HYDROLYTIC ENZYMES OF RABBIT MONONUCLEAR EXUDATE CELLS I. Quantitative Assay and Properties of Certain Proteases, Non-Specific Esterases, and Lipases of Mononuclear and Polymorphonuclear Cells and Erythrocytes

Oil-induccd mononuclear phagocytes (MN) were quantitatively assayed for various hydrolases as unfractionatcd suspensions of frozen and thawed cells. They apparently contain two proteases. The first, measured with ureaor acid-denatured hemoglobin, was similar to purified Proteinase I of lung with respect to pH optimum (pH 4), stability, hydrolytic and polymerizing activities, and reactions to various inhibitors. The second protcasc resembled chymotrypsin in its hydrolysis of glycyl-L-phenylalanine amide, acetyl-i-tyrosine ethyl ester and N-benzoyl-DL-phenylalanine-/3-naphthol ester (BPN). With the latter, its pH optimum was between 5.0 and 5.8, and its action was inhibited by diisopropylphosphorofluoridate (DFP) and p-chloromercuribenzoate. When assayed under the above conditions, polymorphonuclear exudate cells (PMN) and red blood corpuscles (RBC) manifested little or no hydrolysis of either hemoglobin or BPN. MN also contained esterases that split methyl butyrate and /3-naphthyl acetate. The pH optimum with the latter was 7.4, and its hydrolysis was partially inhibited by DFP, fluoride, taurocholate, and eserine. PMN had low esterase activity; RBC had little or none. MN, but not PMN or RBC, contained a stable lipase with a pH optimum of 6.1 in maleate buffer. Protamine, NaC1, heat, p-chloromercuribenzoate, ethylenediamine tetraacetate, taurocholate, and DFP were inhibitory, but no appreciable activation occurred in the presence of heparin or serum. Thus it possessed some of the characteristics of Korn's lipoprotein lipase, but not others. I N T R O D U C T I O N The mononuclear phagocyte (MN) 1 is the functional clement of the reticuloendothelial system, 1 Abbreviations: MN, mononuclear(s) or mononuclear exudate cells (used as a frozen and thawed suspension for enzyme studies); PMN, polymorphonuclear(s) or polymorphonuclear exudate cells ;RBC and WBC, red and white blood corpuscles; urea-Hb and acid-Hb, ureaand acid-denatured hemoglobin; which is the body's major defense against microbial agents and their toxic products. The present EDTA, ethylenediamine tetraacetate; DFP, (diisopropylphosphorofluoridate) ; BPN, N-benzoyl-DLphenylalanine-/3-naphthol ester; NDBB, Naphthanil Diazo Blue B (tetrazotized diorthoanisidine); GPA, glycyl-L-phenylalanine amide; ATEE, acetyl-L-tyrosine ethyl ester; Naph. Ac., ~-naphthyl acetate. on N ovem er 7, 2017 jcb.rress.org D ow nladed fom i nves t i ga t i on was u n d e r t a k e n to assay a n d cha r ac ter ize M N e n z y m e s t h a t hyd ro l yze prote ins , l ipids, po lysacchar ides , a n d nuc le ic acids, w h i c h are the m a j o r c o m p o n e n t s of the m a n y e x o g e n o u s a n d e n d o g e n o u s subs t ances (e.g., bac t e r i a a n d effete b lood corpuscles) t ha t a re d iges ted by these cells. M N proteases , esterases, a n d l ipase a re e v a l u a t e d he re in ; M N lysozyme is e v a l u a t e d in a s u b s e q u e n t r epo r t (1); a n d s tudies on M N n u cleases a re j u s t beg inn ing . S imi l a r e n z y m e s in P M N a n d R B C were also eva l ua t ed . CELL COUNTS : T h e M N exudates conta ined from 10 mill ion to 1 billion cells wi th an average of about 80 million. The R B C present averaged 15 per cent o f the .WBC, varying from 0 to 100 per cent. The differential cell counts usual ly showed 90 to 100 per cent M N (including 2 to 5 per cent lymphocytes) and 0 to 1(1 per cent P M N . For our quant i ta t ive enzyme studies al lowance was made , when necessary,t for bo th the R B C and the P M N present in the M N exudates. C O L L E C T I O N O F P M N E X U D A T E S A N D R B C : P M N exudates were induced by the intraper i toneal FIGUEE 1 Typical oil-induced mononuclear exudate cells from the peritoneal cavity of the rabbit. Wright ' s stain. X 1000. E X P E R I M E N T A L P R O C E D U R E S