Phosphoenolpyruvate-dependent inhibition of collagen biosynthesis, alpha2beta1 integrin and IGF-I receptor signaling in cultured fibroblasts.
ABSTRACT The mechanism of collagen biosynthesis regulation is not fully understood. The finding that prolidase plays an important role in collagen biosynthesis and phosphoenolpyruvate inhibits prolidase activity "in vitro" led to evaluate its effect on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with millimolar concentrations (1-4 mM) of phosphoenolpyruvate monopotassium salt (PEP) for 24 h. It was found that PEP-dependent decrease in prolidase activity and expression was accompanied by parallel decrease in collagen biosynthesis. However, the experiments with inhibitor of PEP production, 3-mercaptopicolinate revealed no direct correlation between collagen biosynthesis and prolidase activity and expression. Since insulin-like growth factor (IGF-I) is the most potent stimulator of both collagen biosynthesis and prolidase activity, and prolidase is regulated by beta(1) integrin signaling, the effect of PEP on IGF-I receptor (IGF-IR) and beta(1) integrin receptor expressions were evaluated. It was found that the exposure of the cells to 4 mM PEP contributed to a decrease in IGF-IR and beta(1) integrin receptor expressions. The data suggest that PEP-dependent decrease of collagen biosynthesis in cultured human skin fibroblasts may undergo through depression of alpha(2)beta(1) integrin and IGF-IR signaling. The hypothetical mechanism of the role of prolidase in IGF-IR, beta(1) integrin receptor expressions, and clinical significance of the process are discussed.
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ABSTRACT: The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 184.108.40.206) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5'-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V(max) values and decreased the K(m) values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5'-triphosphate also caused inhibition. The K(m) values for phosphoenolpyruvate and adenosine 5'-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5'-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5'-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.Journal of Bacteriology 11/1974; 120(1):52-8. · 3.19 Impact Factor
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ABSTRACT: Catalytic pH dependence for the hydrolytic activity of the enzyme prolidase with a series of dipeptide substrates is found to be generally bell-shaped (kcat/Km) or simple sigmoidal (kcat). An enzymic residue with a pKa value of 6.6 is found to be critically involved in the catalytic mechanism, as is the substrate amino group. Significant catalysis at a pH of 6.6 is also observed for prolidase with (alkylthio)acetylprolines and with haloacetylprolines. A reverse-protonation state mechanism for substrate binding and activation is postulated, involving a chelative interaction of the aminoacylamide portion of substrate with a strongly Lewis-acidic active site metal ion.Journal of Biological Chemistry 12/1990; 265(32):19600-5. · 4.65 Impact Factor
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ABSTRACT: The mechanism related to the decrease in the number of fibroblasts in the dermis during aging and the decrease in the biosynthetic capacity of existing fibroblasts to produce collagen is not known. Since prolidase [E. C. 3. 4. 13. 9] has been found to play an important role in the recycling of proline for collagen synthesis and cell growth, we performed comparative studies on prolidase activity and extracellular collagen content in in vitro aged human skin fibroblasts. We found that during aging of human skin fibroblasts there is a correlated decrease of prolidase activity and extracellular collagen content. The correlation is bifunctional. Decrease in prolidase activity contributes to decrease of extracellular collagen content, and removal of extracellular collagen form cultured fibroblasts contributes to a decrease of fibroblast prolidase activity. Using plates coated with different ECM proteins, it was found that extracellular type I collagen in cultured fibroblasts was the most potent activator of intracellular prolidase activity. Our results demonstrate that during in vitro aging of human skin fibroblasts, the prolidase activity of fibroblasts decreases. This phenomenon may be related to the reduced number of fibroblasts and decrease of extracellular collagen content in in vitro aging of the cells.The Tokai journal of experimental and clinical medicine 01/1997; 21(4-6):207-13.