Identification, characterization, and cloning of a novel aminoacylase, L-pipecolic acid acylase from Pseudomonas species.

L-Pipecolic acid is utilized as a vital component of specific chemical compounds, such as immunosuppressive drugs, anticancer reagents, and anesthetic reagents. We isolated and characterized a novel L-aminoacylase, N-acetyl-L-pipecolic acid-specific aminoacylase (LpipACY), from Pseudomonas sp. AK2. The subunit molecular mass of LpipACY was 45 kDa and was assumed to be a homooctamer in solution. The enzyme exhibited high substrate specificity toward N-acetyl-L-pipecolic acid and a high activity for N-acetyl-L-pipecolic acid and N-acetyl-L-proline. This enzyme was stable at a high temperature (60°C for 10 min) and under an alkaline pH (6.0-11.5). The N-terminal and internal amino acid sequences of the purified enzyme were STTANTLILRNG and IMASGGV, respectively. These sequences are highly consistent with those of uncharacterized proteins from Pseudomonas species, such as amidohydrolase and peptidase. We also cloned and overexpressed the gene coding LpipACY in Escherichia coli. Moreover, the recombinant LpipACY exhibited properties similar to native enzyme. Our results suggest that LpipACY is a potential enzyme for the enzymatic synthesis of L-pipecolic acid. This study provides the first description of the enzymatic characterization of L-pipecolic acid specific amino acid acylase.

Changes in Muscle Quality and Body Composition 1 Year After Hand-Assisted Laparoscopic Donor Nephrectomy in Living Kidney Donors.

OBJECTIVES: Our aim was to investigate effects of surgery on living donors' body composition and clarify factors related to it. MATERIALS AND METHODS: We evaluated preoperative computed tomography images of 335 living kidney donors (127 men, 209 women) to calculate 3 body composition parameters and changes with aging by sex: (1) skeletal muscle mass, quantified by skeletal muscle index; (2) fat distribution, calculated by visceral adipose tissue/subcutaneous adipose tissue ratio; and (3) muscle quality, quantified by intramuscular adipose tissue content. Thereafter, with pre- and postoperative computed tomography images from 75 living kidney donors (25 men, 50 women) after hand-assisted laparoscopic donor nephrectomy, we compared pre- and postoperative body composition changes. RESULTS: Annual change in intramuscular adipose tissue content with age was 0.0049 in men and 0.0091 in women. Of 75 patients, 49 had lower quality of muscle, intramuscular adipose tissue content was significantly higher after nephrectomy (P < .001), and median change in intramuscular adipose tissue content was 0.061 (range, 0.018-0.11) in men and 0.052 (range, 0.017-0.18) in women. Univariate analysis revealed that skeletal mass index and visceral adipose tissue/subcutaneous adipose tissue ratio changes were significantly different between the intramuscular adipose tissue content improvement and deterioration groups. Multivariate analysis revealed skeletal mass index change was an independent factor for intramuscular adipose tissue content change (P = .0019). Intramuscular adipose tissue content change was negatively correlated with skeletal mass index change (r = -0.40). CONCLUSIONS: Although muscle quality deteriorates after nephrectomy, maintaining muscle mass is important to retaining muscle quality.

Immunoenzymometric assay for recombinant methioninase in biological fluids.

Immunoassay for recombinant methioninase (rMETase), an anti-cancer agent, in biological sample was developed. Antisera were produced by immunizing rabbits with rMETase. The antisera were evaluated using radioiodine-labeled rMETase and good antisera for sensitive immunoassay were obtained. Horseradish peroxidase (HRP) was coupled to reduced IgG of K232 antiserum through bridging agent, N-(epsilon -maleimidocaproyloxy) sulfosuccinimide ester (sulfo-EMCS), to prepare enzyme-labeled antibody. IgG fraction of K231 antiserum was immobilized on microplate well. Two-site sandwich immunoenzymometric assay (IEMA) was developed using these antibodies and had good standard curve between 0.4 and 12 ng per well. For determination of rMETase in mouse plasma, sample was diluted 100-fold with dilution buffer containing protease inhibitors, because about 10% of rMETase immunoreactivity was lost for 2 h at room temperature. rMETase in mouse plasma could be determined by the proposed method in the range of 0.5-8 microg/ml and the method was validated. This novel IEMA, in substitution for the measurement of its enzyme activity, should be very useful not only for preclinical studies of rMETase but also for the clinical studies.