Rapid degradation of D- and L-succinimide-containing peptides by a post-proline endopeptidase from human erythrocytes

We have been interested in the metabolic fate of proteins containing aspartyl succinimide (Asu) residues. These residues can be derived from the spontaneous rearrangement of Asp and Asn residues and from the spontaneous demethylation of enzymatically methylated L-isoAsp and D-Asp residues. Incubation of the synthetic hexapeptide N-Ac-Val-Tyr-Pro-Asu-Gly-Ala with the cytosolic fraction of human erythrocytes resulted in rapid cleavage of the prolyl-aspartyl succinimide bond producing the tripeptide N-Ac-Val-Tyr-Pro. The rate of this reaction is equal for both L- and D-Asu-containing peptides and is 10-fold greater than the rate of cleavage of a corresponding peptide containing a normal Pro-Asp linkage. When the aspartyl succinimide ring was replaced with an isoaspartyl residue, the cleavage rate was about 5 times that of the normal Pro-Asp peptide. The tripeptide-producing activity copurified on DEAE-cellulose chromatography with an activity that cleaves N-carbobenzoxy-Gly-Pro-4-methylcoumarin-7-amide, a post-proline endopeptidase substrate. These two activities were both inhibited by an antiserum to rat brain post-proline endopeptidase, and it appears that they are catalyzed by the same enzyme. This enzyme has a molecular weight of approximately 80,000 and is covalently labeled and inhibited by [3H]diisopropyl fluorophosphate. The facile cleavage of the succinimide- and isoaspartyl-containing peptides by this post-proline endopeptidase suggests that it may play a role in the metabolism of peptides containing altered aspartyl residues.     

Osteogenesis imperfecta: a heterogeneous morphologic phenotype in cultured dermal fibroblasts

Summary Osteogenesis imperfecta (OI) is a phenotype with clinical and biochemical heterogeneity. We report here that expression of the OI phenotype extends to the level of dermal fibroblast morphology in vitro. Growth characteristics and morphology of control (n=6) and OI cell strains (n=10, representing the four major OI categories, Sillence classification) were compared by measuring the following: (i) days required in culture to reach confluence after plating at uniform density; (ii) cell density at confluence; (iii) width and length of cells (measured on phase contrast micrographs at 300xmagnification). Our results show that: (i) OI fibroblasts take longer (11–27 days, mean 20 days) than control cells (10–19 days, mean 16 days) to reach stationary phase; (ii) all OI phenotypes achieve a lower cell density (0.87x10⁶ cells/P60, range 0.3–1.6x10⁶) at stationary phase relative to control cells (2.2x10⁶ cells/P60, range 1.7–2.6x10⁶; F_4,77=56.1, p<0.01, indicating that OI cells are larger than normal). Cell shape (expressed as the width: length ratio) was also abnormal in OI cells. (F_4,730=37.6, p<0.01), types I and II OI cells have significantly increased ratios (p<0.01) relative to control, type III, and type IV cells. Intra-group phenotypic heterogeneity was also apparent in the OI categories and also within the control population. These findings confirm deviant morphologic phenotypes in OI dermal fibroblasts and further demonstrate interindividual heterogeneity in the expression of genes that determine size and shape of dermal fibroblasts in both OI and normal donors.Publication No. 84013 from the Montreal Children's Hospital Research Institute     

Simple reversed-phase high-performance liquid chromatography quantitation of ganciclovir in human serum and urine

A fast, simple, and cost-effective HPLC method for the quantitation of the antiviral drug ganciclovir is described. The serum samples are extracted with perchloric acid and neutralized with potassium phosphate buffer, and urine samples are diluted with distilled water. A reversed-phase column with isocratic elution by 15 mM potassium phosphate buffer (pH 2.5) containing 0.25% acetonitrile is used to separate ganciclovir; quantitation is by UV absorbance at 254 nm. Total turnaround time is 22 min; more than 3000 samples can be run on a single column without loss of peak quality. The limit of quantitation is 0.05 μg/ml. Recoveries varied from 91 to 10% with coefficients of variation ranging from 0.387 to 7.95%.     

Possible regulation of pyruvate carboxylase fromThiobacillus novellus by hydroxypyruvate

Aspartate, glutamate, or dicarboxylic acids did not inhibit the activity of a highly purified but not homogeneous preparation of pyruvate carboxylase fromThiobacillus novellus. The only effective inhibitors were end-products of the reaction and to a lesser degree hydroxypyruvate. The latter has not been shown previously to regulate the enzyme's activity. Lineweaver-Burk plots revealed that it was uncompetitive with respect to acetyl CoA with a K_ii of 3.6 mM, and noncompetitive with respect to bicarbonate, magnesium ATP, and pyruvate with respective K_ii values of 7.1, 5.5, and 6.47 mM. The corresponding K_is values were 7.02, 5.4, and 4.25 mM. A mathematical model is presented that supports the findings.     

Clinical Utility of Insulin-Like Growth Factor 1 and 2; Determination by High Resolution Mass Spectrometry

Measurement of insulin-like growth factor-1 (IGF-I) has utility for the diagnosis and management of growth disorders, but inter-assay comparison of results has been complicated by a multitude of reference standards, antibodies, detection methods, and pre-analytical preparation strategies. We developed a quantitative LC-MS method for intact IGF-I, which has advantages in throughput and complexity when compared to mass spectrometric approaches that rely on stable isotope dilution analysis of tryptic peptides. Since the method makes use of full-scan data, the assay was easily extended to provide quantitative measurement of IGF-II using the same assay protocol. The validated LC-MS assay for IGF-I and IGF-II provides accurate results across the pediatric and adult reference range and is suitable for clinical use.