No description available.

Prolidase (E.C.3.4.13.9) hydrolyzes imidodipeptides. Prolidase deficiency (PD) (McKusick no. 170100) is an autosomal recessive disorder characterized by a highly variable clinical phenotype. Mutation analysis was performed on a panel of 10 PD cell lines. Single-stranded conformation polymorphism analysis (SSCP) analysis on four overlapping cDNA-PCR products covering the entire coding region of the prolidase gene revealed seven novel mutations: G $ to$ A,551 (R184Q); G $ to$ A,833 (G278D); G $ to$ A, 1342 (G448R); G $ to$ A, 1354 (E452K); delGAG, 1354-1356 (delE452); a deletion of exon 5; and a deletion of exon 7. We used inverse PCR to clone intronic regions flanking exons 5 and 7 and designed primers for conventional PCR of these regions of the genome in patients expressing mRNAs with deleted exons. Two splice acceptor site mutations were identified: a G $ to$ C, nt $-$1 of intron 4 and an A $ to$ G, nt $-$2 of intron 6. To assess the biochemical phenotypes of four of these mutations (R184Q, G278D, G448R, and delE452), we have designed a transient expression system for prolidase in COS-1 cells. The enzyme was expressed as a fusion protein carrying the HA1 epitope of influenza hemagglutinin, allowing its immunological discrimination from the endogenous enzyme. Expression of the R184Q mutation produced 7.4% of control enzymatic activity while the expression of the G278D, G448R and delE452 produced inactive enzymes. Western analysis of the R184Q, G278D and G448R prolidases revealed stable immunoreactive material whereas the delE452 prolidase was not detectable. Pulse-chase experiments revealed that the delE452 mutant protein was synthesized but unstable. / The R184Q allele is carried by an asymptomatic individual, suggesting that its residual activity may be sufficient to prevent the development of symptoms. The other alleles, G278D, G448R, and delE452 which completely abolish enzyme activity associate with the symptomatic form of the disorder. Interestingly, these substitutions are all located at or very close to the putative metal binding residues of prolidase. / We have cloned and sequenced the mouse prolidase cDNA. We have cloned a genomic DNA fragment which carries exons 2, 3, and 4 of the mouse prolidase gene. We constructed a vector for the targeted disruption of the prolidase gene in mouse embryonic stem cells, to create a mouse model for prolidase deficiency.

No description available.

Prolidase deficiency (McKusick 26413) is a rare autosomal recessive disorder characterized by iminodipeptiduria, skin lesions and mental retardation. The enzyme prolidase hydrolyzes dipeptides containing C-terminal proline or hydroxyproline. / The results presented here indicate that prolidase plays a major role in the recycling of dipeptide bound proline. Control fibroblasts were able to use iminodipeptides in lieu of proline to sustain normal growth and protein synthesis whereas prolidase deficient cells were not. / Iminodipeptides added to the media of control and mutant cells showed no adverse effects on protein synthesis or cell growth. These results are consistent with a mechanism of biochemical pathology in which proline deprivation caused by the enzyme deficit is the cause of damage to skin cells. / Prolidase regulation by product and substrate was studied. A two fold decrease of prolidase activity was observed in fibroblasts grown in excess proline. However, cells grown in medium in which iminodipeptides replaced proline showed no significant difference in prolidase activity.

Prolidase deficiency (MIM 26413), an autosomal recessive phenotype, is caused by rare alleles at a locus on chromosome 19cent.-q13.2. The clinical phenotype is pleiotropic (affecting skin, brain, etc.) and of variable expressivity (benign to early death). I established skin fibroblast cultures from 6 homozygous probands and 6 obligate heterozygotes, purified prolidase (E.C. 3.4.13.9, a homodimer) from normal human fibroblasts, raised a monospecific rabbit antiserum to the subunit, and studied its biosynthesis. Pulse-chase immunoprecipitation experiments showed that the subunit is synthesized in the cytosol as a 58 KDa. polypeptide and not processed further. Homozygous prolidase-deficient cell strains expressed 3 classes of mutant alleles which by complementation analysis mapped to one locus. The alleles were designated CRM$-$ (nul), CRM+ activity/size variant, and CRM+ activity variant. Heterozygotes carrying CRM$-$ alleles have heat stable prolidase (50$ sp circ$C, 1hr); heterozygotes carrying CRM+ variant alleles have heat labile enzyme. The finding implies that variant CRM+ allele(s) can confer negative allelic complementation on the dimeric enzyme (dominant relative phenotype). CRM$-$ homozygous cells contain varying amounts of an alternative imidodipeptidase-like activity. The variant prolidase allele (major gene) and amount of alternative "prolidase" activity (modifier gene) are apparently both determinants of the associated clinical phenotype in prolidase deficiency. I obtained and sequenced a tryptic peptide from human kidney prolidase for synthesis of oligonucleotide probes in the future.

No description available.