Measurement of plasma renin concentration in cats by use of a fluorescence resonance energy transfer peptide substrate of renin.

OBJECTIVE: To evaluate the use of a commercially available 5-carboxyfluorescein-based, intramolecularly quenched, fluorescence resonance energy transfer (FRET) peptide substrate of renin for measurement of plasma renin concentration in cats. SAMPLE POPULATION: Plasma samples obtained during a previous study of renal autograft ischemia-reperfusion injury in 10 cats and samples of fetal bovine serum containing recombinant human renin (rh-renin). PROCEDURES: Experiments involving samples of fetal bovine serum containing rh-renin were conducted to identify a suitable control vehicle, optimal substrate concentration, and appropriate duration of incubation. With the use of the identified assay conditions, a standard curve was constructed to allow conversion of relative fluorescent units into values of renin concentration (ng/mL). Subsequently, plasma samples obtained from cats before and after renal autograft ischemia-reperfusion injury were assayed to determine endogenous renin concentration. RESULTS: Under conditions of a 1:50 substrate dilution and 4-hour incubation period, the assay detected small amounts of rh-renin in fetal bovine serum. A linear relationship (R(2) = 0.996) between the relative fluorescent units generated and exogenous rh-renin concentration was evident. The assay detected renin in plasma samples obtained from cats after renal autograft ischemia-reperfusion, and renin concentrations on days 1 and 2 after transplant differed significantly. CONCLUSIONS AND CLINICAL RELEVANCE: The study data indicated that the assay involving the FRET peptide substrate of renin is potentially a rapid and specific method for measurement of plasma renin concentration in cats.

Structure and gene-polypeptide relationships of the region encoding glycerol diffusion facilitator (glpF) and glycerol kinase (glpK) of Pseudomonas aeruginosa.

The glycerol facilitator is one of the few known examples of bacterial solute transport proteins that catalyse facilitated diffusion across the cytoplasmic membrane. A second protein, glycerol kinase, is involved in entry of external glycerol into cellular metabolism by trapping glycerol in the cytoplasm as sn-glycerol 3-phosphate. Evidence is presented that glycerol transport in Pseudomonas aeruginosa is mediated by a similar transport system. The genes encoding the glycerol facilitator, glpF, and glycerol kinase, glpK, were isolated on a 4.5 kb EcoRI fragment from a chromosomal mini-library by functional complementation of an Escherichia coli glpK mutant after establishing a map of the chromosomal glpFK region with the help of a PCR-amplified glpK segment. The nucleotide sequence revealed that glpF is the promoter-proximal gene of the glpFK operon. The glycerol facilitator and glycerol kinase were identified in a T7 expression system as proteins with apparent molecular masses of 25 and 56 kDa, respectively. The identities of the glycerol facilitator and glycerol kinase amino acid sequences with their counterparts from Escherichia coli were 70 and 81%, respectively; this similarity extended to two homologues in the genome sequence of Haemophilus influenzae. A chromosomal delta glpFK mutant was isolated by gene replacement. This mutant no longer transported glycerol and could no longer utilize it as sole carbon and energy source. Two ORFs, orfX and orfY, encoding a putative regulatory protein and a carbohydrate kinase of unknown function, were located upstream of the glpFK operon.