The nucleotide sequence of the gene (pac) coding for penicillin G acylase from E. coli ATCC 11105 was determined and correlated with the primary structure of the two constituent subunits of this enzyme. The pac gene open reading frame consists of four structural domains: Nucleotide positions 1-78 coding for a signal peptide, positions 79-705 coding for the alpha subunit, positions 706-867 coding for a spacer peptide, and positions 868-2538 coding for the beta subunit. Plasmids were constructed which direct the synthesis of a pac gene product lacking the signal peptide, and the synthesis of the alpha subunit or the beta subunit. The following results were obtained: The two dissimilar subunits are processing products of a single precursor polypeptide; the spacer peptide is removed during processing; the precursor polypeptide lacking the signal sequence is accumulated in the cytoplasm; it is not processed proteolytically in the cytoplasm and it does not display enzyme activity. Processing, therefore, requires translocation through the cytoplasmic membrane; processing follows a distinct sequential pathway in vitro.

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The covalent structure of the chymotrypsin II from the larvae of the hornet, Vespa crabro, has been determined. The sequence has been deduced from 3 sets of overlapping peptides generated by trypsin after modification of the lysine or arginine residues and by chymotrypsin. The enzyme is a serine endopeptidase and contains 218 residues in a single polypeptide chain cross-linked by 3 disulfide bonds. Alignment of the sequence of this insect protease with those of chymotrypsin, elastase and trypsin shows about 35% identity with each and a homologous relationship is evident. Serine endopeptidase ChymotrypsinInvertebrate protease Evolution Amino acid sequence

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This enzyme immunoassay for free thyroxin (FT4) involves simultaneous incubation of sample and thyroxin conjugated to horseradish peroxidase (EC 1.11.1.7) in antibody-coated tubes at room temperature. The test can be performed in 90 min. It is not influenced by variations in concentrations of thyroxin-binding globulin (TBG), thyroxin-binding prealbumin, or albumin. With increased concentrations of non-esterified fatty acids, the measured FT4 equivalent concentrations increase immediately, indicating displacement of endogenous thyroxin from its binding sites. FT4 values obtained with this assay for 110 samples, including sera with low albumin and high TBG as well as sera of patients treated with heparin, agreed well with those measured by a two-step radioimmunoassay and with the T4/TBG ratio. The measurable range of FT4 with our assay is 2.5 to 65 ng/L. Intra-assay CVs range from 3.4% to 2.2%, interassay CVs from 6.6% to 2.6% at concentrations of 9 to 45 ng of FT4 equivalent per liter.