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The crystal structure of the Pyrus pyrifolia pistil ribonuclease (S(3)-RNase) responsible for gametophytic self-incompatibility was determined at 1.5-A resolution. It consists of eight helices and seven beta-strands, and its folding topology is typical of RNase T(2) family enzymes. Based on a structural comparison of S(3)-RNase with RNase Rh, a fungal RNase T(2) family enzyme, the active site residues of S(3)-RNase assigned were His(33) and His(88) as catalysts and Glu(84) and Lys(87) as stabilizers of an intermediate in the transition state. Moreover, amino acid residues that constitute substrate binding sites of the two RNases could be superimposed geometrically. A hypervariable (HV) region that has an S-allele-specific sequence comprises a long loop and short alpha-helix. This region is far from the active site cleft, exposed on the molecule's surface, and positively charged. Four positively selected (PS) regions, in which the number of nonsynonymous substitutions exceeds that of synonymous ones, are located on either side of the active site cleft, and accessible to solvent. These structural features suggest that the HV or PS regions may interact with a pollen S-gene product(s) to recognize self and non-self pollen.

Ca2+‐dependent cell‐‐cell adhesion molecules, termed cadherins, are divided into subclasses with distinct tissue distributions and distinct cell‐binding specificities. To elucidate the biochemical relationship of these subclasses, we compared the pattern of tryptic cleavage and the partial amino acid sequence of mouse liver E‐cadherin with those of chicken brain N‐cadherin. Although these two cadherins are distinct in their cell‐binding and immunological specificities, they showed an identical mol. wt and a similar tryptic cleavage pattern. We isolated tryptic fragments of E‐ and N‐cadherin, and determined the sequences of nine amino acid residues of their amino terminus. The results showed that sequences of amino acids from the amino terminus to the 7th residues are identical in these two cadherins. We thus suggest that cadherins with distinct specificities have a common genic origin.

The complete amino acid sequence of Achromobacter lyticusprotease I (EC 3.4.21.50), which specifically hydrolyzes lysyl peptide bonds, has been established. This has been achieved by sequence analysis of the reduced and S-carboxymethylated protease and of peptides obtained by enzymatic digestion with Achromobacterprotease I itself and Staphylococcus aureusV8 protease and by chemical cleavage with cyanogen bromide. The protease consists of 268 residues with three disulfide bonds, which have been assigned to Cys6-Cys216, Cys12-Cys80, and Cys36-Cys58. Comparison of the amino acid sequence of Achromobacterprotease and other serine proteases of bacterial and mammalian origins has revealed that Achromobacterprotease I is a mammalian-type serine protease of which the catalytic triad comprises His57, Asp113, and Ser194. It has also been shown that the protease has 9- and 26-residue extensions of the peptide chain at the N and C termini, respectively, and overall sequence homology is as low as 20% with bovine trypsin. The presence of a disulfide bridge between the N-terminal extension Cys6and Cys216close to the putative active site in the C-terminal region is thought to be responsible for the generation of maximal proteolytic function in the pH range 8.5-10.7 and enhanced stability to denaturation.

The trypsin-sensitive cholecystokinin-releasing peptide is a peptide purified from rat pancreatic juice on the basis of its stimulatory activity toward pancreatic enzyme secretion. We postulate that the peptide acts as a mediator of pancreatic enzyme secretion in response to dietary protein intake and that it (designated as "monitor peptide" from its role in the intestine) could be responsible for the feedback regulation of pancreatic enzyme secretion. About 20 nmol of the highly purified peptide were obtained from 800 ml of rat pancreatic juice by reverse-phase high performance liquid chromatography. It was then sequenced. The peptide comprises 61 amino acid residues (Table I). It has a sequence that closely resembles that of a highly conserved region in pancreatic secretory trypsin inhibitors (PSTIs, Kazal type inhibitor): -Ile-Tyr-Asx-Pro-Val-Cys-Gly-Thr-Asx-Gly-. However, the peptide is less related to other mammalian PSTIs than they are to each other. The additional 5 residues at the NH2 terminus make the peptide larger than the common 56-residue PSTIs. The trypsin-sensitive cholecystokinin-releasing peptide is to be classified as a Kazal-type inhibitor and may be one of the rat PSTIs or a related peptide. The present results and increasing evidence from other laboratories and ours suggest that Kazal-type inhibitors play previously unrecognized multiple physiological roles.

Achromobacterprotease I (API) is a lysine-specific serine protease which hydrolyzes specifically the lysyl peptide bond. A gene coding for API was cloned from Achromobacter lyticusM497-1. Nucleotide sequence of the cloned DNA fragment revealed that the gene coded for a single polypeptide chain of 653 amino acids. The N-terminal 205 amino acids, including signal peptide and the threonine/serine-rich C-terminal 180 amino acids are flanking the 268 amino acid-mature protein which was identified by protein sequencing. Escherichia colicarrying a plasmid containing the cloned API gene overproduced and secreted a protein of Mr50,000 (API‘) into the periplasm. This protein exhibited a distinct endopeptidase activity specific for lysyl bonds as well. The N-terminal amino acid sequence of API’ was the same as mature API, suggesting that the enzyme retained the C-terminal extended peptide chain. The present experiments indicate that API, an extracellular protease produced by gram-negative bacteria, is synthesized in vivoas a precursor protein bearing long extended peptide chains at both N and C termini.

The partial amino acid sequence of the NH2 terminus of a factor named human B-cell differentiation factor or B-cell stimulatory factor 2 (BSF-2) has been determined. Antibodies raised against the synthetic peptide corresponding to residues 1-13 of the NH2-terminal sequence specifically react with BSF-2 generated by a T-cell line and by phytohemagglutinin-stimulated normal T cells. Furthermore, the antipeptide antibodies react with a BSF-2-like factor produced by cardiac myxoma as well as uterine cervical carcinoma cells. The results show that BSF-2 functions in vivo as well and suggest that the constitutive production of BSF-2 may be involved in autoantibody production, since patients with cardiac myxoma and uterine carcinoma showed autoantibody production.

phoS is the structural gene for the phosphate-binding protein, which is localized in periplasm and involved in active transport of phosphate in Escherichia coli. It is also a negative regulatory gene for the pho regulon, and the gene expression is inducible by phosphate starvation. The complete nucleotide sequence of the phoS gene was determined by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). The amino acid sequences at the amino termini of the pre-PhoS and PhoS proteins and at the carboxy terminus of the PhoS protein were determined by using the purified proteins. Furthermore, the amino acid sequence of enzymatically digested peptide fragments of the PhoS protein was determined. The combined data established the nucleotide sequence of the coding region and the amino acid sequence of the pre-PhoS and the PhoS proteins. The pre-PhoS protein contains an extension of peptide composed of 25 amino acid residues at the amino terminus of the PhoS protein, which has the general characteristics of a signal peptide. The mature PhoS protein is composed of 321 amino acid residues, with a calculated molecular weight of 34,422, and lacks the disulfide bond and methionine. The regulatory region of phoS contains a characteristic Shine-Dalgarno sequence at an appropriate position preceding the translational initiation site, as well as three possible Pribnow boxes and one -35 sequence. the nucleotide sequence of the regulatory region of phoS was compared with those of phoA and phoE, the genes constituting the pho regulon.

The venoms of the Najaspecies are known to be cytotoxic. This toxicity has been attributed to the presence of small nonenzymatic polypeptides of 60 amino acid residues, designated as cardiotoxins or cytotoxins. We investigated the cytotoxic potency of Naja nigricollisvenom fractions and isolated another type of cytotoxic component which is even more potent than cardiotoxins. This cytotoxic compound, which was designated as nigexine, was purified to homogeneity and its amino acid sequence was determined. Nigexine is a basic phospholipase A2consisting of a single chain of 118 amino acids. A detailed investigation of the cytotoxic effects on epithelial FL cells, C-13T neuroblastoma cells, and promyelocytic leukemia HL 60 cells revealed that nigexine not only altered cell viability but also prevented cell proliferation. This is a property that was specific to nigexine since other phospholipases A2from various sources had no detectable cytotoxic activity. The cytotoxic activity of nigexine was not dependent on the presence of divalent cations, unlike its enzymatic activity. In particular, the cytotoxic activity of nigexine was identical in the presence or absence of either 2 mMCa2+or Sr2+, or 6 mMEDTA. We also present evidence based on chemical modifications that cytotoxic activity was not correlated with enzymatic activity. Thus, modification with para-bromophenacyl bromide totally abolished the enzymatic activity of nigexine, which nevertheless retained 6–20% of the cytotoxicity of native nigexine. Conversely, treatment with cyanogen bromide gave a compound that retained 7% of the enzymatic activity of the parent molecule but was devoid of detectable cytotoxicity.

Hen oviduct Nα-acetyltransferase was clarified to have a nucleic acid as an existing constituent by the following three results: (i) an ultraviolet absorption spectrum of the purified Nα-acetyltransferase free of S-acetyl coenzyme A (Ac-CoA) had an absorption maximum at 260 nm. (ii) A nucleic acid band stained with ethidium bromide was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (iii) An ethidium bromide band co-migrated with a fluorescent band of the protein treated with N-(7-dimethylamino-4-methylcoumarinyl)maleimide, a reagent specific for thiol groups, on Polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate. Nα-Acetyltransferase lost its activity partially or completely by digestion with bovine pancreatic RNase A, Staphylococcus aureusnuclease, or proteinase K, showing that both the nucleic acid and the protein subunit were necessary for the enzyme activity. The nucleic acid component was identified as an RNA but not a DNA because the RNase T2digest of the nucleic acid was composed of four 3′-ribomononucleotides and completely separated from 3′- and 5′-deoxyribomononucleotides on TLC. The chain length of the nucleic acid of 260 nucleotides estimated by formamide-polyacrylamide gel electrophoresis was calculated to be about 83,000 of the molecular weight. The contents of RNA (35.0%) and protein (65.0%) in Nα-acetyltransferase determined on weight basis corresponded reasonably well to the contents of RNA (34.4%) and protein (65.6%) calculated based on the assumption that Nα-acetyltransferase consisted of one molecule of 7 S RNA (Mr83,000) and two identical Mr79,000 protein subunits. The total molecular weight (241,000) of the holoenzyme calculated based on the above result was identical to the molecular weight (240,000) of Nα-acetyltransferase estimated by Sepharose 6B gel filtration.

Hen oviduct Nα-acetyltransferase was purified to homogeneity by ammonium sulfate fractionation and DEAE-cellulose, Sepharose 6B, hydroxylapatite, and CoA affinity chromatography. The molecular weights of the native Nα-acetyltransferase and its protein subunit were estimated as 240,000 and 79,000, respectively. The purified enzyme exhibited a narrow pH optimum centered at 7.8. The enzyme was activated by dithiothreitol, cysteine, glutathione, and β-mercaptoethanol, but inhibited by Fe2+, Mn2+, Zn2+, Ca2+, Mg2+, and all thiol-specific reagents tested. These findings suggest that a thiol group(s) is essential to the enzyme activity.