Role of the N-terminal hydrophilic domain of acyl-coenzyme A:cholesterol acyltransferase 1 on the enzyme's quaternary structure and catalytic efficiency

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an enzyme involved in cellular cholesterol homeostasis and atherosclerosis. ACAT1 is an allosteric enzyme responding to its substrate cholesterol in a sigmoidal manner. It is a homotetrameric protein that spans the membrane multiple times, with its N-terminal 131 hydrophilic amino acids residing at the cytoplasmic side of the endoplasmic reticulum. This region contains two closely linked putative alpha-helices. Our current studies show that this region contains a dimer-forming motif. Adding this motif to the bacterial glutathione S-transferase (GST) converted the homodimeric GST to a tetrameric fusion protein. Conversely, deleting this motif from the full-length ACAT1 converted the enzyme from a homotetramer to a homodimer. The dimeric ACAT1 remains enzymatically active. Its biochemical characteristics, including the sigmoidal response to cholesterol, the IC(50) value toward a specific ACAT inhibitor, and sensitivity toward heat inactivation, are essentially unaltered. On the other hand, the dimeric ACAT1 exhibits a 5-10-fold increase in the V(max) of the overall reaction and a 2.2-fold increase in the K(m) for oleoyl-coenzyme. Thus, deleting the dimer-forming motif near the N-terminus changes ACAT1 from its tetrameric form to a dimeric form and increases its catalytic efficiency.     

Purification,crystallization and preliminary X-ray studies of human augmenter of liver regeneration

Human augmenter of liver regeneration has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C222, with unit-cell parameters a=51.7 A, b=78.8 A, c=63.7 A. Diffraction data were collected to 2.80 A with a completeness of 99.9% (99.9% for the last shell), a R(sym) value of 0.092(0.236) and an I/sigma(I) value of 6.2(2.7).     

Crystallization and preliminary x-ray crystallographic studies of trichosanthin delta C7

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. TCS has various pharmacological properties. It is possible to reduce the antigenicity of TCS by deleting up to seven C-terminal residues of TCS (TCS-C7) with minimal effect on its activity. TCS-C7 has been crystallized and the crystal diffracted to 1.8 A. It belongs to space group P2(1), with unit-cell parameters a=71.6A, b=74.4A, c=87.6A, beta=97.0 degrees. It is given that there are four molecules per asymmetric unit.     

Crystallization and preliminary X-ray diffraction analysis of FKBP12 complexed with a novel neurotrophic ligand

A novel neurotrophic ligand, (3R)-4-(p-Toluenesulfonyl)-1,4-thiazane-3-carboxylic acid-L-Leucine ethyl ester, has been complexed with FKBP12 and crystallized using the hanging-drop vapor-diffusion method. Crystals belong to P2(1) space group, with unit cell parameters a=41.2, b=29.6, c=41.5 A, beta=114.0 degrees. The crystals diffract to 1.8 A resolution limit.     

An antiport mechanism for a member of the cation diffusion facilitator family: divalent cations efflux in exchange for K+ and H+

Members of the cation diffusion facilitator (CDF) family of membrane transport proteins are found in eukaryotes and prokaryotes. The family encompasses transporters of zinc ions, with cobalt, cadmium and lead ions being additional substrates for some prokaryotic examples. No transport mechanism has previously been established for any CDF protein. It is shown here that the CzcD protein of Bacillus subtilis, a CDF protein, uses an antiporter mechanism, catalysing active efflux of Zn2+ in exchange for K+ and H+. The exchange is probably electroneutral, energized by the transmembrane pH gradient and oppositely oriented gradients of the other cation substrates. The data suggest that Co2+ and Cd2+ are additional cytoplasmic substrates for CzcD. A second product of the same operon that encodes czcD has sequence similarity to oxidoreductases and is here designated CzcO. CzcO modestly enhances the activity of CzcD but is not predicted to be an integral membrane protein and has no antiport activity of its own.     

Sol-gel encapsulation of lactate dehydrogenase for optical sensing of L-lactate

Sol-gel encapsulation of lactate dehydrogenase and its cofactor can be employed as a disposable sensor for L-lactate. The sensor utilized the changes in absorbance or fluorescence from the reduced cofactor nicotinamide adenine dinucleotide (NADH) upon exposure to L-lactate. Although, problems such as diminished enzymatic activity and/or leaching of enzyme from the sol-gel matrix occurred, the sol-gel process is sufficiently mild to permit retention of enzymatic activity. The apparent activity of LDH in the sensor is at least 10% of that of the dissolved enzyme. The sensor has a linear dynamic range over the normal physiological L-lactate level and has a long-term storage stability of at least 3 weeks.     

Determination of andrographolide,deoxyandrographolide and neoandrographolide in the Chinese herb Andrographis paniculata by micellar electrokinetic capillary chromatography

In this paper a micellar electrokinetic capillary chromatographic (MEKC) method has been developed for determining the active components (andrographolide, deoxyandrographolide and neoandrographolide) in water:ethanol extracts of the Chinese crude herb Andrographis paniculata and its preparations (Chuanxinlian and Xiaoyan Lidan tablets). The optimum separation conditions were 15 mM sodium dodecyl sulphate in 30 mM borate buffer (pH 9.5) with UV detection wavelength at 214 nm and a constant voltage of 16 kV. An HPLC method was employed in order to validate the MEKC method with respect to separation efficiency, sensitivity, linearity and repeatability. The two methods are shown to be complementary because of their different selectivity and thus are very suitable for cross-validation studies. The MEKC method is demonstrated to be more appropriate for the analysis of the active compounds in A. paniculata in that it is easier and less expensive to use and does not suffer from contamination of the chromatographic column.     

Slit proteins: molecular guidance cues for cells ranging from neurons to leukocytes

Recent studies of molecular guidance cues including the Slit family of secreted proteins have provided new insights into the mechanisms of cell migration. Initially discovered in the nervous system, Slit functions through its receptor, Roundabout, and an intracellular signal transduction pathway that includes the Abelson kinase, the Enabled protein, GTPase activating proteins and the Rho family of small GTPases. Interestingly, Slit also appears to use Roundabout to control leukocyte chemotaxis, which occurs in contexts different from neuronal migration, suggesting a fundamental conservation of mechanisms guiding the migration of distinct types of somatic cells.     

O-glycosylation of EGF repeats: identification and initial characterization of a UDP-glucose: protein O-glucosyltransferase

O-Glucose is an unusual form of posttranslational modification consisting of glucose directly attached to protein through O-linkage. Several serum proteins (factor VII, factor IX, protein Z, and thrombospondin) contain this unique modification on their epidermal growth factor (EGF)-like repeats. Comparison of the glycosylation sites on these proteins revealed a putative consensus sequence for O-glucose modification: C(1)XSXPC(2), where C(1) and C(2) are the first and second conserved cysteines of the EGF repeat. We identify and characterize an enzymatic activity capable of adding glucose to EGF repeats: UDP-glucose: protein O-glucosyltransferase. Using extracts of Chinese hamster ovary cells as the enzyme source, recombinant factor VII EGF repeat as the acceptor, and UDP-[(3)H]glucose as the donor, we show that the activity is linearly dependent on time, enzyme amount, and substrate concentration. As with most glycosyltransferases, metal ions (such as manganese) are required for activity. Analysis demonstrated that the glucose is added in O-linkage to the EGF repeat. Mutation of the serine to alanine in the predicted glycosylation site abrogates glycosylation, as does reduction and alkylation of the EGF repeat, suggesting that the enzyme recognizes not only the consensus sequence but also the 3D structure of the EGF repeat. Detection of O-glucosyltransferase activity in extracts of cell lines from insects to humans and a variety of rat tissues suggests that O-glucose modification is widespread in biology. These studies lay the foundation for future work on the biological role of the O-glucose modification.     

2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.