In-vitro biosynthesis of multiple preproglucagons results from acetylation of the primary translation products

ABSTRACT In-vitro translation of anglerfish islet mRNA revealed three glucagon precursors (preproglucagons): one with Mr 16 000 and two with Mr 14 000. The two Mr 14 000 precursors were well separated upon isoelectric focusing gels (pI values of 7·2 and 7·3), but had identical peptide maps. Translation of hybrid-selected Mr 14 000 preproglucagon mRNA in the presence of microsomal vesicles revealed that both precursors were processed to the same proglucagon. Northern blot analysis detected two mRNA species encoding Mr 14 000 precursor. A full-length Mr 14 000 preproglucagon cDNA was subcloned into a transcription vector, and coupled in-vitro transcription—translation was performed; surprisingly, both Mr 14 000 precursors were synthesized. To test whether acetylation of the free amino terminus generated the more acidic precursor, acetylase activity was partially inactivated with the inhibitor S-acetonyl-CoA, and acetyl-CoA was depleted by addition of oxaloacetate and citrate synthetase. Under these conditions, the level of the most basic preproglucagon was greatly enhanced, but when exogenous acetyl-CoA was added, the acidic form predominated. We conclude that acetylation generates the acidic precursor, and we discuss the implications of our findings for the biogenesis of other peptide hormones.

Acetoin and Diacetyl Formation by Streptococcus lactis subsp. diacetylactis DRC3

Cells of Streptococcus lactis subsp. diacetylactis DRC3 which had been grown on lactose in the presence of citrate were unable to form acetoin and diacetyl (AD) from citrate when suspended in succinate buffer, pH 4.4. Inclusion of both a source of energy and nitrogen in the buffer was necessary for AD formation. Concentrations of AD and D at pH 4.4 were about five and three times the concentrations at pH 6.4. The amounts of AD and D from citrate were about eight and two times those from pyruvate, calculated on a molar basis. It appears that D, at least, is formed not only from pyruvate arising during citrate cleavage, but also from acetyl-CoA resulting from a probable citrate breakdown in a reversible reaction of a citrate synthetase. The rate of AD formation, under optimum conditions, was 0.047 μmoles mg (dry wt−1) cells ml−1 min−1. Pyruvate-grown cells produced little AD from pyruvate. AD production was inhibited partly by acetate and completely by acetaldehyde. Cetylpyridinium chloride at a concentration higher than μg ml−1 suppressed AD production from citrate because of the absence of interfering compounds normally present in milk.

Acetoin and Diacetyl Production by Lactobacillus plantarum Able to Use Citrate

Whole-cell suspensions of Lactobacillus plantarum grown on lactose in the presence of citrate did not produce acetoin and diacetyl (AD) (D) from citrate in succinate buffer, pH 4.4 unless both a source of energy and nitrogen was present, but did from pyruvate. The total AD and the amount of D, produced by citrate-grown cells, from citrate were about two times the amounts formed from pyruvate, calculated on a molar basis. It appears, that AD are formed not only from pyruvate resulting from cleavage of citrate but also from acetyl-coenzyme A arising during a probable breakdown of citrate in a reversible reaction of citrate synthetase. Neither acetate nor acetaldehyde had any effect on the total AD or the amount of D produced from pyruvate by pyruvate-grown cells. The rates of AD production from pyruvate by whole-cell suspensions of pyruvate-grown L. plantarum and Streptococcus subsp. diacetylactis represented only 69.7 and 6.6%, respectively, of that produced by Lactobacillus casei. These were 0.075 μmoles/mg dry wt−1 ml−1 min−1 for L. casei, 0.053 for L. plantarum and 0.005 for S. lactis subsp. diacetylactis.

Effect of Phenylalanine and Its Metabolites on the Metabolism of Leucocytes and Lymphocytes

1. The pathogenesis of the mental retardation in phenylketonuria remains obscure. Leucocytes have proved of value in the study of other inborn errors of metabolism. The lymphocyte is a suitable model cell for the study of mammalian metabolism, because of its ability to divide in vitro in response to various stimuli. 2. We have examined the effects of phenylalanine, phenylpyruvate, phenyl-lactate and phenylacetate on the human leucocyte and the resting and phyto-haemagglutinin-stimulated rabbit lymphocyte. 3. Phenylpyruvate and phenyl-lactate reduced acetate incorporation into leucocyte lipid by 38% and 48% respectively. Only phenyl-lactate reduced acetate incorporation into the resting and stimulated lymphocyte, by 20% and 34% respectively. 4. Glucose incorporation into leucocyte lipid was unaffected by phenylalanine, phenylpyruvate and phenyl-lactate. Only phenyl-lactate inhibited (46%) the production of CO2 from glucose. 5. Phenylalanine and leucine incorporation into trichloroacetic acid-insoluble material of resting and stimulated lymphocytes was inhibited by phenyl-lactate (10–42%), phenylpyruvate (27–57%) and phenylacetate (19–39%). 6. Uridine incorporation into resting and stimulated cells was inhibited by phenyl-lactate (22–26%), phenylpyruvate (42–52%) and phenylacetate (20%). 7. Thymidine incorporation into resting lymphocytes was reduced by phenyl-lactate, phenylpyruvate, phenylacetate and phenylalanine by 12–26%. Incorporation into the stimulated cell was inhibited by phenylpyruvate and phenyl-lactate (90%) and phenylacetate (66%). 8. Phenylalanine inhibited lymphocyte pyruvate kinase and phenylpyruvate inhibited citrate synthetase. 9. These results are compared with published data relating to experimental hyperphenylalaninaemia and the effects of these metabolites on nervous tissue in vitro.

Radiometric Assay of Citrate Condensing Enzyme (Citrate Synthetase) in Liver Tissue and Mitochondria

Citrate synthetase activity was determined in samples of commercially available citrate condensing enzyme. We measured the amount of radioactive citrate formed from the condensation of carbon-14 labeled acetyl CoA and oxaloacetate. The labeled citrate was oxidized by oxidative bromination; 14CO2 evolved was collected and represented the amount of citrate synthesized. Enzyme activity as low as 10-4 µmol/min was measured reproducibly with this procedure. Results obtained with this assay compared closely with those of a widely used spectrophotometric assay in which Ellman’s SH-coupling reagent [5,5-dithio-bis-(2-nitrobenzoate)] is coupled with CoA released during the reaction. Accuracy of labeled citrate determination in the presence of labeled acetate or pyruvate was excellent. The method can be used to determine citrate synthetase activity in skeletal muscle, liver, and isolated liver mitochondria, or to study the fate of labeled citrate injected into animals.