Recently, the genes of two isoenzymes of phospholipase D from white cabbage (PLD1 and PLD2) with molecular masses of 91.7 and 91.9 kDa, respectively, have been sequenced and expressed in Escherichia ...coli Schäffner, I., Rücknagel, K.-P., Mansfeld, J., and Ulbrich-Hofmann, R. (2002). Eur. J. Lipid Sci. Technol. 104: 79-87. Both enzymes are highly homologous (91% identity) and behave very similarly. Phospholipase D purified from white cabbage leaves (PLDcab) is compared with the two recombinant enzymes in sodium dodecylsulfate and native polyacrylamide gel electrophoresis, isoelectric focusing, N-terminal sequencing, and mass spectrometry after tryptic digestion. As a result, PLDcab clearly can be assigned to PLD2. In contrast to recombinant PLD2, however, PLDcab is N-terminally acetylated.
10 N-aminoacyl-O-4-nitrobenzoyl hydroxamates were investigated as potential inhibitors of aminopeptidases. While the metal-depending enzymes aminopeptidase M, aminopeptidase P and leucine ...aminopeptidase were inhibited reversibly by the compounds, the thiol enzyme cathepsin H was inhibited efficiently in time-dependent reactions according to its substrate specificity. N-phenylalanyl-O-4-nitrobenzoyl hydroxamate was shown to be most effective, exhibiting a second-order-rate constant of inhibition of 31,766 M-1 s-1.
The synthesis of six new glycerophospholipids with choline-analogous head groups by enzymatic transphosphatidylation with phospholipase D is described. Starting from 1,2-dioleoyl-
...sn-glycero-3-phosphocholine, primary or secondary N-heterocyclic alcohols and (2-hydroxyethyl) trimethylarsonium were substituted for choline in two-phase systems.
The synthesis of six new glycerophospholipids with choline-analogous head groups by enzymatic transphosphatidylation with phospholipase D (PLD) is described.
The molecular mass of the native FK506-binding peptidyl-prolyl
cis/trans isomerase (PPIase) FKBP25mem from
Legionella pneumophila (Mip (macrophage infectivity potentiator) protein) was determined by ...two methods. By gel-permeation chromatography we found no indication of the presence of the monomeric enzyme. However, an oligomeric state with a molecular mass of about 62 kDa was detected. By cross-linking with dimethyl pimelimidate and subsequent SDS-PAGE of either the surface proteins of intact
L. pneumophila cells or the purified recombinant FKBP25mem in solution, we observed an immunoreactive band indicative of a mass in the dimer range. In contrast to human recombinant FKBP12, the enzymatic activity of
Legionella FKBP25mem was strongly dependent on the protein concentration, pointing to a dimer as the most active species. However, the inhibition by FK506 yielded a nearly constant value of
K
i of about 250 nM when measured in the same range of FKBP25mem concentration. These results may be explained by the fact that monomeric FKBP25mem has little, if any, influence on enzymatic activity when compared with the homodimer.
Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires' disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to ...the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPlase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPlases. The Icy gene (Legionella cyclophilin) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17968 Da called L. pneumophila cyclophilin 18 (L.p.Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coli with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histidine tag and an enterokinase cleavage site exhibits PPlase activity when produced at high levels in E. coli K-12. After removal of the extension by enterokinase, the properties of the recombinant Cyp18 were indistinguishable from those of the authentic enzyme. In order to investigate the influence of Cyp18 on intracellular survival of L. pneumophila an Icy-negative L. pneumophila strain was constructed. Compared with the wild-type strain, the mutant did not exhibit a significant phenotype but was 10-fold less invasive for Acanthamoeba castellanii. Like human cyclophilin, the L. p. Cyp18 exhibits nuclease activity, but this enzymatic activity does not appear to be linked with the native structure of the protein.
The influence of glycosylation on proteolytic degradation was studied by comparing cleavage sites in ribonuclease A (RNase A) and ribonuclease B (RNase B), which only differ by a carbohydrate chain ...attached to Asn34 in RNase B. Primary cleavage sites in RNase B were determined by identifying complementary fragments using matrix-assisted laser desorption/ionization mass spectrometry and compared with those in RNase A Arnold et al. (1996), Eur. J. Biochem. 237, 862-869. RNase B was cleaved by subtilisin even at 25 degrees C at Ala2-Ser21 as known for RNase A. Under thermal unfolding, the peptide bonds Asn34-Leu35 and Thr45-Phe46 were identified as primary cleavage sites for thermolysin and Lys31-Ser32 for trypsin. These sites are widely identical with those in RNase A. Treatment of reduced and carbamidomethylated RNase A and RNase B with trypsin led to a fast degradation and revealed new primary cleavage sites. Therefore, the state of unfolding seems to determine the sequence of degradation steps more than steric hindrance by the carbohydrate moiety does.
In Alzheimer's disease (AD), one of the cardinal neuropathological signs is deposition of amyloid, primarily consisting of the amyloid beta-peptide (Abeta). Structural variants of AD-associated Abeta ...peptides have been difficult to purify by high-resolution chromatographic techniques. We therefore developed a novel chromatographic protocol, enabling high-resolution reverse-phase liquid chromatography (RPLC) purification of Abeta variants displaying very small structural differences. By using a combination of size-exclusion chromatography and the novel RPLC protocol, Abeta peptides extracted from AD amyloid were purified and subsequently characterized. Structural analysis by microsequencing and electrospray-ionization mass spectrometry revealed that the RPLC system resolved a complex mixture of Abeta variants terminating at either residue 40 or 42. Abeta variants differing by as little as one amino acid residue could be purified rapidly to apparent homogeneity. The resolution of the system was further illustrated by its ability to separate the structural isomers of Abeta1-40. The present chromatography system might provide further insight into the role of N-terminally and posttranslationally modified Abeta variants, because each variant can now be studied individually.
Oligopeptides derived from the gag polyprotein (Pr55
gag) of human immunodeficiency virus type 1 (HIV-1) segment were used to evaluate the extension of the putative binding region for the complex of ...Pr55
gag and the human cytosolic peptidyl prolyl
cisltrans isomerase (PPIase) 18 kDa cyclophilin (Cyp18). Five N-terminally acetylated, C-terminally amidated oligopeptides containing one (HIV-1 Gag
218–224; 1), two (HIV-1 Gag
218–226 and HIV-1 Gag
217−224; 2 and 3, respectively), three (HIV-1 Gag
217–226; 4) or four (HIV-1 Gag
213−237; 5) proline residues were synthesized. Using competition experiments with a standard substrate the binding affinities to Cyp18 of the synthesized peptides were determined. The IC
50 value of 184 μ.M for the 25-mer peptide 5 was fivefold or more lower than those of the peptides 1–4 lacking one or more prolines. Failure of competition in assays containing enzymes of other PPIase families by millimolar concentrations of 5 revealed a Cyp18 specific interaction involving the active site of the enzyme. In its far UV circular dichroism, aqueous solutions of 5 display properties of random coil conformation, but spectra were also consistent with a small contribution of proline specific secondary structures. However, a proline-rich peptide typical of forming left-handed polyproline II helices did not compete for the active site of Cyp18. The results demonstrate that the putative binding region of HIV-1 gag polyprotein has a certain degree of binding affinity to the PPIase site of Cyp18, and may add a previously unrecognized topological component to the known subsite specificity of cyclophilins.
Ribonuclease (RNase) A and the more stable glycosylated RNase B differ by a carbohydrate moiety (GlcNAc2Man5–9) attached to Asn34. As previously shown, the first proteolytic cleavage sites to appear ...on thermal denaturation of both enzymes are in the structural region around Asn34. To discriminate the contribution of the modifying moiety to the stabilization toward thermal unfolding, on the one hand, and proteolytic fragmentation, on the other hand, the carbohydrate chain of RNase B was shortened by treatment with glycosidases to obtain GlcNAc‐RNase and (GlcNAc)2Man3‐RNase and extended by binding to concanavalin A or concanavalin A‐agarose. The results show a saltatory increase of the thermal unfolding constants and transition temperatures of GlcNAc‐RNase in comparison to RNase A, whereas the extension of the modification at Asn34 in the other RNase species does not further increase thermal stability. Therefore, the stability difference between RNase A and RNase B derivatives is attributed to the first carbohydrate unit. In contrast, the rate of proteolysis decreases gradually with increasing volume of the modifying moiety. As concluded from the analysis of the primary cleavage fragments, the main degradation pathway is shifted from the Asn34‐Leu35 to the Thr45‐Phe46 peptide bond due to increasing shielding effects.
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