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Polyadenylated mRNA was purified from the aleurone cells of Cyamopsis tetragonoloba (guar) seeds germinated for 18 h and used for the construction of a cDNA library. Clones with the alpha-galactosidase encoding gene were identified using oligo-nucleotide mixed probes based on the NH2 terminal amino acid sequence and on the sequence of an internal peptide. The nucleotide sequence of the cDNA clone showed that the enzyme is synthesized as a precursor with a 47 amino acid NH2 terminal extension. This pre-sequence most likely functions to target the protein outside the aleurone cells into the endosperm. Based upon structural features, it is proposed to divide the precursor into a pre-(signal sequence) part and a glycosylated pro-part comparable with those of the yeast mat A/alpha factor and killer factor. A comparison of the derived amino acid sequence of this alpha-galactosidase from plant origin revealed significant stretches of homology with respect to the amino acid sequences of the enzymes from Saccharomyces cerevisiae and from human origin but only to a minor extent compared with the alpha-galactosidase from Escherichia coli.

Experiments are described which were designed to obtain insight into the question why Escherichiu coli K12 induces the PhoE protein pore in its outer membrane under conditions of phosphate limitation. Growth experiments showed that the presence of PhoE protein is advantageous for the growth rate of cells growing in a medium with large linear polyphosphates, but not with Pi, as the only phosphorus source. Further experiments, in which rates of permeation of p-lactam antibiotics through the outer membrane were determined, showed that the most likely explanation for this result is that the PhoE protein pore is a relatively efficient channel for negatively charged solutes in general. Comparison of the rates of permeation of the two p-lactam antibiotics cephaloridine and cefsulodin through PhoE protein pores and through OmpF protein pores in vivo suggested that the relatively efficient permeation of cefsulodin through PhoE protein pores must be attributed to the negative charge of a sulphate residue which is present in cefsulodin but absent in cephaloridine. Subsequent experiments showed that the rate of uptake of cefsulodin through PhoE protein pores, but not through OmpF protein pores, was reversibly inhibited by linear polyphosphates whereas phosphorylated compounds and other negatively charged solutes were also found to be inhibitory. The largest inhibition was observed with solutes containing several phosphate residues. Dixon plots showed that inhibition by Pi and linear polyphosphates was of the competitive type. The results are interpreted in terms of a recognition site on the PhoE protein pore for phosphorus-containing solutes and for other negatively charged compounds.

Antisera were raised against the purified Esckerichia coli K 12 outer membrane proteins ompA-, ompC-and ompF proteins and protein e. Several immunological methods were used to investigate the specificity of the antisera and the immunological relationship between the major outer membrane proteins. Although the antisera had been raised against highly purified proteins, several of them contained activity against lipopolysaccharide and lipoprotein due to minor impurities in the immunogens. The three general porins ompF protein, ompC protein and protein e were shown to be cross-reactive. Anti-(ompA protein) serum only reacted with the homologous protein. None of these antisera reacted with the phage lambda receptor protein or with protein 111. Pore protein preparations isolated from SaImonella typhimurium, Klebsiella aerogenes, Enterobacter cloaceae and Proteus mirabilis were found to be structurally related to the E. coli K12 porins as they reacted with the antisera raised against E. coli K12 porins.The outer membrane of Enterobacteriaceae contains phospholipids, which are mainly or exclusively present in the inner monolayer [1,2], lipopolysaccharide, which is exclusively located in the outer monolayer [3] and proteins [4-71, many of which transverse the lipid bilayer [8]. The most abundant protein in the outer membrane of Escherichia coli is the lipoprotein (7 x lo5 copies per cell) [9, lo]. Wildtype cells of E. coli K12 contain several other major proteins. For the proteins designated as b, c and d in our laboratory [5], other nomenclatures were also used [4,7,11], but it is now agreed that the proteins will be named according to the designation of their structural genes [12], which are ompF, ompC, and ompA respectively. Although these proteins are often called 'major outer membrane proteins', this term can be misleading, as the relative amounts of these proteins are dependent on the growth conditions [ 13 -181.The ompA protein has a function in the F-pilusmediated conjugation [19,20] and might play a role in the uptake of colicin L [21] and ferrichrome iron [22]. The ompF and ompC proteins form tight complexes with the peptidoglycan layer, which resists incubation in 2 % sodium dodecyl sulphate at 60°C [15,23,24]. These proteins form aqueous pores through which small molecules up to a molecular weight of about 700 can pass the outer membrane [25-291. can be present in pseudo revertants of mutants lacking the ompF and ompC proteins [21,26,30,31]. We have recently observed that this protein is induced in wildtype E. coli K12 cells by phosphate limitation [17] and is coregulated with alkaline phosphatase [32]. Also protein e is peptidoglycan-associated and has porin properties [31]. The ompF and ompC proteins and protein e are very similar with respect to their total amino acid composition [31,33,34] and their amino termini [30], but considerable differences were found in the fragment patterns obtained by incubation of the purified proteins with cyanogen bromide and proteolytic enzymes [31,33,34]. Protein I11 usuall...