Assessing analytical variability of measurement of vitamin A in corn-soy blend.

Two multilaboratory investigations were conducted by SUSTAIN to assess variability in the measurement of vitamin A, the marker used to verify levels of vitamin premix addition to enriched/fortified food aid products, including the widely distributed corn-soy blend (CSB). CSB specifications identify AACC Approved Method 86-06 or equivalent methods for vitamin A analysis, however there is no requirement to demonstrate equivalency. CSB samples with known and blinded levels of vitamin A and a reference standard were analyzed by 16 laboratories using their respective methods. Calculated coefficients of variation across all laboratories and methods for unknown samples and reference standard were 35 and 7.1%, respectively, suggesting the largest source of variation is the vitamin extraction procedure. Laboratories generally overestimated low levels and underestimated high levels of vitamin A within the range of 6000 and 16 000 IU/lb. Only two laboratories demonstrated excellent internal precision (+/- 300 IU vitamin A/lb) and reported values within 95% confidence interval for all blinded samples. Results of this study have implications both for quality control in food aid products (due to the use of vitamin A as a marker) and for regulatory oversight of vitamin A content in commercial food products.

Dissimilarity in protein chain elongation factor requirements between yeast and rat liver ribosomes.

Factor requirements for yeast and rat liver ribosomes were determined in several different reactions using either yeast or liver factors. In polymerization assays yeast ribosomes required a factor in addition to elongation factor 1 (EF-1) and elongation factor 2 (EP-2). The third factor (EF-3) requirement was observed with EFs from either yeast or liver for both poly(U)-directed polyphenylalanine synthesis and elongation of endogenous peptidyl-tRNA. No significant effect of EF-3 was observed with liver risomes in either assay. In contrast to results with polypeptide synthesis EF-3 was not required for EF-1 dependent binding of [3H]Phe-tRNA or the translocation-dependent formation of N-acetylphenylalanylpuromycin. Up to 2-fold stimulation of the binding reaction was observed with saturating levels of either yeast or liver EF-1. No effect of EF-3 was observed on ribosome-EF-2-GDP-fusidic acid complex formation. The data suggest that the yeast EF-3 may be a loosely bound ribosomal protein which is not required for a specific step in the elongation cycle but is involved in the coordination of the partial reactions required for polymerization.

Cryptopleurine--an inhibitor of translocation.

Ribosomes from a cryptopleurine-resistant mutant of yeast were analyzed to determine the subunit localization of the resistance alteration. As was previously reported (Grant, P., Sanchez, L., and Jiminez, A. (1974), J. Bacteriol. 120, 1308), in vitro resistance of polyphenylalanine synthesis to cryptopleurine was conferred by 40S subunits from the mutant. Binding studies with sensitive ribosomes were carried out in order to identify the subunit binding site for cryptopleurine. Over the range of concentrations which inhibited polyphenylalanine synthesis, binding was proportional to concentration, so that a unique binding site could not be detected. Furthermore, binding to isolated subunits was about fourfold greater than to 80S ribosomes, suggesting that non-specific binding was sensitive to the condition of the particles. Model systems were developed in order to determine which step of the elongation cycle was inhibited by cryptopleurine. Elongation factor 1 dependent binding of Phe-tRNA to ribosomes was not inhibited by cryptopleurine concentrations, which inhibited polyphenylalanine synthesis. The initial rate of N-acetylphenylalanylpuromycin formation was inhibited when 10(-5) M cryptopleurine was added prior to translocation, but not when added after. Little inhibition was observed in either case when mutant ribosomes were used. These results suggest that cryptopleurine primarily inhibited translocation.

A cycloheximide sensitivity factor from yeast required for N-acetylphenylalanylpuromycin formation.

A protein (factor P) has been isolated from yeast, which was required for sensitivity to cycloheximide of a partially purified polyphenylalanine synthesis system. In the absence of factor P, 10(-3) M cycloheximide was required for 50% inhibition of polyphenylalanine synthesis, while in its presence, 10(-6) M gave 50% inhibition. Coincident with cycloheximide sensitivity was an activity required for EF-2 dependent N-acetylphenylalanylpuromycin (N-AcPhePuro) formation. Transfer of N-AcPhe to puromycin from the tRNA bound in the presence of 26 mM MgCl2 required factor P, as well as EF-2. Studies with antibody against EF-2 demonstrated that P factor was not required during the EF-2 translocation step but for some subsequent step.

A ribosome-dependent GTPase from yeast distinct from elongation factor 2.

Three proteins required for poly(U)-directed polyphenylalanine synthesis have been separated from yeast. Two of the factors correspond to the elongation factors 1 and 2 described for other eukaryotic systems, according to the criteria of phenylalanyl-tRNA binding and diphtheria toxin-catalyzed ADP-ribosylation. The third protein, while absolutely required for polyphenylalanine synthesis, was a more active ribosome-dependent GTPase than elongation factor 2.

Modification of ribosomes in cryptopleurine-resistant mutants of yeast.

Cryptopleurine-resistant mutants of Saccharomyces cerevisiae were isolated. A single, recessive nuclear gene, very closely linked to the mating locus (2.1 centimorgans), is responsible for resistance. Ribosomes from the mutants were found to be resistant to cryptopleurine when analyzed by poly(U)-directed polyphenylalanine synthesis. Analysis of the distribution of ribosomes between monosomes and polysomes in sensitive cells exposed to cryptopleurine suggests that some step is inhibited during the elongation phase of protein synthesis.

Characterization of the initial peptide of Q-beta RNA polymerase and control of its synthesis.

Bacteriophage Qbeta RNA directs the cell-free synthesis of two fMet-containing dipeptides prior to the first round of ribosomal translocation. One of these, fMet-Ala, corresponds to the initial segment of the Qbeta coat protein. In the present work we take advantage of the fact that translation of the Qbeta RNA polymerase cistron is repressed by its coat protein to correlate the other peptide, fMet-Ser, with the Qbeta RNA polymerase gene. Our experiments show that repression affects translation of the first two codons of the polymerase cistron, thereby isolating the effect of Qbeta coat repressor. Further studies, using single rounds of translocation of the Qbeta mRNA and codon-specific tRNAs, allow us to predict the first three amino acids of the Qbeta RNA polymerase protein, fMet-Ser-Lys, and to suggest that the initiation region of the Qbeta RNA polymerase cistron has the sequence ...AUG.UC[unk].AA[unk]...

Translocation of mRNA codons. II. Properties of an anti-translocase antibody.

A purified preparation of translocase, one of several enzymes required for protein biosynthesis, has been used to prepare a specific anti-translocase antibody. This antibody provides an extremely useful tool not only for detecting the enzyme independent of its activity, but for inhibiting the translocase-mediated reaction and, thus, protein biosynthesis. Though the antibody very rapidly inhibits elongation of the peptide chain, it fails to effect initiation, binding, or peptide bond formation, which strongly suggests that translocase is not a direct participant in these reactions. It also arrests translation of the defined tricodon AUG(U(6)) at the second codon, permitting formation only of the dipeptide, fMet-Phe, rather than the tripeptide, fMetPhe-(Phe)(2), which is formed in the presence of control serum. We have shown previously that the third codon becomes available only in the presence of translocase, thereby defining the site of action of the antibody. The antibody also has been used to demonstrate that translocase is antigenically distinct from the other proteins required for initiation and protein synthesis in E. coli.

Translocation of mRNA codons. I. The preparation and characteristics of a homogeneous enzyme.

This report describes a convenient scheme for the further purification of an E. coli enzyme which is required for the translocation step in protein biosynthesis. The homogeneous enzyme translocase appears to be a relatively large monomeric protein, having a molecular weight of approximately 72,000, and acts in a catalytic fashion during protein synthesis. It is also one of the major soluble macromolecular constituents of rapidly growing E. coli, comprising more than 2 per cent of the protein in ribosome-free extracts. Further, the rate of in vitro protein synthesis is linearly dependent upon the concentration of the pure enzyme until approximately one molecule of translocase is present per ribosome in reaction mixtures.