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A Recktenwald

Publications and source records attributed to A Recktenwald.

12 recordsLinked to original sources
PubMed1998

Heparinase III limits rat arterial smooth muscle cell proliferation in vitro and in vivo.

Heparinase III degrades heparan sulfate proteoglycans, which are co-receptors for growth factors that stimulate arterial proliferation. We assessed the ability of locally-delivered heparinase III to limit medial vascular smooth muscle cell proliferation induced by balloon catheter injury in rat carotid arteries. Whereas vehicle-treated arteries showed 12% of smooth muscle cells proliferating after 2 days, heparinase III (0.022-5.7 mg/kg) treated arteries showed 0.8-4%. Chemically-inactivated heparinase III did not limit proliferation. In isolated rat A10 vascular smooth muscle cells, heparinase III (1 IU/ml) inhibited both PDGF-BB and bFGF mediated increases in proliferation and migration. These results suggest that heparinase III can limit proliferation by affecting heparan sulfate proteoglycan binding growth factors following arterial injury.

Animals↗
PubMed1997

Cloning, sequencing and overexpression of the leucine dehydrogenase gene from Bacillus cereus.

The L-leucine dehydrogenase gene from Bacillus cereus (DSM 626) was cloned from a partial genomic library and sequenced. The open reading frame has 1101 bp and codes for a protein of 39.9 kDa. The deduced amino acid sequence of the LeuDH from B. cereus shares 70-80% identity with LeuDH's from the thermophilic strains B. stearothermophilus and Thermoactinomyces intermedius. The active protein was overexpressed in Escherichia coli to yield approximately 30% of the total soluble protein.

Amino Acid Oxidoreductases↗
PubMed1995

Reduction of strong lipase-polyclonal antibodies binding by limited proteolysis.

Initial attempts to purify Geotrichum candidum lipase (EC 3.1.1.3) using immunoaffinity chromatography have been hampered by the tight binding of the lipase to the immobilized rabbit anti-GCL IgG. Stringent elution conditions were unable to release more than a few percent of the antigen. To decrease the tight binding, the immunosorbent was treated with minute amounts of different proteases, of which elastase proved to be most effective. Using the elastase-treated immunosorbent both natural and recombinant GCL II were purified to homogeneity with 30% of lipase recovered from the immunoaffinity chromatography step.

Antibodies, Fungal↗
PubMed1995

Redesigning the active site of Geotrichum candidum lipase.

Attempts to engineer enzymes with unique catalytic properties have largely focused on altering the existing specificities by reshaping the substrate binding pockets. Few experiments have aimed at modifying the configuration of the residues essential for catalysis. The difference in the topological location of the triad acids of Geotrichum candidum lipase (GCL) and the catalytic domain of human pancreatic lipase (HPL), despite great similarities in their topologies and 3-D structures, suggest that these are related enzymes whose catalytic triads have been rearranged in the course of evolution (Schrag et al., 1992). In this study we prepared a double mutant GCL in which the catalytic triad acid is shifted to the position equivalent to the location of the triad acid of HPL. The double mutant maintains approximately 10% of the wild type activity against triglycerides and the fluorogenic ester 4-methylumbelliferyl-oleate. The only significant differences between the 3-D structures of the double mutant and wild type GCL are at the mutated sites. Even the water structure in the region of the triad is unchanged. The hydrogen bonding pattern of the catalytic triad of the double mutant is very similar to that of pancreatic lipase. The acid of the double mutant is stabilized by only two hydrogen bonds, whereas three hydrogen bonds are observed in the wild type enzyme. These results strongly support the hypothesis that the pancreatic lipases are evolutionary switchpoints between the two observed arrangements of the catalytic triads supported by the alpha/beta hydrolase fold and suggest that this fold provides a stable protein core for engineering enzymes with unique catalytic properties.

Amino Acid Sequence↗
PubMed1993

Cloning and expression of Geotrichum candidum lipase II gene in yeast. Probing of the enzyme active site by site-directed mutagenesis.

The three-dimensional structure of lipase II of Geotrichum candidum strain ATCC34614 (GCL II) has provided insights with respect to the nature of the catalytic machinery of lipases. To support these structural observations, we have carried out an analysis of GCL II by mutagenesis. The gene encoding lipase II of Geotrichum candidum strain ATCC34614 (GCL II) was amplified using the polymerase chain reaction, cloned, and sequenced. The intronless lipase gene was expressed and secreted from Saccharomyces cerevisiae at approximately 5 mg/liter of culture. Recombinant GCL II was purified by immunoaffinity chromatography and characterized using a combination of substrates and independent analytical methods. The recombinant enzyme and the enzyme isolated from its natural source have comparable specific activities against triolein of about 1000 mumol of oleic acid released/min/mg of protein. The putative catalytic triad Ser217-His463-Glu354 was probed by site-directed mutagenesis. The substitution of Ser217 by either Cys or Thr and of His463 by Ala led to a complete elimination of the activity against both triolein and tributyrin. Substitution of Glu354 by either Ser, Ala or Gln renders the enzyme inactive and also perturbs the enzyme stability. However, the enzyme with the conservative replacement Glu354 Asp is stable and displays only a small decrease of triolein activity but a 10-fold decrease in activity against tributyrin. There was no appreciable difference in esterase activity between the native, recombinant wild type, and Glu354 Asp mutant. These results confirm that the triad formed by Ser217-Glu354-His463 is essential for catalytic activity. They also show that the active site of GCL II is more tolerant to a conservative change of the carboxylic side chain within the triad than are other hydrolases with similar catalytic triads.

Aspartic Acid↗
PubMed1992

Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V.

T4 endonuclease V [endodeoxyribonuclease (pyrimidine dimer); deoxyribonuclease (pyrimidine dimer), EC 3.1.25.1] initiates repair of damaged DNA by hydrolysis of the N-glycosyl bond at the 5' side of a pyrimidine photodimer in double-stranded DNA. To study one of the active sites of T4 endonuclease V, systematic site-directed mutagenesis was performed on the synthetic T4 endonuclease V gene, in parallel with three-dimensional structure analysis by x-ray crystallography. The mutant proteins were evaluated for DNA glycosylase activity using an oligonucleotide duplex (14-mer) containing a single thymidine dimer as a substrate. Replacement of either Glu-23 with glutamine or asparatic acid or Arg-3 with glutamine completely abolished DNA glycosylase activity. Mutation of Arg-3 to lysine or of Arg-26 to glutamine or lysine in a basic amino acid cluster caused serious defects in DNA glycosylase activity, which are reflected in the increases in Km and decreases in kcat of DNA glycosylase activity. On the other hand, substitutions of lysine for Arg-22 or of glutamine for Arg-117 or Lys-121 resulted in increases in the Km value. The completely inactive mutant proteins, E23Q and R3Q, in which glutamine was substituted for Glu-23 and Arg-3, respectively, were further investigated by CD spectroscopy for their ability to bind the oligonucleotide substrate. It was found that the E23Q protein retained specific substrate-binding ability, whereas the R3Q protein did not. These results indicate that Glu-23 plays an important role in catalysis of the DNA glycosylase reaction, and that Arg-3 is a crucial residue for substrate binding. In addition, Arg-22, Arg-26, Arg-117, and Lys-121 in the basic amino acid cluster also participate in substrate binding. We conclude that the basic amino acid cluster in T4 endonuclease V is an essential structure for DNA glycosylase activity.

Amino Acid Sequence↗
PubMed1988

Interactions between bladder tumor cells as tumor spheroids from the cell line J82 and human endothelial cells in vitro.

Multicellular tumor spheroids (MCTS) are a reliable model of nonvascularized tumor cell aggregates showing a well defined three-dimensional growth pattern and are comparable with small metastatic cancer cell complexes in blood circulation. In the present study we have established a co-culture system of multicellular bladder tumor spheroids with human endothelial cells on extracellular matrix (ECM) in order to investigate morphological and proliferative changes of endothelial and tumor cells within a defined time of cell-cell interaction. The MCTS--endothelial cell--extracellular matrix complex was observed within coculture periods from 1/2 to seven days. Morphological changes (light microscopy, scanning and electron microscopy) indicated that MCTS are not influenced by cocultured endothelial cells. The tumor cells invaded into the ECM after degradation of endothelial cells in the center of the contact zone. Endothelial cells, however, showed degenerative changes as well as a complex reaction in their proliferation activities. We could recognize an initial increase of proliferation of endothelial cells next to the MCTS. Later on, endothelial cells next to invading tumor cells showed changes in morphological polarity. The model system used has the advantage of using human tumor tissue. It distinguishes between basic cellular mechanisms like adherence, migration, DNA synthesis and proliferation in the study of the contact of tumor cells and vascular endothelial cells as an important event in hematogenous tumor spread.

Cell Communication↗
PubMed1987

Multicellular bladder tumor spheroids in coculture with human endothelial cell monolayers.

Multicellular tumor spheroids (MCTS) are avascular spherical tumors grown systematically out of single cells in vitro. The coculture of MCTS of the human bladder carcinoma cell line J 82 with confluent human endothelial cell layers serves as a model to study cell-cell interactions as an important feature in hematogenous tumor spread. Basic information is obtained by phase contrast microscopy and histochemistry. More detailed information is given by the application of scanning and transmission electron microscopy. Immunohistochemistry helps to differentiate endothelial cells from tumor cells (factor VIII-related antigen). The application of this coculture model and of the various methods of evaluation enables us to study cell-to-cell contact phenomena systematically.

Carcinoma, Transitional Cell↗