Biomedical subjects

S Gräfe

Publications and source records attributed to S Gräfe.

5 recordsLinked to original sources

PubMed2005

Instantaneous dynamics and quantum control fields: principle and numerical applications.

The relation between laser pulses serving the purpose of controlling elementary molecular processes and the instantaneous dynamics of the perturbed system is investigated. The application of the conditions assuring a controlled change of the expectation value of an observable directly links the internal motion to the external perturbation. Several numerical applications document that the derived control fields are efficient and can be interpreted clearly on physical grounds.

Journal Article↗

PubMed1999

Mg-chelatase of tobacco: the role of the subunit CHL D in the chelation step of protoporphyrin IX.

The Mg-chelation is found to be a prerequisite to direct protoporphyrin IX into the chlorophyll (Chl)-synthesizing branch of the tetrapyrrol pathway. The ATP-dependent insertion of magnesium into protoporphyrin IX is catalyzed by the enzyme Mg-chelatase, which consists of three protein subunits (CHL D, CHL I, and CHL H). We have chosen the Mg-chelatase from tobacco to obtain more information about the mode of molecular action of this complex enzyme by elucidating the interactions in vitro and in vivo between the central subunit CHL D and subunits CHL I and CHL H. We dissected CHL D in defined peptide fragments and assayed for the essential part of CHL D for protein-protein interaction and enzyme activity. Surprisingly, only a small part of CHL D, i.e., 110 aa, was required for interaction with the partner subunits and maintenance of the enzyme activity. In addition, it could be demonstrated that CHL D is capable of forming homodimers. Moreover, it interacted with both CHL I and CHL H. Our data led to the outline of a two-step model based on the cooperation of the subunits for the chelation process.

Chlorophyll↗

PubMed1999

Characterization of recombinant Arabidopsis thaliana threonine synthase.

Threonine synthase (TS) catalyses the last step in the biosynthesis of threonine, the pyridoxal 5'-phosphate dependent conversion of L-homoserine phosphate (HSerP) into L-threonine and inorganic phosphate. Recombinant Arabidopsis thaliana TS (aTS) was characterized to compare a higher plant TS with its counterparts from Escherichia coli and yeast. This comparison revealed several unique properties of aTS: (a) aTS is a regulatory enzyme whose activity was increased up to 85-fold by S-adenosyl-L-methionine (SAM) and specifically inhibited by AMP; (b) HSerP analogues shown previously to be potent inhibitors of E. coli TS failed to inhibit aTS; and (c) aTS was a dimer, while the E. coli and yeast enzymes are monomers. The N-terminal region of aTS is essential for its regulatory properties and protects against inhibition by HSerP analogues, as an aTS devoid of 77 N-terminal residues was neither activated by SAM nor inhibited by AMP, but was inhibited by HSerP analogues. The C-terminal region of aTS seems to be involved in dimer formation, as the N-terminally truncated aTS was also found to be a dimer. These conclusions are supported by a multiple amino-acid sequence alignment, which revealed the existence of two TS subfamilies. aTS was classified as a member of subfamily 1 and its N-terminus is at least 35 residues longer than those of any nonplant TS. Monomeric E. coli and yeast TS are members of subfamily 2, characterized by C-termini extending about 50 residues over those of subfamily 1 members. As a first step towards a better understanding of the properties of aTS, the enzyme was crystallized by the sitting drop vapour diffusion method. The crystals diffracted to beyond 0.28 nm resolution and belonged to the space group P222 (unit cell parameters: a = 6.16 nm, b = 10.54 nm, c = 14.63 nm, alpha = beta = gamma = 90 degrees).

Amino Acid Sequence↗

PubMed1997

Mg-chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two-hybrid system, and reconstitution of the enzyme activity by co-expression of recombinant CHL D, CHL H and CHL I.

Mg-protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin IX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg-protoporphyrin IX. In Rhodobacter sphaeroides, and Synechocystis, the three open reading frames bchD/chID, bchH/chIH and bchI/chII encode proteins which are required for in vitro Mg-chelatase activity. In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchI have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full-length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide. Sequence comparison of tobacco CHL D revealed similarities to the D subunit of Rhodobacter and Synechocystis of 44% and 75%. The amino terminal half of CHL D shows significant similarity (46%) to the entire CHL I peptide sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are linked with a glutamine/asparagine/proline-rich region flanked by a highly acid-rich segment. Protein-protein interaction among the three subunits CHL D, H and I was studied using the yeast two-hybrid system. Physical interaction was demonstrated between CHL D and CHL I indicating that CHL D is part of the Mg-chelatase. Heterodimer formation of CHL H with CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicated in the more sensitive assay on X-Gal-containing agar plates. In vitro Mg2+ insertion into protoporphyrin IX was demonstrated in protein extracts of yeast strains expressing the three subunits of tobacco Mg-chelatase. The reconstitution of the recombinant enzyme activity required additional ATP.

Amino Acid Sequence↗

PubMed1996

Structural dissection and functional analysis of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A.

The overlapping tandem promoters of the streptokinase gene, P1 and P2, identified previously by S1 nuclease transcript mapping were functionally dissected by mutagenesis of their -10 regions and fused transcriptionally with or without the 202-bp upstream region (USR) to the luciferase reporter gene (luc) from Photinus pyralis to analyze the contribution of the different sequence elements to promoter activity in Escherichia coli and the homologous Streptococcus equisimilis strain H46A. In E. coli, virtually the entire promoter activity derived from the upstream promoter P1. In S. equisimilis, luc expression increased in the following order of the involved sequence elements: P2 approximately equal to P2 + USR < P1 < P1 + P2 < P1 + USR < P1 + P2 + USR. This shows that (1) in the homologous system, P1 and P2 alone are extremely weak, (2) in the USR-less arrangement, only the combined core promoters have substantial activity, and (3) the USR stimulates only P1 and the combination of P1 + P2. Thus, the tandem promoters presumably function by mutual contributary action and their full activity strongly depends on the AT-rich and statically bent upstream region. The distinctive feature determining the strength of P1 in both hosts appears to be its extended -10 region which matches the consensus TRTGN established for strong S. pneumoniae and Bacillus subtilis promoters.

Base Sequence↗