Calcitonin biosynthesis: evidence for a precursor.

Calcitonin biosynthesis has been studied in chicken ultimobranchial glands incubated in vitro in the presence of radioactive amino acids. The results obtained suggest the existence of a biosynthetic precursor of higher molecular weight or procalcitonin. This precursor has been identified by pulse-chase experiments, molecular weight determinations, biological activity measurements and analysis of tryptic peptides. Its molecular weight is about 13000 (calcitonin, about 3500) as determined by polyacrylamide gel electrophoresis. Procalcitonin is present in small amounts in chicken ultimobranchial glands and it is biologically active in rats.     

Isobutyrate as a precursor of n-butyrate in the biosynthesis of tylosine and fatty acids

Labelled sodium isobutyrate [(CD3)2-CHCOONa] was added to the culture medium of Streptomyces fradiae and up to 14 atoms of deuterium were found to be incorporated into a molecule of tylosin aglycone (tylactone). This observation is in accordance with the data in the literature. When fatty acids were analyzed, as much as 34% of the isobutyrate incorporated into the cell was formed to be transformed into butyrate that was used for the synthesis of even, straight-chain fatty acids; 57% of the labelled isobutyrate was incorporated into the even isoacids, whereas 9% was degraded to propionate and further used for the synthesis of the odd acids.     

Evidence for biosynthesis of lactase-phlorizin hydrolase as a single-chain high-molecular weight precursor

Precursor forms of lactase-phlorizin hydrolase, sucrase-isomaltase and aminopeptidase N were studied by pulse-labelling of organ-cultured human intestinal biopsies. After labelling the biopsies were fractionated by the Ca2+-precipitation method and the enzymes isolated by immunoprecipitation. The results indicate that the lactase-phlorizin hydrolase is synthesized as a Mr 245 000 polypeptide, which is intracellularly cleaved into its mature Mr 160 000 form. Sucrase-isomaltase is shown to be synthesized as a single chain precursor (Mr 245 000 and 265 000) while the precursor of aminopeptidase N is shown to be of apparently the same size as the mature enzyme (Mr 140 000 and 160 000).     

Glutamyl-transfer RNA: a precursor of heme and chlorophyll biosynthesis.

In green plants, archaebacteria and many eubacteria, the porphyrin ring that is common to both chlorophyll and heme is synthesized from 5-aminolevulinic acid (ALA) via an interesting pathway. This two-step process involves the unusual enzymes glutamyl-tRNA reductase and glutamate-1-semialdehyde 2,1-aminomutase. Interest in this pathway has increased since it was discovered that a tRNA cofactor was required for the formation of ALA. This tRNA(Glu) is common to the biosyntheses of both porphyrins and proteins.     

Combinatorial biosynthesis of 5-O-desosaminyl erythronolide A as a potent precursor of ketolide antibiotics

Ketolides, characterized by possessing a 3-keto group in place of the l-cladinose moiety of erythromycin A, are the recent generation of antimicrobials derived semi-synthetically from the 14-membered ring macrolide erythromycin A. The multi-step synthetic route to ketolides can be shortened by using 5-O-desosaminyl erythronolide A as a precursor, which reduces the steps for the removal of l-cladinose attached at the C-3 position in erythromycin A. Deletion of an eryBV gene encoding mycarosyl glycosyltransferase in the erythromycin-producer Saccharopolyspora erythraea resulted in the accumulation of 5-O-desosaminyl erythronolide B. In vivo expression of the cytochrome P450 gene pikC, which encodes the substrate-flexible hydroxylase from the pikromycin biosynthetic pathway of Streptomyces venezuelae, in the eryBV deletion mutant strain of Sac. erythraea led to 5-O-desosaminyl erythronolide A production.     

Proteolytic precursor processing in the biosynthesis of mitochondria

Essentially all polypeptides synthesized in the cytoplasm and imported into either the matrix or into the inner or outer membrane of mitochondria are made as larger molecular weight precursors. All known examples of in vivo or in vitro synthesized precursors are summarized. Little information on the nature of the proteolytic enzymes involved in the processing of the larger precursor polypeptides exists. The biosynthesis of rat liver cytochrome c oxidase is discussed in detail. In contrast to reported data, the cytoplasmic subunits of rat liver cytochrome c oxidase are synthesized as larger molecular weight precursors and not as a polyprotein. Precursors to subunits IV and V show an extra-peptide sequence of about 3000 daltons. Evidence against the existence of a polyprotein precursor was also obtained, when messenger RNAs for the individual subunits IV and V were isolated and analyzed in respect to their size. A length of 990 +/- 80 and 830 +/- 70 nucleotides was estimated for the poly(A)+-RNA of cytochrome c oxidase subunits IV and V, respectively. In experiments on the site of synthesis, it was found that cytochrome c oxidase subunits IV and V are made on free, loosely and tightly membrane-bound polyribosomes.     

L-arginine is a precursor for nitrate biosynthesis in humans

Nitrogen from L-arginine was incorporated into urinary nitrate in human subjects. Two subjects given an oral dose of [15N2]L-arginine excreted 24 and 17 umol [15N]nitrate/24 hr, respectively, in their urine in the 24 hr period following the dose. This work demonstrates that L-arginine, a nitrogen source for biosynthesized nitrate in cultured cells and research animals, is a precursor for endogenously synthesized nitrate in humans.     

Indole alkaloid biosynthesis : An investigation into leucine as a possible precursor of the monoterpene portion of vindoline

Because of the nonspecific incorporation of acetate into the monoterpenoid portion of the indole alkaloids, the amino acid leucine has been investigated as an alternative precursor. Incorporations of leucine into the alkaloids vindoline and catharanthine in Vinca rosea are found to be 0.07 and 0.02%, respectively, levels comparable with those of acetate. An equation is developed to calculate the quantity of [¹³C]leucine needed to be fed in order to obtain alkaloids with sufficient ¹³C enrichment to be analysed by ¹³C nmr spectroscopy. This equation assumes that percentage incorporations do not decrease with increasing quantities of leucine fed, an assumption found to be true. [2-¹³C]Leucine was synthesized from [2-¹³C]acetic acid in an eight-step procedure in 11.5% overall yield, and fed to Vinca rosea plants. Incorporations into vindoline obtained using both ¹⁴C and ¹³C were in reasonable agreement, and the activity was found to be spread over seven carbon atoms, none of which corresponded to the two atoms at which activity was expected. It is concluded that the leucine → mevalonate → monoterpene route does not operate in indole alkaloid biosynthesis, leaving open the question of the origin of the monoterpene portion of the alkaloids. An attempt to confirm these results by degradation of ¹⁴C-labelled alkaloids was unsuccessful, vindoline proving to be unexpectedly resistant to oxidation.     

L-tyrosine is the precursor of PQQ biosynthesis in Hyphomicrobium X

A method was developed to study amino acids as possible precursors of PQQ biosynthesis. Cultures of Hyphomicrobium X, growing on [13C]methanol, were supplemented with unlabelled amino acids. Uptake and participation in metabolism were determined via gas chromatography/mass spectrometry of derivatized amino acids, obtained from hydrolyzed cellular protein, by measuring their 12C content. Several amino acids appeared to be incorporated into the protein to a significant extent, without degradation or conversion. Among these were the aromatic amino acids, L-tyrosine and L-phenylalanine. Using the same replacement approach, their incorporation into PQQ was determined by 1H- and 13C-NMR spectroscopy of purified PQQ obtained from the culture medium. It appeared that the complete carbon skeleton of tyrosine was present, forming the o-quinone and pyrrole-2-carboxylic acid moieties in PQQ, while phenylalanine was not incorporated at all. Starting with L-tyrosine, possible biosynthetic routes to PQQ are discussed.     

Cholesterol arachidonate as a prostaglandin precursor in adrenocortical cells.

Rat adrenocortical cells were incubated with labeled arachidonate, and the radioactivity in unesterified fatty acids was reduced by washing with 2% albumin solutions. These cells were then incubated for two hours in the absence and presence of 7.1 x 10(-10)M ACTH. During subsequent incubation of prelabeled cells with ACTH, both the mass and radioactivity of arachidonate in adrenocortical cholesteryl esters was depleted to the same extent (30--32%). The released arachidonate was in part incorporated into phospholipids, and there was also a significant increase in unesterified arachidonic acid. During this period, there was also increased incorporation of arachidonate into labeled prostaglandins. Of this increase, 92% by isotope analysis, and 88% by radioimmunoassay techniques was attributable to prostaglandins of the E pathway. These data demonstrate that prostaglandin E synthesis is specifically increased during ACTH stimulation of rat adrenocortical cells and suggest that a major source of the arachidonate substrate for this synthesis is derived from hormone-dependent hydrolysis of cortical cholesteryl esters.