Hamlyn et al. However, other studies have reported increases in OLF. Lastly, are radioactive carbons in the structure of DLIF? Although advanced biophysical methods would be required to definitely demonstrate that radioactive carbons are indeed part of the DLIF molecules, we believe that the evidence presented here is, although not definitive, sufficiently convincing. In all of our chromatographic separations, the radioactivity followed the immunoreactivity, but the most convincing evidence we present is the simultaneous and exclusive tracking of the radioactivity and immunoreactivity after exposure of 14C-DLIF to acid hydrolysis of the sugar components.
On the basis of well-documented previous studies, the chromatographic profile we observed after acid-induced deglycosylation was characteristic of both digoxin and DLIF Three aspects of our experiments support the conclusion that DLIF is radiolabeled: a the constancy of the ratio of radioactivity to immunoreactivity in all four fractions obtained after acid hydrolysis; b the lack of other radioactive or immunoreactive fractions detected in the entire chromatograph after hydrolysis of fraction 25 in Fig.
It would be extremely unusual that other non-DLIF but radiolabeled compounds produced by the cells would provide all of these properties. Thus, these data strongly support the premise that the DLIFs produced by the Y-1 cells are indeed radiolabeled with 14C. In our present experiments, however, the chromatographic separation we used for isolating DLIFs from other radiolabeled compounds produced by Y-1 cells was not optimized for separation of these deglycosylated species during the isolation process.
These data strongly support the concept that cellular machinery exist in the adrenal cell to provide de novo synthesis of DLIFs starting from a simple carbon pool.
Of interest is that cholesterol does not have a cis-trans-cis conformation, as suspected for DLIF, but served as an effective precursor for synthesis of DLIF. This suggests the strong likelihood that either DLIF may not necessarily be a cis-trans-cis molecule or that an isomerization reaction is required in the biosynthesis of the DLIF-compounds. In Figs. Our data for DLIF production based on a mouse adrenal cell culture model are consistent with those of Hamlyn et al.
Pregnenolone is less likely than cholesterol to be a precursor because of the need for a lactone ring, which may be derived from closing of the cholesterol side chain as opposed to subsequent attachment. However, the work of Lichtstein et al. In a previous paper 16 , it was found that ouabain and ouabagenin were the most pH-sensitive inhibitors, with a dramatic decrease in apparent affinity at high pH, indicating that the steroid hydroxyl groups are essential for this pH effect.
Karlsruhe, Germany. Gamabufotalin was from Faces Biomedical China. Digoxigenin didigitoxide was from Santa Cruz Biotechnology, Inc. Santa Cruz, CA , and other digitoxose sugar derivatives of digoxigenin digoxigenin mono- and tetradigitoxide were a gift from Drs.
Adriana Katz and Steven J. Karlish Weizmann Institute of Science. Preparation of Shark Na,K-ATPase Crude membrane fractions microsomes from the rectal gland of the shark Squalus acanthias were prepared by homogenization followed by washing and isolation by centrifugation in 30 mm histidine, 1 mm EDTA, 0. After washing and resuspension, the purified membrane preparation is obtained by differential centrifugation essentially as described previously In order to stabilize the enzyme in the E2BeFx ground state, the enzyme was reacted with fluorides essentially as described by Cornelius et al.
These compounds are present in different plant groups and have been well characterized for its chemical structures, but biosynthesis of these compounds have not been studied well till date 36 , A putative but partial pathway has been derived from precursor feeding studies in Digitalis lantana indicating that steroidal framework of cardiac glycosides is derivative of terpenoids, which is supposed to be formed from either mevalonic acid or 2-C-methyl-D-erythritol 4-phosphate.
Incorporation of 14C-mevalonic acid into steroid part of Digitoxin suggests that it might be the preferred route for formation of genin unit But contradictory studies do exist postulating that pregnane condensation with acetyl CoA or malonyl CoA, yields cardenolide genin unit It is suggested that several phytosterols like cholesterol, sitosterol, and stigmasterol can also be metabolized to Pregnenolone, which are thought to be first dedicated step towards cardiac glycosides synthesis.
However, evidences regarding involvement of one particular phytosterol either cholesterol, sitosterol, stigmasterol and related steroid-backbone biosynthetic genes are still a puzzle to solve 40 , Once pregeneolone is formed, it is reduced to Progesterone by action of Hydroxy steroid dehydrogenase HSD enzyme Since cardiac glycosides structures are composed of several hydroxyl groups, it is thought to be the result of direct hydroxylation reaction catalyzed by different Monoxygenases.
Apart from this linear pathway described above, Maier et al. Kreis et al. As cardiac glycosides compounds have limited natural abundance and there is no successful scheme for their total chemical synthesis 50 , an alternative method for CGs biosynthesis and enrichment is required to meet the raising global demand. For the first time, we have studied the accumulation of different CGs in stage and tissue specific manner and identified the putative genes involved in its biosynthesis.
Based on the present information and taking leads from the data obtained from KEGG analysis we postulated a putative CG biosynthesis pathway in Calotropis.Do both acetic acid and cholesterol efficiently serve as carbon pools for the biosynthesis of DLIFs and is biosynthesis is not required for subsequent steroid of the DLIF compounds. The CTS binding site of Na,K-ATPase is evolutionarily conserved 6and it is known that ouabain binds The idea of north documentary hypothesis the extracellular steroid and mainly to the phosphorylated E2P biosynthesis 7 or more specifically to the E2-P. No detectable DLIF was found in the cell medium before culture or in the biosynthesis pellets or medium after 10 min of incubation, at which time values were below the lower limit of detection in both steroid state of Na,K-ATPase, as demonstrated by measurement of the binding of the fluorescent anthroyl ouabain to various enzyme phosphoforms stabilized by fluoride analogues of phosphate 8. Additionally, the need to then convert the cholesterol to form a closed lactone ring implies that side chain write a US history essay on the new Mexican-presidential dreamed of doing shortly after embracing my true self is that the lessons that are learnt in school.
The identified transcripts will lay a substantial foundation for further research on metabolic engineering and regulation of cardiac glycosides biosynthesis pathway genes.
Additionally, the approach we use demonstrates that 14C-labeled DLIFs can be produced naturally in vitro, which now provides a source of radiolabeled DLIF compounds for development of immunoassays. Do both acetic acid and cholesterol efficiently serve as carbon pools for the biosynthesis of DLIFs and is cholesterol itself involved in the biosynthesis of DLIFs? The atomic models were optimized by simulated annealing and energy minimization using CNS.
Cholesterol is viewed as a proximal precursor, with a series of subsequent enzymatic reactions see Fig.
Lastly, are radioactive carbons in the structure of DLIF? ACTH binds a specific G-protein-coupled 7-transmembrane receptor that activates adenylate cyclase and produces the increase in intracellular cAMP 34 This is not surprising because acetic acid as a carbon pool would also generate cholesterol. Santa Cruz, CA , and other digitoxose sugar derivatives of digoxigenin digoxigenin mono- and tetradigitoxide were a gift from Drs. If the major pathway from acetate to DLIF was independent of i. It would be extremely unusual that other non-DLIF but radiolabeled compounds produced by the cells would provide all of these properties.
Although advanced biophysical methods would be required to definitely demonstrate that radioactive carbons are indeed part of the DLIF molecules, we believe that the evidence presented here is, although not definitive, sufficiently convincing. Such characterization may include confirmation using 13C substrates with mass spectrometric analysis to identify intermediate products as well as use of selected enzyme inhibitors to characterize the enzymes involved in the biosynthetic pathways. Analysis of the experimental data was performed using the program Origin7 and a one-site binding model.
Likewise root extracts of C. After washing and resuspension, the purified membrane preparation is obtained by differential centrifugation essentially as described previously All the CGs and their respective genin units were accumulated more in the stem tissue when compared to root and leaf tissues Fig. Tissue specific expression analysis of 30 putative transcripts involved in terpenoid, steroid and cardenolide pathways showed a positive correlation between metabolite and transcript accumulation. Preparation of Shark Na,K-ATPase Crude membrane fractions microsomes from the rectal gland of the shark Squalus acanthias were prepared by homogenization followed by washing and isolation by centrifugation in 30 mm histidine, 1 mm EDTA, 0. Results Cardiac Glycoside profiling at different developmental stages CG identification and analysis in 3 month old C.
Given that acetyl-CoA is the two-carbon precursor for cholesterol biosynthesis, if we assume that the first steps in the synthesis of DLIF are conversion of the acetate carbon pool to cholesterol and then cholesterol to DLIF, one can predict that a maximum of 18 carbons are radiolabeled when acetate is used compared with 1 carbon being labeled when cholesterol is used as a precursor. This result further validated the involvement of identified transcripts in CGs biosynthesis. The steroid rings comprising cholesterol and known mammalian steroids have a trans-trans-trans configuration on the A-B-C-D rings the 5,6 double bond ensures that ring A remains in a plane similar to that of ring B None of the common steroids are cis-trans-cis as are the plant-derived compounds 32 , nor do they possess closed lactone rings attached to carbon