Plasma membrane-bound and lysosomal peptidases in human alveolar macrophages.

Abstract

Alveolar macrophages protect the lungs against noxious agents. Proteases and peptidases are essential for this defense and many metabolic activities. Human alveolar macrophages were evaluated for the presence of six important peptidases. Deamidase, a serine peptidase identical with the lysosomal protective protein and possibly with cathepsin A, had high specific activity in alveolar macrophages and is also present in cultured mouse J774A.1 and human U937 cells, used for the sake of comparison. In fractionated J774A cells, most of the deamidase activity was in the lysosomal fraction and in the final supernatant. Deamidase in human alveolar macrophages, obtained by bronchoalveolar lavage from 23 patients, cleaved dansyl-Phe-Leu-Arg at a rate of 2.26 mumol/h/mg protein and hydrolyzed the chemotactic peptide N-f-Met-Leu-Phe even faster, at a rate of 53.1 mumol/h/mg protein, the highest activity for this enzyme with any of the cells we tested. Rabbit antiserum, elicited with the recombinant partial sequence of the enzyme, immunoprecipitated 77-88% of the macrophage deamidase. In immunocytochemistry, this antiserum localized deamidase within the human macrophages. The enzyme was inhibited by diisopropylfluorophosphate (DFP; 1 mM) and by ebelactone B (10 microM), noncompetitively. The mRNA of deamidase was detected in mouse macrophages by Northern blot; the two protein chains of deamidase were shown in human macrophages by Western blot. In addition, two other serine peptidases were also highly active in macrophages: dipeptidyl peptidase IV (1.38 mumol/h/mg protein) and prolylcarboxypeptidase (0.72 mumol/h/mg protein). The activity of plasma membrane zinc metallopeptidases, neutral endopeptidase 24.11 and carboxypeptidase M, in contrast, was low or absent (angiotensin I converting enzyme; kininase II).(ABSTRACT TRUNCATED AT 250 WORDS)

Cathepsin A Is the Major Hydrolase Catalyzing the Intracellular Hydrolysis of the Antiretroviral Nucleotide Phosphonoamidate Prodrugs GS-7340 and GS-9131

“…An important function of CatA in this multienzyme complex is to protect both glycosidases from rapid proteolysis in the lysosomes (10,33,34,52). Further studies revealed that CatA is a multifunctional enzyme possessing deaminase and esterase activities at neutral pH and carboxypeptidase activity at pH 5.0 to 5.5 (18)(19)(20)(21)50). Peptide substrates with hydrophobic amino acid residues in the P1Ј position are cleaved with high efficiency; however, CatA also shows high affinity for charged residues in this position (13,35,37,38).…”

Angiotensin 1-9 and 1-7 Release in Human Heart

“…38 We purified the enzyme first from human platelets and found it highly concentrated in macrophages and endothelial cells. 19,27,36 Besides Ang I and BK, CATA cleaves endothelin fastest below neutrality. 36 Because it breaks a C-terminal amide bond at neutral pH, we called it deamidase.…”

“…The DNA of the expression vector DA-pVL1392 was cotransfected into High Five cells (Invitrogen) with linearized BaculoGold baculovirus DNA. Eighty percent of rCATA proenzyme was secreted, 22,24 then activated with trypsin, measured with Dansyl-Phe-LeuArg, 20,27 and purified on S-Sepharose column.…”

“…The activity was inhibited by serine peptidase inhibitors, diisopropylfluorophosphate (DFP) 63% and ebelactone B 54%. 20,27,31 The metallopeptidase inhibitor, 1,10-phenanthroline, instead of inhibiting, enhanced the reaction by 13% and even more at pH 5.5 (45%). At that pH, the rate of conversion of enkephalinamide increased to 29.3 (Table 1), but inhibitors of CATA were much less effective ( Table 1).…”

On the Mechanism of Action of Prolylcarboxypeptidase

“…Wallingford and associates described that Prcp-null mice had elevated levels of alpha-MSH1-13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet. Prcp null mice were also resistant to high-fat diet-induced obesity Soisson and associates described the crystal structure of human PRCP E112D polymorphism in the prolylcarboxypeptidase gene was found to be associated with blood pressure response to benazepril in Chinese hypertensive patients Javerzat and associates described overexpression of PRCP during the intermediate phase of the chick chorio-allantoic membrane Recombinant PRCP (rPRCP) metabolized BK1-8 to BK1-7, whereas rPRCP was ineffective in metabolizing BK 1-9 PRCP was found to be active in human cerebrospinal fluid Overexpression of angiotensin type 2 (AT2) receptor in mouse coronary artery endothelial cells was found to increase expression of PRCP, which may contribute to kinin release PRCP is a 4OHTAM resistance factor in estrogen receptor-positive breast cancer cells PRCP gt/gt mice were found to be hypertensive and prothrombotic (Sorrells and Erdos, 1971) (Odya et al, 1978) (Skidgel et al, 1981) (Deddish et al, 1990) (Tan et al, 1993) (Tamaoki et al, 1994) (Suzawa et al, 1995) (Suga et al, 1995) (Jackman et al, 1995) (Watson, Jr. et al, 1997) To deal with proper delivery of blood to the tissues as well as many types of insults, including chemical and physical damage as well as infection, the cardiovascular system has evolved an intricate multilayer of specialized biochemical pathways. Multiple feedback mechanisms controlling blood flow with contrasting effects regulate normal vascular function, which have either stimulatory or inhibitory effects.…”