Elizabeth K. Wilk

Purification, Characterization, and Cloning of a Cytosolic Aspartyl Aminopeptidase

An aminopeptidase with a preference for N-terminal aspartyl and glutamyl residues but distinct from glutamyl aminopeptidase (EC 3.4.11.7) was purified to near homogeneity from rabbit brain cytosol. Its properties were similar to an enzyme described previously (Kelly, J. A., Neidle, E. L., and Neidle, A. (1983) J. Neurochem. 40, 1727–1734). Aspartyl aminopeptidase had barely detectable activity toward simple aminoacyl-naphthylamide substrates. Its activity was determined with the substrate Asp-Ala-Pro-naphthylamide in the presence of excess dipeptidyl-peptidase IV (EC 3.4.14.5). The native enzyme has a molecular mass of 440 kDa and migrates as a single band of 55 kDa after SDS-polyacrylamide gel electrophoresis. The sequences of three tryptic peptides were used to screen the GenBankTM data base of expressed sequence tags. Human and mouse clones described as “similar to a yeast vacuolar aminopeptidase” and containing full-length cDNAs were identified and sequenced. The human cDNA was expressed in Escherichia coli. The amino acid sequence has significant homology to yeast aminopeptidase I, placing it as the first identified mammalian member of the M18 family of metalloproteinases. Homologous sequences in Caenorhabditis elegans and in prokaryotes revealed three conserved histidines, three conserved glutamates and five conserved aspartates. Aspartyl aminopeptidase is found at relatively high levels in all mammalian tissues examined and is likely to play an important role in intracellular protein and peptide metabolism.

Identification of histidine residues important in the catalysis and structure of aspartyl aminopeptidase.

Aspartyl aminopeptidase (DAP), a widely distributed and abundant cytosolic enzyme, removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. DAP is a member of the M18 family of the MH clan of cocatalytic metallopeptidases. The human and mouse enzymes have been cloned. We have identified 8 highly homologous eukaryotic sequences that are probable aspartyl aminopeptidases. Eight histidine residues of human DAP were sequentially mutated to phenylalanine. Mutation of His94, His170, and His440 abolished enzymatic activity. His94 and His440 are postulated to be involved in binding cocatalytic zinc atoms by homology with other members of the MH clan. Mutation of His352 dramatically reduced enzyme activity. Gel-filtration analysis of the His352 mutant revealed destabilization of the quaternary structure and dissociation of the native 440-kDa enzyme. Mutation of His33 and of histidines residing in a cluster at residues 349, 359, and 363 all decreased k(cat). These studies reveal an important role for histidine residues both in catalysis and in the structural integrity of DAP.

Human norepinephrine metabolism: Its evaluation by administration of tritiated norepinephrine

It has become increasingly apparent that evaluation of human norepinephrine metabolism simply by assay of catecholamines in urine is inadequate for differentiation of many physiological or pathological states. In an attempt to examine norepinepherine metabolism in the human subject, tritium-labeled d,l-norepinephrine was administered to 11 normal adults and the definitive turnover rates and relative specific activities of norepinephrine and its major catabolites, vanillylmandelic acid, 3-methoxy-4-hydroxyphenylethyleneglycol, and normetanephrine, as well as the cumulative 24 hr isotope excretion were determined. The major endogenous norepinephrine catabolites were also quantitatively assayed. In order to verify the reliability of the isotope label, parallel studies were carried out in two patients to whom norepinephrine-(14)C was administered. Metabolic studies were repeated after the administration of reserpine to gain further insight into the distribution of the label.All studies demonstrated a consistent difference between the relative specific activities of the amines and their deaminated congeners, thereby indicating an uneven distribution of the labeled material. The marked decrease in the relative specific activities of the deaminated catabolites after the administration of reserpine showed that the present experimental technique succeeded in labeling, though to a limited extent, the storage or reserpine-releasable pool. A dose of reserpine known to interfere with sympathetic activity but failing to elicit a change in excretion of endogenous catecholamine catabolites, nonetheless resulted in a marked abnormality in the metabolic handling of labeled norepinephrine. It is anticipated that such studies may not only be of value in measuring sympathetic activity in the intact human subject during physiologic variations and pathologic states associated with abnormalities in catecholamine metabolism, but may serve as a technique whereby drugs that affect human norepinephrine metabolism may undergo precise pharmacologic evaluation.

Occurrence of pyroglutamyl peptidase II, a specific TRH degrading enzyme in rabbit retinal membranes and in human retinoblastoma cells

Pyroglutamyl peptidase II, a highly specific thyrotropin releasing hormone (TRH)-degrading enzyme is found in highest concentration in brain where it is localized to synaptic membranes. Retina contains relatively high concentrations of both immunoreactive TRH and TRH receptors. We report that the specific activity of pyroglutamyl peptidase II in rabbit retinal membranes exceeds that of all non-CNS tissues thus far studied. Nine clonal cell lines were screened for this enzymatic activity. The specific activity of pyroglutamyl peptidase II in Y79 retinoblastoma cells was greater than the highest activity found in other cell lines by approximately one order of magnitude. These studies further support a functional relationship between pyroglutamyl peptidase II and TRH and identify a cell line suitable for studies on the regulation of this enzyme.

Modification of the taniguchi method for the determination of normetanephrine and metanephrine

Abstract Using tritium-labeled normetanephrine enables one to determine the exact point at which this substance is completely eluted from the adsorbing resin into borate buffer. With this refinement the Taniguchi method gave good results for the determination of normetanephrine and metanephrine.