Osteoinduction in human fat-derived stem cells by recombinant human bone morphogenetic protein-2 produced in Escherichia coli.

Bioactive recombinant human bone morphogenetic protein-2 (rhBMP-2) was obtained using Escherichia coli pET-25b expression system: 55 mg purified rhBMP-2 were achieved per g cell dry wt, with up to 95% purity. In murine C2C12 cell line, rhBMP-2 induced an increase in the transcription of Smads and of osteogenic markers Runx2/Cbfa1 and Osterix, measured by semi-quantitative RT-PCR. Bioassays performed in human fat-derived stem cells showed an increased activity of the early osteogenic marker, alkaline phosphatase, and the absence of cytotoxicity.

Characterization of recombinant human endothelial nitric-oxide synthase purified from the yeast Pichia pastoris.

Human endothelial nitric-oxide synthase (eNOS) was expressed in the methylotrophic yeast Pichia pastoris, making use of the highly inducible alcohol oxidase promoter. The recombinant protein constituted approximately 3% of total protein and was largely soluble (>75%). About 1 mg of purified eNOS was obtained from 100-ml yeast cell cultures by affinity chromatography of crude cell supernatants. The purified enzyme had a V(max) of 192 +/- 18 nmol of L-citrulline x mg(-1) x min(-1), had a K(m) for L-arginine of 3.9 +/- 0.2 microM, and showed an absolute requirement for tetrahydrobiopterin (H(4)biopterin). NADPH oxidase activity was 136 +/- 9 and 342 +/- 24 nmol x mg(-1) x min(-1) in the absence and presence of 0.1 mM L-arginine, respectively, and not affected by H(4)biopterin. The protein contained 0.56 +/- 0.06 equivalents of FAD and 0.79 +/- 0.08 equivalents of FMN. On-line gel filtration/inductively coupled plasma mass spectrometry analysis confirmed that both iron (0.80 +/- 0.09 mol/subunit) and zinc (0.43 +/- 0.03 mol/subunit) were bound to the enzyme. Graphite furnace-atomic absorption spectroscopy yielded a value for bound iron of 0.84 +/- 0.04 mol/subunit. The absorbance of the enzyme at 398 nm implied a heme content of 0.85 +/- 0.09 mol/subunit, and the high pressure liquid chromatography heme assay gave an estimate of 0.71 +/- 0.02 mol heme/subunit. Gel permeation chromatography yielded one single peak with a Stokes radius of 6.62 +/- 0.7 nm, indicating that the native protein is dimeric. Upon low temperature gel electrophoresis the untreated protein appeared mainly as a monomer (88 +/- 3%), but pretreatment with H(4)biopterin and L-arginine led to a pronounced shift toward dimers (77 +/- 4%). Thus, in contrast to bovine eNOS (List, B. M., Klösch, B., Völker, C., Gorren, A. C. F., Sessa, W. C., Werner, E. R., Kukovetz, W. R., Schmidt, K., and Mayer, B. (1997) Biochem. J. 323, 159-165; Rodriguez-Crespo, I., Gerber, N. C., and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 11462-11467), the human eNOS appears to be markedly stabilized by H(4)biopterin.

The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases.

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774-777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC50 for nNOS was 18 +/- 6 microM GST-PIN with 63 nM nNOS after 30 min at 37 degrees C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K(M) for L-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.

Characterization of bovine endothelial nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of haem in dimerization.

The fatty-acylation-deficient bovine endothelial NO synthase (eNOS) mutant (Gly-2 to Ala-2, G2AeNOS) was purified from a baculovirus overexpression system. The purified protein was soluble and highly active (0.2-0.7 micromol of l-citrulline. mg-1.min-1), contained 0. 77+/-0.01 equivalent of haem per subunit, showed a Soret maximum at 396 nm, and exhibited only minor uncoupling of NADPH oxidation in the absence of l-arginine or tetrahydrobiopterin. Radioligand binding studies revealed KD values of 147+/-24.1 nM and 52+/-9.2 nM for specific binding of tetrahydrobiopterin in the absence and presence of 0.1 mM l-arginine respectively. The positive co-operative effect of l-arginine was due to a pronounced decrease in the rate of tetrahydrobiopterin dissociation (from 1.6+/-0.5 to 0. 3+/-0.1 min-1). Low-temperature SDS gel electrophoresis showed that approx. 80% of the protein migrated as haem-containing dimer after preincubation with l-arginine and tetrahydrobiopterin. Gel-filtration chromatography yielded one peak with a Stokes radius of 6.8+/-0.04 nm, corresponding to a hydrodynamic volume of 1. 32x10(-24) m3, whereas haem-deficient preparations (approx. 0.3 equivalent per subunit) contained an additional protein species with a hydrodynamic radius of 5.1+/-0.2 nm and a corresponding volume of 0.55x10(-24) m3, suggesting that haem availability regulates eNOS dimerization.

ERG1, encoding squalene epoxidase, is located on the right arm of chromosome VII of Saccharomyces cerevisiae.

The ERG1 gene of Saccharomyces cerevisiae encodes squalene epoxidase, a key enzyme in the ergosterol pathway. ERG1 is an essential gene. Disruption of the gene with URA3 results in a lethal phenotype when cells are grown under aerobic conditions, even in the presence of ergosterol. However, cells are viable in the presence of ergosterol under anaerobic growth conditions during which ergosterol is taken up by cells. Physical and genetic mapping data reveal that ERG1 is located on the right arm of chromosome VII proximal to QCR9 at a distance of 14.6 cM from ADE3.