displaced by unlabelled rat biotin binding protein at comparable concentration range (0.5-10 ng). Significant fractions of radioiodinated rat biotin binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin binding protein. In immature rats, the circulating half-lives of rat biotin binding protein and rat serum albumin were determined to be 12 and 17 h respectively. The rat biotin binding protein and rat serum albumin were analysed by techniques that exploit their physicochemical properties. They displayed similar electrophoretic mobilities in alkaline as well as denaturing sodium dodecyl sulphate-polyacrylamide gels. However, in nonequilibrium pH gradient polyacrylamide gel electrophoresis, they resolved clearly. In two-dimensional tryptic peptide map analysis, the two proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. These results showed that the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunological cross-reactivity

Characterization and hormonal modulation of immunoreactive thiamin carrier protein secreted by adult rat Leydig cells in vitro

Leydig cells isolated from adult rats and maintained under defined conditions in culture secrete a protein of molecular weight (Mr) 70 000 which is immunologically similar to chicken thiamin carrier protein (TCP). Synthesis of immunoreactive TCP by these cells is demonstrated by immunoprecipitation of [35S]methionine incorporated, newly synthesized proteins with monoclonal and polyclonal antibodies to chicken TCP. The amount of immunoreactive TCP secreted into the culture supernatant is quantitated by using a specific radioimmunoassay. Under the influence of LH, secretion of immunoreactive TCP is enhanced 3-fold and can be inhibited by up to 70% with aromatase inhibitor (1,4,6-androstatrien-3,17-dione). Cyclic AMP acts as a second messenger in the sequence of events involved in LH-induced elevation of immunoreactive TCP in Leydig cells. The effects of exogenous estradiol-17beta and diethylstilbestrol are comparable in terms of stimulation of secretion of immunoreactive TCP by these cells. Tamoxifen brought about a 70% decrease in the elevated levels of immunoreactive TCP. These results suggest that estrogen mediates immunoreactive TCP induction in hormonally stimulated adult rat Leydig cells

Diamine oxidase of Lathyrus sativus seedlings. Purification and properties

Diamine oxidase (EC 1.4.3.6) was purified from 5-day-old etiolated seedlings of Lathyrus sativus by MnCl2 treatment, (NH4)2SO4 and acetone fractionations, DEAE-Sephadex chromatography followed by gel filtration on Sephadex G-200. A single step purification of the enzyme was achieved by using an immunoaffinity column, wherein rabbit antibodies to the homogeneous diamine oxidase were coupled to CNBr-activated Sepharose. The enzyme thus obtained was homogeneous by electrophoretic, immunological and ultracentrifugal criteria. It had an Mr of 148,000 (6.46S) and was a dimer with similar sub-units (Mr 75,000). Amino acid analysis showed the absence of cysteine residues although it contained five disulphide bonds. The enzyme had copper (2.7 g atom/mol enzyme) but was not a glycoprotein. No absorption maximum in the visible region was detectable. Ethylenediamine 1,3-diaminopropane and histamine were potent competitive inhibitors for the substrate putrescine. The addition of monospecific antibodies to the enzyme increased the Km for benzyl amine without any change in the Vmax Diamine oxidase from pea seedling, partially purified, exhibited complete crossreactivity with the antibodies to the L. sativus enzyme

Hormonal modulation of reproduction-specific thiamin carrier protein in the rat

The hormonal modulation of thiamin carrier protein in the plasma and uterine luminal secretion during the normal reproductive phases of the animal (estrous cycle and pregnancy) as well as during experimental estrogenisation was investigated in the rat using a specific and sensitive homologous radioimmunoassay procedure developed for this purpose. Following a single injection of estrogen to immature male rats, thiamin carrier protein rapidly accumulated in plasma attaining peak concentration at 48 h and declining thereafter. A 1.5-fold amplification of the inductive response was observed on secondary stimulation with the hormone. The magnitude of the response exhibited a clear dependency on the dose of the steroid hormone, whereas the time at which peak levels of thiamin carrier protein production was remained unaltered in the concentration range of the steroid tested. The inductive effect of estrogen was severely curtailed by the antiestrogens,viz., En- and Zu-clomiphene citrates, while progesterone was incapable of either modulating the estrogen-induced response or eliciting an induction by itself. Cycloheximide drastically blocked the response to estrogen. Evidence for the ability of uterus to serve as yet another independent site of thiamin carrier protein synthesis was obtained by in vitro incorporation of radioactive amino acids into immunoprecipitable thiamin carrier protein in the tissue explants of estrogenised female rats. The levels of thiamin carrier protein in uterine luminal fluid measured during estrous cycle, pregnancy and experimental estrogenisation exhibited remarkable similarity to the plasma thiamin carrier protein profiles

Arginine decarboxylase is a component activity of the multifunctional enzyme putrescine synthase in cucumber seedlings

A homogenous PreParation of Putrescine synthase, the versatile multifunctional enzyme involved in agmatine →Putrescine conversion inCucumis sativus was found to catalyze enzymatic decarboxylation of arginine also. Similarly, the Purified arginine decarboxylase mediated the comPonent as well as the comPlete set of couPled reactions harboured by Putrescine synthase. Both the enzyme PreParations exhibited identical electroPhoretic and chromatograPhic behaviour and were immunologically indistinguishable. All the enzymic activities are stabilized concurrently by feeding arginine to the intact seedlings. Therefore, it is concluded that the multifunctional Putrescine synthase inCucumis sativus seedlings also harbours arginine decarboxylase activity unlike its counterPart in Lathyrus sativus

Purification and characterization of arginine decarboxylase from cucumber (Cucumis sativus) seedlings

A simple, reproducible and rapid protocol for the purification of arginine decarboxylase fromCucumis sativus seedlings has been standardised. The purification steps involved ion-exchange chromatography on diethylaminoethyl-cellulose followed by gel filtration on Sephadex G-l 50. The purified enzyme preparation migrated as a single stainable band on Polyacrylamide gels at both basic and acidic pH, but under denaturing and reducing conditions on sodium dodecyl sulphate-polyacrylamide gels resolved into polypeptides of molecular weight 48,000,44,000 and 15,000. However, in the absence of 2-mercaptoethanol on electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, the enzyme moved as single band with a molecular weight of 150,000. Evidence was obtained to indicate that these three polypeptides were probably derived from a single larger molecular weight enzyme. On storage of the purified protein, the 48,000 species was preferentially degraded to smaller polypeptides. The preliminary data suggested that the 48,000 and 44,000 species shared many common tryptic peptides as revealed by finger printing of the [125I ]-labelled protein. The purified enzyme was a glycoprotein and had a Km of 0.5 mM for arginine. Its activity was stimulated by dithiothrietol and pyridoxal phosphate. EDTA did not inhibit the enzyme activity. Mn2+ at 1 mM stimulated arginine decarboxylase activity but was inhibitory at higher concentration

Correlation between riboflavin carrier protein induction and its mRNA activity in estrogen stimulated chicken liver and oviduct

Poly A enriched RNA from either liver or oviduct of estradiol-17β treated immature chicks supported [3H]-leucine incorporation into immunoprecipitable riboflavin carrier protein in a dose-dependent manner when translated in the rabbit reticulocyte lysate system. Primary translation product of riboflavin carrier protein had a molecular weight of 38,000 which on incubation with a stripped hepatic microsomal preparation was processed to a product with a size comparable to native riboflavin carrier protein. Poly A enriched RNA from both the liver and the oviduct of estrogen-treated birds stimulated [3H]-leucine incorporation into riboflavin carrier protein and this was 2-3 fold higher during secondary stimulation vis-a-vis primary stimulation with the steroid. Poly A enriched RNA from the liver of progesteronetreated birds during secondary stimulation did not support riboflavin carrier protein synthesis. In contrast, poly A enriched RNA from the oviduct of the birds treated with progesterone during secondary (but not primary) stimulation did exhibit riboflavin carrier protein-mRNA activity which was comparable to that stimulated by estradiol-17β

Enzymic conversion of agmatine to putrescine in Lathyrus sativus seedlings. Purification and properties of a multifunctional enzyme (Putrescine synthase)

The participation of a multifunctional enzyme (a single polypeptide with multiple catalytic activities (14)) has been demonstrated in the conversion of agmatine to putrescine in Lathyrus sativus seedlings. This enzyme (putrescine synthase) with inherent activities of agmatine iminohydrolase, putrescine transcarbamylase, ornithine transcarbamylase, and carbamate kinase has been purified to homogeneity and has Mr = 55,000. In the presence of inorganic phosphate, the enzyme catalyzed the stoichiometric conversion of agmatine and ornithine to putrescine and citrulline, respectively. The different activities associated with the enzyme copurified with near constancy in their specific activity. The enzyme catalyzed phosphorolysis and arsenolysis of N-carbamyl putrescine. The multifunctionality of putrescine synthase was also supported by 1) activity staining, 2) intact transfer of the ureido-14C group from labeled N-carbamyl putrescine to ornithine to form citrulline, and 3) the affinity of the enzyme toward structurally and functionally related affinity matrices. An agmatine cycle is proposed wherein N-carbamyl putrescine arising from the agmatine iminohydrolase reaction is converted to putrescine and citrulline, with the ureido group of N-carbamyl putrescine being transferred intact to ornithine. Preliminary results indicate that this series of reactions is also present in other plants

Biosynthesis and regulation of polyamines in higher plants

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Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α -difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3-4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein