Abstract
The neutral leukocyte proteases involved in the release of free amino acids in incubation mixtures of blood cell lysates and of human serum albumin with leukocyte lysates were characterized by several techniques including 1H nuclear magnetic resonance spectroscopy, electrophoresis, high performance liquid chromatography, gel filtration, amino acid analysis, and NH2-terminal analysis. The data suggested that the enzymes which contributed significantly to the extensive protein hydrolysis observed were two endopeptidases and three exopeptidases. Identification and analysis of the products obtained in incubation mixtures of human serum albumin with elastase and leucine aminopeptidase were compared with the products obtained in incubation mixtures of the protein with leukocyte lysates.
Highlights
Classical studies of specific isolated enzyme activities have provided much information about metabolic machinery; metabolic maps do not constitute the final picture of metabolism
After identifying the resonances observed in the ‘H NMR spectrum of incubated bloodcell lysates asarising from methyl moieties of free aliphatic amino acids produced as products of putative neutral protease action in leukocytes (l), we investigated the enzyme or enzymes which take part in the hydrolysis
The “alkaline earths” do not appear to have much effect in The specificity of low molecular weight protease inhibitors the formation of proteolysis products in the incubations of was employed as an aid to identify the enzymes involved in leukocytes with human serum albumin; only the addition of the release of free amino acids from polypeptides
Summary
Erythrocyte Preparation-Human bloodwas collected by venipuncture and transferred into tubes containing 3volumes of physiological saline (0.154 M). Cell suspensions were centrifuged for 10 min a t 350 X g, 277 K. The platelet-rich supernatant was decanted, the cells were resuspended ( 5 volumes) in physiological saline, and centrifuged under the same conditions. Cells were washed four times with Krebs bicarbonate buffer (2) with antibiotics, pH 7.4, 277 K, 5volumes, bycentrifuging at 3600 X g for 5 min. Blood cell lysates, including the buffy coat, were prepared by twice freezing (78 K ) and thawing the samples. The filtered cells were washed in Krebs bicarbonate buffer as above. Erythrocyte lysates were made by twice freezing and thawing the cells. Andthe buffy coat was collected and washed five times (room temperature, 5 volumes) in. The abbreviations used are: TLCK, Nu-p-tosyl-L-lysinechloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; HPLC, high pressure liquid chromatography; PMN, polymorphonuclear; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl
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