Klaus P. Schäfer

Helicobacter pylori cadA encodes an essential Cd(II)-Zn(II)-Co(II) resistance factor influencing urease activity.

Inactivation of Helicobacter pylori cadA, encoding a putative transition metal ATPase, was only possible in one of four natural competent H. pylori strains, designated 69A. All tested cadA mutants showed increased growth sensitivity to Cd(II) and Zn(II). In addition, some of them showed both reduced 63Ni accumulation during growth and no or impaired urease activity, which was not due to lack of urease enzyme subunits. Gene complementation experiments with plasmid (pY178)‐derived H. pylori cadA failed to correct the deficiencies, whereas resistance to Cd(II) and Zn(II) was restored. Moreover, pY178 conferred increased Co(II) resistance to both the cadA mutants and the wild‐type strain 69A. Heterologous expression of H. pylori cadA in an Escherichia coli zntA mutant resulted in an elevated resistance to Cd(II) and Zn(II). Expression of cadA in E. coli SE5000 harbouring H. pylori nixA, which encodes a divalent cation importer along with the H. pylori urease gene cluster, led to about a threefold increase in urease activity compared with E. coli control cells lacking the H. pylori cadA gene. These results suggest that H. pylori CadA is an essential resistance pump with ion specificity towards Cd(II), Zn(II) and Co(II). They also point to a possible role of H. pylori CadA in high‐level activity of H. pylori urease, an enzyme sensitive to a variety of metal ions.

Molecular characterization of metal-binding polypeptide domains by electrospray ionization mass spectrometry and metal chelate affinity chromatography.

Metal ion-binding of synthetic peptides containing HxH and CxxC motifs was investigated by electrospray ionization mass spectrometry (ESI-MS) and metal chelate affinity chromatography. A high affinity of Ni2+ and Cu2+ to HxH containing sequences was found. Based on their natural metal ion-binding potential it was possible to include metal affinity chromatography in the purification process of two proteins without using an additional His-tag sequence: ATPase-439, a P type ATPase from Helicobacter pylori and the amyloid precursor protein (APP).

Synthesis and structural characterization of human-identical lung surfactant SP-C protein.

An efficient synthesis for human‐identical lung surfactant protein SP‐C is described with a semi‐automated solid phase synthesizer using Fmoc chemistry. Double coupling and acetic anhydride capping procedures were employed for synthetic cycles within the highly hydrophobic C‐terminal domain of SP‐C. Isolation of the protein was performed by mild cleavage and deprotection conditions and subsequent HPLC purification yielding a highly homogeneous protein as established by sequence determination, electrospray, plasma desorption and MALDI mass spectrometry. A general method has been employed for the preparation of Cys‐palmitoylated protein by using temporary Cys(tButhio) protection, in situ deprotection with β‐mercaptoethanol and selective palmitoylation of resin‐bound SP‐C. The mild synthesis and isolation conditions provide SP‐C with a high α‐helical content, comparable to that of the natural SP‐C, as assessed by CD spectra. Furthermore, first biophysical data indicate a surfactant activity comparable to that of the natural protein.

Lung surfactant: A biotechnological challenge

The genes for all three of the bona fide surfactant associated proteins have been cloned, allowing their production by recombinant DNA technology. In addition, improved protocols for the isolation of the natural surfactant proteins (NSP) made them available in larger quantities. Whereas, the NSP are often mixtures of allelic variants or functional isomers from gene families, the recombinant proteins (RSP) are obtained as single pure protein species. Antibodies directed against the N/RSP in combination with DNA probes have allowed new approaches to analyze the formation, location, transport, structure and functional capacities of these molecules as well as their interactions with one another and the phospholipids.

HNRNP core protein A1 is a member of a complex multigene family: Gene structure and evidence for the existence of two expressed genes in human cells

Until recently, no sequence information concerning hnRNP core proteins was available. To date, this situation has well changed. Sequence data of core protein A1 in mammals (Williams et al; Proc. Natl. Acad. Sci. USA 82 5666, 1985; Cobianchi et al; J. Biol. Chem. 261 3536, 1986; Riva et al; EMBO J. 5 2267, 1986), and the hnRNP C proteins (Lahiri and Thomas; J. Biol. Chem. 260, 598, 1986; Swanson et al; Mol. Cell. Biol. 1731, 1987) and Drosophila (Dawid et al; Proc. Natl. Acad. Sci. 84, 1819) lead to insights into hnRNP protein structure and function. We report here the isolation and characterization of a novel human cDNA encoding the core protein AI. Furthermore human genomic clones apparently coding for A1 were isolated and characterized. The results indicate that A1 is a member of a complex gene family.