P. Radhakantha Adiga

Regulation of arginine decarboxylase and putrescine levels in Cucumis sativus cotyledons

Arginine decarboxylase (arginine carboxy-lyase EC 4.1.1.19) of Cucumis sativus cotyledons, has a pH optimum of 8.3 and a temperature optimum of 40°. Among the various plant hormones administered to excised cotyledons in culture, benzyladenine and its riboside were most effective in increasing the arginine decarboxylase activity and putrescine content. The enzyme activity and putrescine content were significantly increased on acid feeding of the cotyledons and decreased by KCl treatment. The KCl effect could be only partially reversed by benzyladenine. Abscisic acid inhibited cotyledon growth and also reduced arginine decarboxylase and putrescine levels. This effect was overcome by cytokinins. The half life of the enzyme using cycloheximide was 3.7 hr. Dibutyryl cyclic AMP and 5′-AMP also marginally stimulated the enzyme and putrescine levels. Mixing experiments indicate that there is neither a non-dialysable activator nor inhibitor of the enzyme.

Estrogen-induced synthesis of riboflavin-binding protein in immature chicks. Kinetics and hormonal specificity.

The kinetics of estrogen-induced elevation in the plasma concentration of riboflavin-binding protein, a minor yolk constituent, was investigated in immature male chicks, using a specific and sensitive radioimmunoassay procedure. Following a single injection of the hormone, the plasma riboflavin-binding protein content was enhanced several-fold at 6 h, reaching peak levels around 48 h and declining thereafter. A two-fold amplification of the response was evident on secondary stimulation with the hormone. A 4-h lag phase prior to onset of induction was noticed during both primary and secondary stimulations with the steroid hormone. The magnitude of the response was dependent on the hormonal dose whereas the initial lag phase and the time of peak riboflavin-binding protein accumulation were unaltered within the range of hormonal doses tested. The half-life of riboflavin-binding protein in the circulation was 10 h, as calculated from measurement of the rate of disappearance of exogenously administered 125I-labelled protein. Simultaneous administration of progesterone did not affect the kinetics of riboflavin-binding protein production. On the other hand, the antiestrogens, cis- and trans-clomiphene citrates, given 30 min prior to estrogen and cycloheximide, effectively counteracted the hormone-induced riboflavin-binding protein elaboration. Both progesterone and the antiestrogens per se were completely ineffective in substituting for estrogen in the inductive process.

The interaction of riboflavin with a protein isolated from hen's egg white: A spectrofluorimetric study

The interaction of riboflavin with a protein isolated from egg white has been studied spectrofluorimetrically at different pH values. In 0.1 M phosphate buffer pH 7.0; 1:1 complex formation occurs with the association constant Ka = 7.7-10(7) M-1. In the presence of 0.033% sodium dodecyl sulphate, the complex dissociated with a rate constant of 4-10(-2) sec-1 at 29 degrees C. The binding was sensitive to pH and to the antibodies produced against the protein. On lowering the pH from 7 to 4 the binding affinity decreased approximately 100-fold and below pH 4, the binding could not be detected at all. These data, together with those obtained by measuring the fluorescence intensities of riboflavin in presence of N-bromosuccinimide oxidized- and disulphide reduced apoprotein, suggest that carboxyl functions, 1-2 tryptophan residues and 2-3 disulphide bridges are essential for binding. The emission spectra of the protein under different conditions upon excitation at 280 and 295 nm were analyzed to calculate the quantum yield (Q) and the efficiency of energy transfer (e) from tyrosine to tryptophan residues. From these data it was concluded that the energy transfer did not occur with equal efficiency under all conditions and that the tryptophan residues responsible for the riboflavin binding are more accessible to N-bromosuccinimide oxidation than others.

Riboflavin carrier protein: A serum and tissue marker for breast carcinoma

We have earlier shown that the estrogen‐modulated riboflavin carrier protein (RCP) first isolated from the chicken egg is evolutionarily conserved in mammals and is elaborated by lactating mammary gland as demonstrated with rat mammary epithelial cells in culture and confirmed by isolation of the vitamin carrier from bovine milk. In view of several earlier reports that many milk proteins as well as other estrogen‐inducible proteins are up‐regulated and secreted into circulation in animal models and in women with neoplastic breast disease, we analyzed serum RCP levels in a double‐blind study using a specific radioimmunoassay in pre‐ and post‐menopausal women with clinically diagnosed breast cancer at early and advanced stages of the disease and compared these levels with those in normal age‐matched control volunteers. Our data reveal that the serum RCP levels in cycling breast cancer patients are 3‐ to 4‐fold higher (p < 0.01) than those in their normal counterparts. This difference in circulatory RCP levels between cancer patients and their age‐matched normal counterparts is further magnified to 9‐ to 11‐fold (p < 0.005) at the post‐menopausal stage. In addition, there seems to be a good correlation between rising RCP levels and disease progression, since significantly higher RCP concentrations (p < 0.005) are encountered in patients with advanced metastasizing breast cancer versus those with early disease. Using specific monoclonal antibodies, RCP could be localized immunohistochemically in the cytoplasm of invading neoplastic cells of lobular and ductal carcinomas of the breast, indicating that the malignant cells are probably the source of the elevated serum RCP levels in breast cancer. These findings suggest that measurement of circulatory RCP and the immunohistochemical staining pattern of RCP in biopsy specimens could be exploited as an additional marker in diagnosis/prognosis of breast cancer in women.

Immunological characterization of riboflavin carrier proteins using monoclonal antibodies

Monoclonal antibodies to chicken riboflavin carrier protein have been produced by fusing immunized mouse spleen cells with myeloma SP2/O-Ag 14. The three different monoclonal antibodies specifically bound 125I-labelled chicken riboflavin carrier protein and were characterized with respect to their affinities to bind the antigen, subclass and isotype. These three monoclonal antibodies had similar affinities for holo-, apo- and SDS-denatured riboflavin carrier protein but were unable to recognize the reduced and carboxymethylated protein indicating that they were directed to specific conformational epitopes on the native avian protein. Succinylation of the vitamin carrier protein while still retaining flavin binding characteristics totally abolished the cross-reactivity with all the three monoclonal antibodies indicating that lysine residues were involved at the antigenic sites of the protein. This shows that the antigenic loci may be distinct from the flavin binding sites in the protein. All three antibodies were able to recognize riboflavin carrier protein present in the sera of pregnant rats, monkeys and humans indicating that the epitopes to which they are directed are conserved throughout evolution. These antibodies can therefore be effectively used for radioimmunoassays and further studies on the functional aspects of this protein in higher mammals.

Amine levels in Lathyrus sativus seedlings during development

In growing Lathyrus sativus seedlings, the levels of DNA, RNA and protein markedly decreased in the cotyledons and progressively increased in the embryo-axis. In cotyledons, spermidine and spermine contents were substantially reduced while those of agmatine and putrescine were sharply increased. By contrast the embryo-axis progressively accumulated relatively larger amounts of agmatine, homoagmatine. putrescine, cadaverine, spermidine and spermine in parallel with similar changes in its DNA, RNA and protein content. While the cotyledons contained ca 50% of the total agmatine and putrescine present in the plant embryo by day 10, the embryo-axis, though representing less than 20% of the dry wt, contained 90 and 75% of total cadaverine and homoagmatine respectively of the seedlings. Spermidine and spermine levels of this tissue were also comparatively higher, being of the order of 80 and 50% respectively of the total. The root and shoot portions of the embryo-axis also exhibited a similar relationship between changes in DNA, RNA and protein and all the above amines during development. However, the polyamine content of the shoots was relatively higher than those of the roots during the growth period.

Purification of a circulatory riboflavin carrier protein from pregnant bonnet monkey (M. radiata): comparison with chicken egg vitamin carrier

A specific protein exhibiting immunological cross-reactivity with chicken egg-white riboflavin carrier protein was detected by radioimmunoassay in the pregnancy sera of bonnet monkeys (Macaca radiata). This protein, which is capable of binding [14C]riboflavin, was purified by gel filtration on Sephacryl S-300, fast protein liquid chromatography on a Mono Q anion exchanger and chromatofocusing on Mono P columns. The isolated primate carrier protein was similar to its avian counterpart in terms of physicochemical characteristics, such as isoelectric point (pI less than or equal to 4), electrophoretic mobility, molecular weight (approx. 36,000) and ligand binding. These findings may account for the extensive immunological cross-reactivity observed between the two proteins and suggest that the two vitamin carriers may have similar function in terms of embryonic vitamin nutrition.

A monoclonal antibody recognizing the C-terminal region of chicken egg white riboflavin carrier protein terminates early pregnancy in mice

In order to identify the functionally relevant epitopes on chicken riboflavin carrier protein, we have raised monoclonal antibodies to the vitamin carrier. One of these, 6B2C12, was found to interact specifically with a synthetic oligopeptide corresponding to the C-terminal 17 amino acid residues of the chicken egg white riboflavin carrier protein, which is missing in part in the egg yolk riboflavin carrier protein. This epitope is conserved through evolution in mammals including humans. Administration of the ascites fluid of 6B2C12 to pregnant mice intraperitoneally, resulted in the termination of pregnancy indicating that this epitope is involved in or closely associated with the transplacental transport of the vitamin from the maternal circulation to the growing fetus.

Mechanism of foetal wastage following immunoneutralization of riboflavin carrier protein in the pregnant rat: disturbances in flavin coenzyme levels

Immunoneutralization of maternal RCP results in a >90% decrease in the content and the incorporation of [2‐14C]riboflavin into embryonic FAD as well as a percentage redistribution of both embryonic FMN and riboflavin. This is unaccompanied by any discernible changes in flavin distribution pattern in the maternal liver. Embryonic α‐glycerophosphate dehydrogenase and NADPH‐cytochrome c reductase register significant decreases in activities in the RCP antiserum‐treated rats. These alterations readily explain the arrest of foetal growth culminating in pregnancy termination in the antiserum‐treated animals.

Decarboxylation of homoarginine and lysine by an enzyme from Lathyrus sativus seedlings

Abstract Homoarginine decarboxylase has been purified ca 110-fold from Lathyrus sativus seedlings and resolved from arginine decarboxylase by DEAE-Sephadex column chromatography. The enzyme was less active than arginine decarboxylase and was highly labile. This preparation decarboxylated l -lysine in addition to L-homoarginine. The purified enzyme preparation had an absolute requirement for exogenous Mn2+ or Fe2+ for both the enzyme activities. The pH and temperature optima for decarboxylation of both homoarginine and lysine were the same viz. 8·4 and 41° respectively. The Km value l -homoarginine was 3·33 mM and for l -lysine was 0·88 mM. Arginine and homoarginine decarboxylases appear to be different and separable entities having different physico-chemical characteristics, despite the fact that their respective guanido amino acid substrates undergo similar metabolic conversion to guanido- and diamines in this plant system.