Proteolytic activity and free amino acid concentrations in polymorphonuclear leucocytes.
Polymorphonuclear granulocytes (PMN) are valuable tools for evaluating amino acid (AA) metabolism in nucleated cells, although variations of free amino acid concentrations due to the methods used for the separation of the cells and the procedures used for lysis have been reported. Furthermore, analytical variations in PMN AA concentration may be induced by protease activation during preparation, so that free AA detected in cells could originate from proteolysis other than from the physiological metabolic pathways and transport systems. To study this possibility we measured granulocyte protease activity and AA concentrations in cell suspensions processed with and without the addition of antiproteolytic agents. Granulocyte AA concentrations and protease activity in samples treated with antiproteolytics were 8-15 times lower than in samples processed without antiproteolytics. The use of protease inhibitors throughout the sample preparation is necessary for reliable estimation of free AA in granulocytes.
Carrea
A
A
Nephrology Department, Istituto Giannina Gaslini, Genoa, Italy.
Canepa
A
A
Perfumo
F
F
Ancarani
P
P
Verrina
E
E
Gusmano
R
R
D000596:Amino Acids / Q000097:blood*; D001391:Azo Compounds; D002851:Chromatography, High Pressure Liquid; D003094:Collagen; D004247:DNA / Q000097:blood; D010450:Endopeptidases / Q000097:blood*; D006801:Humans; D009504:Neutrophils / Q000737:chemistry* / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology; D012044:Regression Analysis
D000596:Amino Acids; D001391:Azo Compounds; D011480:Protease Inhibitors; C028470:Azocoll; D003094:Collagen; D004247:DNA; D010450:Endopeptidases