[41] Human liver aminopeptidase

Little, Gwynne H.; Starnes, Willis L.; Běhal, Francis J.

doi:10.1016/s0076-6879(76)45044-9

Cited by 43 publications

(12 citation statements)

References 11 publications

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“…Human liver aminopeptidase (Little et al, 1976) and purified enzymes from Bacillus lichenijiormis (Rodriguez-Absi & Prescott, 1978) and Bacillus subtilis (Wagner et al, 1979) are representatives of one-zinc enzymes whereas the hexameric leucine aminopeptidases from bovine lens (Carpenter & Vahl, 1973) and from swine kidney (Van Wart & Lin, 1981) contain two metal binding sites per subunit. Aeromonas aminopeptidase also is a two-metal enzyme but differs in several respects from the latter two.…”

Section: Discussionmentioning

confidence: 99%

Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates

Bayliss¹,

Prescott²

1986

Biochemistry

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Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 99%

Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates

Bayliss¹,

Prescott²

1986

Biochemistry

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“…A 10-l aliquot of sample was added to 190 l of 0.1 M phosphate buffer (pH 6.8) and then, to start the reaction, 3 mM of the amino acid-␤NA substrate was added in a volume of 100 l. Upon incubation at 37°C, released ␤NA was recorded fluorometrically (SpectraMAX GeminiXS; Molecular Devices) over time at 410 nm with an excitation wavelength of 340 nm (45,66,80). One unit of aminopeptidase was defined as that which yielded 1 M of pNI or ␤NA in 1 min, based upon standard curves generated with pNA and pNI (66,80).…”

Section: Methodsmentioning

confidence: 99%

The Type II Secretion System ofLegionella pneumophilaElaborates Two Aminopeptidases, as Well as a Metalloprotease That Contributes to Differential Infection among Protozoan Hosts

Rossier

Dao

Cianciotto

2008

Appl Environ Microbiol

109

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Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic amoebae and human macrophages. A key factor for L. pneumophila in intracellular infection is its type II protein secretion system (Lsp). In order to more completely define Lsp output, we recently performed a proteomic analysis of culture supernatants. Based upon the predictions of that analysis, we found that L. pneumophila secretes two distinct aminopeptidase activities encoded by the genes lapA and lapB. Whereas lapA conferred activity against leucine, phenylalanine, and tyrosine aminopeptides, lapB was linked to the cleavage of lysineand arginine-containing substrates. To assess the role of secreted aminopeptidases in intracellular infection, we examined the relative abilities of lapA and lapB mutants to infect human U937 cell macrophages as well as Hartmannella vermiformis and Acanthamoeba castellanii amoebae. Although these experiments identified a dispensable role for LapA and LapB, they uncovered a previously unrecognized role for the type II-dependent ProA (MspA) metalloprotease. Whereas proA mutants were not defective for macrophage or A. castellanii infection, they (but not their complemented derivatives) were impaired for growth upon coculture with H. vermiformis. Thus, ProA represents the first type II effector implicated in an intracellular infection event. Furthermore, proA represents an L. pneumophila gene that shows differential importance among protozoan infection models, suggesting that the legionellae might have evolved some of its factors to especially target certain of their protozoan hosts.Legionella pneumophila is the agent of Legionnaires' disease pneumonia (29). In fresh waters, this gram-negative bacterium exists in protozoan hosts and as a part of biofilms. Disease occurs when inhaled legionellae, possibly including those still associated with protozoa or protozoan vesicles (11, 12), invade alveolar macrophages. Although many factors promote the ecology and pathogenesis of L. pneumophila, protein secretion stands out for its multifaceted significance. L. pneumophila possesses type II secretion, type IVB secretion, and type IVA secretion (7, 17), and genome sequencing suggests the existence of type I and type V secretion systems (14,40). Among these pathways, the Lsp type II system is arguably implicated in the broadest array of phenotypes (17). Indeed, this pathway is required for secretion during growth in bacteriologic media at 37°C, extracellular survival in broth/water at 12 to 25°C, colony morphology, intracellular infection of protozoa (acanthamoebae and hartmannellae), optimal intracellular infection of human macrophages and monocytes, and virulence in a murine model of pneumonia (37,44,55,61,62,68,69). Although type II secretion exists in many gram-negative organisms, including various animal and plant pathogens, L. pneumophila is the only intracellular pathogen known to possess a functional type II system (17). Type II secretion is a two-step process in which nascent proteins ar...

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“…Aminopeptidase Activity-This was measured by the fluorometric method described by Little et al (1976). Briefly, 1% Triton X-100 eluates from 1F8 or IgG beads were mixed with 0.5 ml of 3 mM L-alanyl-␤-naphthylamide in a total volume of 1.5 ml of PBS with 0.5% Triton X-100 to a final substrate concentration of 1 mM.…”

Section: Methodsmentioning

confidence: 99%

Glut4 Is Targeted to Specific Vesicles in Adipocytes of Transgenic Mice Overexpressing Glut4 Selectively in Adipose Tissue

Tozzo

Kahn

Pilch

et al. 1996

Journal of Biological Chemistry

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(1993) J. Biol. Chem. 268, 22243-22246) have 15-20-fold more Glut4 than normal adipocytes. To study compartmentalization of intracellular Glut4 in these cells, we fractionated light microsomes prepared from transgenic and normal adipocytes in velocity and density sucrose gradients. Glut4-containing intracellular membranes from both cell types have a specific and narrow distribution in these gradients, i.e. behave as homogeneous vesicles with identical sedimentation coefficients and different buoyant densities. Immunoadsorption of Glut4-containing vesicles with covalently immobilized monoclonal anti-transporter antibody demonstrated that the total polypeptide composition of these vesicles from transgenic and normal cells was identical, with the exception of Glut4 itself, which was much more abundant in the transgenic cells. Both preparations also had comparable levels of secretory carrier membrane proteins and of aminopeptidase activity (gp160). Glut4-containing vesicles from both normal and transgenic adipocytes excluded Glut1, which in both cell types formed a different vesicle population. Thus, even under conditions of high level overexpression, Glut4 is still specifically targeted to the same unique type of structurally defined insulinsensitive vesicles as in normal cells.Insulin-sensitive adipose and skeletal muscle tissues express a specific isoform of glucose transporter protein, Glut4, which under basal conditions, i.e. in the absence of insulin, resides in intracellular microsomal vesicles. After insulin administration, Glut4-containing vesicles fuse with the plasma membrane and deliver the transporter to the cell surface (for recent reviews, see

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[41] Human liver aminopeptidase

Cited by 43 publications

References 11 publications

Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates

Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates

The Type II Secretion System ofLegionella pneumophilaElaborates Two Aminopeptidases, as Well as a Metalloprotease That Contributes to Differential Infection among Protozoan Hosts

Glut4 Is Targeted to Specific Vesicles in Adipocytes of Transgenic Mice Overexpressing Glut4 Selectively in Adipose Tissue

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