A set of 8 proteins (SSI, sulfate-starvation-induced proteins) was observed by comparative two-dimensional electrophoresis to be induced when Escherichia coli were grown using compounds other than sulfate or cysteine as the sole sulfur source. These proteins were isolated after two-dimensional gel electrophoresis, digested with trypsin and the masses of the resulting peptides determined by mass spectrometry. The list of peptide masses served as a protein fingerprint which was used to search the databases, allowing four of the SSI proteins (SSI2, 5, 7, 8) to be identified with a high degree of confidence.To identify the other SSI proteins, and to obtain sequence information for as inany of the proteins as possible, automated on-line HPLC MS/MS (fragmentation analysis using coupled mass scanning devices) data collection was performed. The uninterpreted MS/MS spectra were used as peptide fingerprints to search the databases. Genes encoding two further proteins (SSI 1 and 3) were identified in the 8.5' region of the Escherichia coli genome. N-terminal sequencing of all of the proteins confirmed the results of protein and peptide fingerprinting and in addition showed that SSI 6 shows 50% similarity to the Bacillus suhtilis oij&l gene product. SSI 4 was not found in the databases by any of these methods. The ,methods described are of general use for the rapid analysis of complex cell responses. MS data accumulation takes about 5 min/protein for protein fingerprinting and 30 min for peptide fingerprinting and requires approximately 100 fmol of material. N-terminal sequencing however, takes about 5 h/protein and approximately 1 pmol to obtain a 10 amino acid sequence for a search.

An arylsulfatase (EC 3.1.6.1) was extracted from Pseudomonas aeruginosa PA01 and purified 2700-fold to homogeneity. Synthesis of this enzyme was repressed when sulfate, cysteine or thiocyanate was supplied as the sole sulfur source for growth, but derepressed with all other sulfur sources tested. The apparent molecular mass was determined by SDSPAGE to be 57 kDa, and the enzyme was presumed to be a monomer after gel filtration chromatography. The arylsulfatase showed maximal activity at 57°C and pH 8.9, and a K , of 105 pM for 4-nitrocatecholsulfate. Despite previous reports that both inducible and derepressible forms of arylsulfatase exist in f? aeruginosa, we found only one enzyme under a variety of growth conditions: a sulfate-repressed enzyme with a native isoelectric point of 4.76. The gene encoding this enzyme (atsA) was isolated by complementation of a Tn5-751 mutant of I? aeruginosa PAO1.Sequencing revealed a 1602-bp reading frame encoding a 534-amino-acid protein with sequence similarity to known bacterial and eukaryotic arylsulfatases (30-40% and 25-30 % identity, respectively), but lacking the signal peptide which is present in all known sequences. The lack of this signal peptide suggests that the I? aeruginosa arylsulfatase is neither periplasmic nor membrane-associated, unlike other known arylsulfatases. The atsA gene was located at 15-17' on the I? aeruginosa genome by Southern hybridization. Only a single copy was observed under moderate stringency conditions.

SummaryCytochrome c 552 is the terminal component of the formate-dependent nitrite reduction pathway of Escherichia coli. In addition to four 'typical' haem-binding motifs, CXXCH-, characteristic of c-type cytochromes, the N-terminal region of NrfA includes a motif, CWSCK. Peptides generated by digesting the cytochrome from wild-type bacteria with cyanogen bromide followed by trypsin were analysed by on-line HPLC MS/MS in parent scanning mode. A strong signal at mass 619, corresponding to haem, was generated by fragmentation of a peptide of mass 1312 that included the sequence CWSCK. Neither this signal nor the haemcontaining peptide of mass 1312 was detected in parallel experiments with cytochrome that had been purified from a transformant unable to synthesize NrfE, NrfF and NrfG: this is consistent with our previous report that NrfE and NrfG (but not NrfF) are essential for formate-dependent nitrite reduction. Redox titrations clearly revealed the presence of high and low midpoint potential redox centres. The best fit to the experimental data is for three n ¼ 1 components with midpoint redox potentials (pH 7.0) of þ45 mV (21% of the total absorbance change), ¹90 mV (36% of the total) and ¹210 mV (43% of the total). Plasmids in which the lysine codon of the cysteine-lysine motif, AAA, was changed to the histidine codon CAT (to create a fifth 'typical' haem c-binding motif), or to the isoleucine and leucine codons, ATT and CTT, were unable to transform a Nrf ¹ deletion mutant to Nrf þ or to restore formate-dependent nitrite reduction to the transformants. The presence of a 50 kDa periplasmic c-type cytochrome was confirmed by staining proteins separated by SDS-PAGE for covalently bound haem, but the methyl-viologen-dependent nitrite reductase activities associated with the mutated proteins, although still detectable, were far lower than that of the native protein. The combined data establish not only that there is a haem group bound covalently to the cysteine-lysine motif of cytochrome c 552 but also that one or more products of the last three genes of the nrf operon are essential for the haem ligation to this motif.

An arylsulfatase (EC 3.1.6.1) was extracted from Pseudomonas aeruginosa PA01 and purified 2700-fold to homogeneity. Synthesis of this enzyme was repressed when sulfate, cysteine or thiocyanate was supplied as the sole sulfur source for growth, but derepressed with all other sulfur sources tested. The apparent molecular mass was determined by SDSPAGE to be 57 kDa, and the enzyme was presumed to be a monomer after gel filtration chromatography. The arylsulfatase showed maximal activity at 57°C and pH 8.9, and a K , of 105 pM for 4-nitrocatecholsulfate. Despite previous reports that both inducible and derepressible forms of arylsulfatase exist in f? aeruginosa, we found only one enzyme under a variety of growth conditions: a sulfate-repressed enzyme with a native isoelectric point of 4.76. The gene encoding this enzyme (atsA) was isolated by complementation of a Tn5-751 mutant of I? aeruginosa PAO1.Sequencing revealed a 1602-bp reading frame encoding a 534-amino-acid protein with sequence similarity to known bacterial and eukaryotic arylsulfatases (30-40% and 25-30 % identity, respectively), but lacking the signal peptide which is present in all known sequences. The lack of this signal peptide suggests that the I? aeruginosa arylsulfatase is neither periplasmic nor membrane-associated, unlike other known arylsulfatases. The atsA gene was located at 15-17' on the I? aeruginosa genome by Southern hybridization. Only a single copy was observed under moderate stringency conditions.

α and β parvalbumins are Ca2+‐binding proteins of the EF‐hand type. We determined the protein sequence of human brain α parvalbumin by mass spectrometry and cloned human β parvalbumin (or oncomodulin) from genomic DNA and preterm placental cDNA. β parvalbumin differs in 54 positions from α parvalbumin and lacks the C‐terminal amino acid 109. From MS analyses of α and β parvalbumins we conclude that parvalbumins generally lack posttranslational modifications. α and β parvalbumins were differently expressed in human tissues when analyzed by immunoblotting and polymerase‐chain‐reaction techniques. Whereas α parvalbumin was found in a number of adult human tissues, β parvalbumin was restricted to preterm placenta. The pattern of α parvalbumin expression also differs in man compared to other vertebrates. For example, in rat, α parvalbumin was found in extrafusal and intrafusal skeletal‐muscle fibres whereas, in man, α parvalbumin was restricted to the muscle spindles. Different functions for α and β parvalbumins are discussed.