Paracatalytic inactivation of L-2-haloacid dehalogenase from Pseudomonas sp. YL by hydroxylamine. Evidence for the formation of an ester intermediate

Asp10 of L-2-haloacid dehalogenase from Pseudomonas sp. YL was proposed to act as a nucleophile to attack the alpha-carbon of L-2-haloalkanoic acids to form an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon (Liu, J.-Q, Kurihara, T., Miyagi, M., Esaki, N., and Soda, K. (1995) J. Biol. Chem. 270, 18309-18312). We have found that the enzyme is paracatalytically inactivated by hydroxylamine in the presence of the substrates monochloroacetate and L-2-chloropropionate. Ion spray mass spectrometry demonstrated that the molecular mass of the enzyme inactivated by hydroxylamine during the dechlorination of monochloroacetate is about 74 Da greater than that of the native enzyme. To determine the increase of the molecular mass more precisely, we digested the inactivated enzyme with lysyl endopeptidase and measured the molecular masses of the peptide fragments. The molecular mass of the hexapeptide Gly6-Lys11 was shown to increase by 73 Da. Tandem mass spectrometric analysis of this peptide revealed that the increase is due to a modification of Asp10. When the enzyme was paracatalytically inactivated by hydroxylamine during the dechlorination of L-2-chloropropionate, the molecular mass of the hexapeptide was 87 Da higher. Hydroxylamine is proposed to attack the carbonyl carbon of the ester intermediate and form a stable aspartate beta-hydroxamate carboxyalkyl ester residue in the inactivated enzyme.     

Suppressors of the unc-73 gene of Caenorhabditis elegans

The unc-73 gene of Caenorhabditis elegans is necessary for proper axon guidance. Animals mutant in this gene are severely uncoordinated and also exhibit defects in cell migration and cell lineages. We have isolated coordinated revertants of unc-73 (e936). These fall into three classes: intragenic revertants, extragenic dominant suppressors (sup-39), and a single apparently intragenic mutation that is a dominant suppressor with a linked recessive lethal phenotype. sup-39 mutations cause early embryonic lethality, but escapers have a wild-type movement phenotype as larvae and adults. Gonads of sup-39 mutant animals show a novel defect: normal gonads have a single row of oocytes, but sup-39 gonads often have two rows of oocytes. This result suggests that the mutant gonad is defective in choosing on its surface only a single site form which nuclei will emerge to form oocytes. These results are interpreted in terms of an effect of unc-73 on determination of cell polarity.     

Fine structure of the semicompact formation of the nucleus ambiguus of the rat

We investigated the fine structure of the semicompact formation of the nucleus ambiguus (AmS), which was identified by retrogradely labeled pharyngeal (PH) motoneurons. When cholera toxin subunit B-conjugated horseradish peroxidase was injected into the lower pharyngeal muscle, many retrogradely labeled PH neurons were found throughout the AmS. Besides the PH neurons, two types of neurons were recognized in the AmS: unlabeled medium-sized neurons and unlabeled small neurons. The PH neuron was large (27.6 x 44.1 microm) and polygonal, and contained many Nissl bodies and well-developed cell organelles with a prominent spherical nucleus. The medium-sized neuron was dark and oval (19.3 x 33.2 microm), and contained many free ribosomes and much swollen rough endoplasmic reticulum with a distorted oval nucleus. The small neuron was spindle-shaped (12.3 x 20.2 microm), and had poorly developed cell organelles with an irregularly shaped nucleus. The average number of axosomatic terminals in a sectional plane was largest in the PH neurons (32.8), smaller in the medium-sized neurons (23.1), and smallest in the small neurons (6.3). The number of axo-somatic terminals containing round vesicles (Gray's type I) was almost equal to that of terminals containing pleomorphic vesicles (Gray's type II) in the PH neuron, and slightly smaller in the small and the medium-sized neurons. About 60% of the axodendritic terminals were Gray's type I, and 40% were type II. These results indicate that there are two different types of interneurons besides the PH motoneurons in the AmS.