The amino acid sequence of jack bean urease has been determined. The protein consists of a single kind of polypeptide chain containing 840 amino acid residues. The subunit relative molecular mass calculated from the sequence is 90770, indicating that urease is composed of six subunits. Out of 25 histidine residues in urease, 13 were crowded in the region between residues 479 and 607, suggesting that this region may contain the nickelbinding site. Limited tryptic digestion cleaved urease at two sites, Lys-128 and Lys-662. Proteolytic products were not dissociated and retained full enzymatic activity. Five tryptic peptides containing the reactive cysteine residues were isolated and characterized with the aid of sulfhydryl-specific reagents, N-iodoacetyl-N'-(5-sulfo-lnaphthy1)ethylenediamine and N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide. The reactive cysteine residues were located at positions 59,207, 592,663, and 824. The possibility that Cys-59, Cys-207, Cys-663, and Cys-824 are involved in the urease activity of the enzyme has been eliminated. Cys-592, which is essential for enzymatic activity, is located in the above-mentioned histidine-rich region.Jack bean urease was the first enzyme isolated as a crystalline protein by Sumner in 1926 [l]. However, the subunit structure of this enzyme has been somewhat ambiguous. The relative molecular mass reported first for the native urease was 480000 [2] while recently the value of 590 000 was reported [3]. The values reported for the monomer range over 30000-97000 [3-61. On the other hand, the subunits of ureases from microorganisms appear to be smaller than jack bean urease in size and number [7-91. Recently it was suggested that one large and two small polypeptides may be generally associated with microbial ureases [lo].Jack bean urease is also the first example of a nickel metalloenzyme and contains two nickel ions per 96600-M, subunit [ l l , 121. Spectrophotometric studies showed that the nickel ion has an essential role in catalysis and that the substrate and inhibitors of urease bind to the nickel ion [13, 141. A model for the mechanism of action of urease, taking into consideration the nickel ion, has been proposed [15].The presence of sulfhydryl groups in a protein was first described for jack bean urease [16]. The sulfhydryl groups in the enzyme are classified into the exposed ones which are titrated in the native conformation and the buried ones which can be titrated only in the presence of denaturant. Urease has been reported to have 27 -35 reactive exposed cysteine residues per urease molecule [17 -191. These cysteine residues were divided into the inessential ones, which readily react with sulfhydryl reagents like 5,5'-dithiobis(2-nitrobenzoic acid) without activity loss, and the essential ones, which react more

The amino acid sequence of two nonspecific lipid-transfer proteins (nsLTP) B and C from germinated castor bean seeds have been determined. Both the proteins consist of 92 residues, as for nsLTP previously reported, and their calculated M , values are 9847 and 9593 for nsLTP-B and nsLTP-C, respectively. The sequences of nsLTP-B and nsLTP-C, compared to the known sequence of nsLTP-A from the same source, are 68% and 35% similar, respectively. No variation was found at the positions of the cysteine residues, indicating that they might be involved in disulfide bridges.Nonspecific lipid-transfer proteins (nsLTP), purified from rat and bovine liver, facilitate the transfer of all common phospholipids, cholesterol, neutral glycosphingolipids, and gangliosides between membranes [l -71. The isoelectric point of bovine nsLTP is 9.55 [3] and its complete primary structure has been elucidated [8]. The protein consists of 121 residues with an M , of 13021 and contains one cysteine residue. This single cysteine residue is essential for the transfer of lipids between membranes [9]. The mode of action of nsLTP remains to be elucidated. Although it was believed that nsLTP did not act as a carrier, because of its inability to bind a lipid [9 -121, Nichols [13] has recently reported that rat liver nsLTP binds a fluorescently labeled phosphatidylcholine.On the other hand, nsLTPs from plant sources were purified from maize seedlings, spinach leaves, and germinated castor bean seeds [14-161. These proteins transfer various phospholipids as well as galactolipids. The amino acid sequences of nsLTPs from germinated castor bean seeds [17] and spinach leaves [18] were determined.In this paper, we report the amino acid sequences of two nsLTP isoproteins from germinated castor bean seeds, indicating the positions of disulfide bridges. MATERIALS AND METHODSMost of this information is presented in the miniprint supplement. Designations T, L, S, CH and C mean the peptides derived by trypsin, lysyl endopeptidase, Staphylococcus aureus V8 protease, chymotrypsin and clostripain digestions, respectively. RESULTS Isolation of the digestedpeptides of nsLTP-BThe amino acid sequence of nsLTP-B was established by sequencing the whole protein, and the peptides obtained, by digestion with trypsin, chymotrypsin and S. aureus V8 protease. The tryptic peptides of intact nsLTP-B were first subjected to reverse-phase HPLC on a column of Synchropak RP-P (Fig. Sl). T4 and T6 in peak 1 were purified on a YMC AM 303 column (data not shown). Peaks 2, 3, and 4 were pure T3, T7 and T8, respectively. Peak 5 contained three peptides T1, T5 and T10 which were linked by two disulfide bridges, and these peptides were purified on a column of M & S pack CI8 after reduction and carboxymethylation (Fig. S2). Peak 6 contained T2 and T9 which were linked by a disulfide bridge, and was likewise chromatographed on Prote 8 (Fig. S3). Complete sets of tryptic peptides, except for Arg at position 37, were obtained. Amino acid compositions of these tryptic peptides are shown in...

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Four kinds of nonspecific lipid transfer proteins (nsLTP) were purified from different organs of castor bean (Ricinus communis L.) seedlings. Amino acid compositions and amino-terminal sequences of the four nsLTPs were determined and compared with those of castor bean isoforms, nsLTP-A, -B, and -C, previously reported [Takishima et al. (1986) Biochim. Biophys. Acta 870, 248-255; Takishima et al. (1988) Eur. J. Biochem. 177, 241-249]. Two isoforms from the cotyledons were identified as nsLTP-A and -C, one isoform from the endosperms as nsLTP-B, and the other was a new isoform from the axes. This new isoform was named nsLTP-D and its amino acid sequence was determined. These results demonstrated organ-specific occurrence of the nsLTP isoforms in castor bean seedlings. The isoforms nsLTP-A, -B, -C, and -D showed similar transfer activity not only for phosphatidylcholine and phosphatidylethanolamine but also for monogalactosyldiacylglycerol, although the homology among their amino acid sequences ranged from 70 to 30%. Two cDNA clones (pnsLTP-C and pnsLTP-D) for nsLTPs of castor bean seedlings were isolated and sequenced. pnsLTP-C was the cDNA clone for nsLTP-C expressed in the cotyledons, and pnsLTP-D was that for nsLTP-D in the axis. A coupled in vitro transcription-translation analysis of both cDNA clones revealed that pnsLTP-C encodes the full-length of nsLTP-C precursor (pro-nsLTP-C), while pnsLTP-D encodes a part of nsLTP-D precursor. PronsLTP-C contained a 24-amino acid pre-sequence preceding the mature nsLTP-C (92 amino acids).(ABSTRACT TRUNCATED AT 250 WORDS)