Partial purification and properties of a rat brain phospholipase.

At least 90% of a membrane-bound phospholipase D was solubilized by extraction of freeze-dried rat brain with 0.8% Miranol H2M and 0.5% cholate. The bulk of base exchange reaction enzymes remained firmly bound to the particulate fraction under these conditions. The phospholipase D specific activity was enriched 240-fold by a purification protocol employing ammonium sulfate precipitation, and both Sepharose 4B and DEAE-cellulose column chromatography. The approximate molecular weight of the partially purified enzyme was calculated to be 200,000 based upon the elution profile from Sepharose 4B and Sephadex G-200 columns. The optimum pH was 6.0, and Km values for phosphatidylcholine and phosphatidylethanolamine were 0.75 mM and 0.91 mM, respectively. The enzyme activity was not dependent on the presence of divalent cation although Ca2+ and Fe2+ showed stimulatory effects.     

Folate-dependent enzymes in cultured Chinese hamster ovary cells: evidence for mutant forms of folylpolyglutamate synthetase

A Chinese hamster ovary auxotroph requiring glycine + adenosine + thymidine (CHO AUXB1) was shown by us previously to lack several folylpolyglutamate synthetase (FPGS) type activities. Two revertants of AUXB1 (one spontaneous and one Pt(S0₄)₂ induced) have been isolated and found to contain altered forms of this enzyme. The revertant enzymes are more sensitive to heat inactivation (37 °C, pH 7.4 or 9.0) than the parent CHO enzyme. Increased sensitivity of revertant FPGS is observed irrespective of whether one assays the specific catalysis of radioactive tetrahydropteroyldi- or tetraglutamate synthesis. ATP and MgCl₂ protect both revertant and parent CHO FPGS against rapid heat denaturation at pH 9.0, but not at pH 7.4. A genetically related auxotroph (CHO AUXB3) contains one-fifth the parent amount of FPGS. AUXB3 FPGS shows a normal sensitivity to 37 °C heat inactivation, but it has an altered substrate saturation and specificity pattern when assayed for tetrahydropteroyldi[U-¹⁴C]glutamate synthesis. Also, unlike the FPGS from parent CHO and a genetically unrelated mutant requiring only glycine (CHO AUXB2), the AUXB3 enzyme specifically lacks tetrahydropteroyltetra[U-¹⁴C]glutamate synthetase activity. These findings and polyethylene glycol fusion data with AUXB2 indicate that AUXB1 and AUXB3 each carry a mutation in the structural gene for a CHO FPGS that catalyzes tetrahydropteroyldi- as well as tetraglutamate formation. The altered form of FPGS in AUXB3 is responsible for its glycine + adenosine auxotrophy under standard culture conditions.     

Neuronal precursor cells in the rat hippocampal formation contribute to more than one cytoarchitectonic area.

We have tested the hypothesis that cell lineage restriction boundaries define the borders between cytoarchitectonic areas in the cerebral cortex. Clonally related cells were identified using a retroviral marking technique, and the dispersion of neuronal clones was examined with respect to the transitions between cortical areas. We chose to study the hippocampal formation because we found that clones of hippocampal neurons, unlike those in neocortex, are compact and readily identifiable in the adult and that transitions between areas in the hippocampus are sharp relative to the spread of a typical clone. We conclude, contrary to the hypothesis, that clones of neurons transgress the boundaries between areas in the hippocampal formation, that border-crossing clones are observed as frequently as would be expected if clones spread freely over the hippocampus with no constraint imposed by area borders, and that different types of pyramidal neurons, characteristic of different areas, may appear to a single clone. different areas, may appear in a single clone.     

ATPase-defective derivatives of Escherichia coli DnaK that behave differently with respect to ATP-induced conformational change and peptide release.

We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of Escherichia coli DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The dnaK alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a DeltadnaK mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell.     

An approach to chemotaxonomy of the Asarum subgenus

A survey of chemical composition of 23 species of Asarum subgenus Heterotropa showed that the five groups could be distinguished on the basis of the presence or absence of asatone, phenol ethers and terpenes.     

Bcl-2 phosphorylation is required for inhibition of oxidative stress-induced lysosomal leak and ensuing apoptosis.

B-cell leukemia/lymphoma 2 (Bcl-2) blocks oxidant-induced apoptosis at least partly by stabilizing lysosomes. Here we report that phosphorylation of Bcl-2 may be required for these protective effects. J774 cells overexpressing wild-type Bcl-2 resist oxidant-induced lysosomal leak as well as apoptosis, and this protection is amplified by pretreatment with phorbol 12-myristate 13-acetate (which promotes protein kinase C (PKC)-dependent phosphorylation of Bcl-2). In contrast, cells overexpressing the Bcl-2 mutant S70A (which cannot be phosphorylated) are not protected in either circumstance. Transfection with Bcl-2(S70E), a constitutively active Bcl-2 mutant which does not require phosphorylation, is protective independent of PKC activation. In contrast, C(2)-ceramide, a putative protein phosphatase 2A activator, abolishes the protective effects of wild-type Bcl-2 overexpression but does not diminish protection afforded by Bcl-2(S70E). Additional results suggest that, perhaps as a consequence of lysosomal stabilization, Bcl-2 may prevent activation of phospholipase A2, an event potentially important in the ultimate initiation of apoptosis.     

Preliminary X-ray study of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida N.C.I.B. 9869

Single crystals of p-cresol methylhydroxylase, a flavocytochrome c from Pseudomonas putida, have been prepared. The crystals are orthorhombic, space group P212121 with unit cell parameters; a = 140.3 A, b = 130.6 A and c = 74.1 A. They contain a single non-symmetric dimer per asymmetric unit and diffract to at least 2.5 A resolution.