The influence of the nucleotide at position ‐3 relative to the AUG initiation codon on the initiation of protein synthesis was studied in two different in vitro translation systems using synthetic mRNAs. The four mRNAs, transcribed from cDNAs directed by an SP6 promoter, were identical except for mutations at nucleotide ‐3. In each case, translation of mRNAs produced a single protein of Mr = 12,600. Relative translational efficiencies showed a hierarchy in the reticulocyte lysate system (100, 85, 61 and 38% for A, G, U and C in position ‐3, respectively) but no differences in the wheat germ system. Differential mRNA degradation or polypeptide chain elongation were excluded as causes of the differences observed in translation in the reticulocyte lysate. mRNA competition increased the differences observed in translational efficiencies in reticulocyte lysate but showed no effect in wheat germ. Analysis of 61 plant and 209 animal mRNA sequences revealed qualitative and quantitative differences between the consensus sequences surrounding AUG initiation codons. Whereas the consensus sequence for animals was CACCAUG that for plants was AACAAUGGC. Both the structural and functional findings suggest that the factors which select AUG initiation codons in plants and animals differ significantly.

The mechanism by which digestive zymogens become activated during acute pancreatitis remains poorly understood. Given the ability for cholecystokinin (CCK) to induce pancreatitis in vivo, the effects of high dose CCK on preparations of isolated pancreatic acini were examined. Using an immunologic technique for the detection of zymogen activation, CCK was found to stimulate the conversion of procarboxypeptidase Al to a 35-kD form having the same net charge and electrophoretic mobility as purified recombinant carboxypeptidase Al. This enhanced conversion was proportional to the dose of CCK (maximal at 100 nM), and time dependent. CCK also produced changes in the electrophoretic mobility of procarboxypeptidase B and chymotrypsinogen 2 immunoreactivity, consistent with activation of these zymogens. These events were detectable only within acinar cell pellets and not in the incubation medium, suggesting an intracellular site of conversion. The conversion of procarboxypeptidase Al to its active form was inhibited by pretreatment with the weak base chloroquine (40MM) and the protonophore monensin (10 AM). This conversion was also inhibited by pretreatment with the serine protease inhibitor benzamidine (10 mM) but not the cysteine protease inhibitor E64 (100 MM). The results suggest that high dose CCK stimulates the intracellular activation of digestive zymogens within isolated pancreatic acini. This event appears to require an acidic subcellular compartment and serine protease activity. (J. Clin. Invest.

Sequence determination of up to 24 aminoterminal residues of several putative precursors for dog pancreas secretory proteins, synthesized in vitro by translation of their mRNAs in the presence of radioactively labeled amino acids, revealed extensive sequence homology in the 16 amino-terminal residues. It is suggested that this common sequence constitutes a metabolically short-lived peptide extension which precedes the amino-terminal sequences of all pancreatic secretory proteins and that it functions in the transfer of these proteins across the microsomal membrane. This sequence was found to contain an unusually large percentage of hydrophobic residues. In the recently formulated signal hypothesis (1, 2) it was proposed that segregation of specific proteins, (e.g., secretory, lysosomal, and peroxisomal proteins) in intracellular membrane-bounded compartments is accomplished by a metabolically short-lived "signal" sequence in the nascent polypeptide chain. This unique sequence would result from the translation of a sequence of codons which is located immediately to the 3' end of the initiation codon and is common to all mRNAs coding for proteins that are to be segregated from the cytosol by transfer across a membrane. According to this scheme (2), the signal sequence of the nascent chain, emerging from a space within the ribosome, causes specific receptor proteins in the membrane to aggregate, thereby forming a proteinaceous tunnel in the membrane; consequent binding of the ribosome to the aggregated proteins would then provide the topological conditions for chain transfer. Subsequently the signal sequence is removed and is therefore no longer present in the parent molecule. Recent work from this laboratory has provided evidence for certain aspects of the signal hypothesis (2-4). In this paper we report the amino-acid sequences of up to 24 amino-terminal residues of several polypeptides resulting from in uitro translation of mRNAs isolated from dog pancreas rough microsomes. The sequence data are consistent with the predictions made in the signal hypothesis. METHODSThe following experimental procedures have been or will be described elsewhere: (a) the subcellular fractionation of dog pancreas (Scheele and Blobel, in preparation); (b) the isolation of mRNAs from dog pancreas rough microsomes by sodium dodecyl sulfate-phenol extraction and subsequent ohgo(dT) (2); (e) the elution of the electrophoretically separated translation products from gel regions by electrophoresis (Devillers-Thiery and Blobel, unpublished); (f) the precipitation of the eluted polypeptides by acetone-HCI (5), with myoglobin serving as a coprecipitant; (g) the identification of gel bands derived from pancreatic secretion with known enzymes and zymogens of the exocrine pancreas (Scheele, in preparation).Sequence Determination. The radioactively labeled polypeptides eluted from the gels, as well as the carrier myoglobin, were dissolved in 20% acetic acid and applied to the Beckman 890 B sequencer. An improved DMAA program (no. 111374...

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