Purification and characterization of a serine protease (esterase B) from rat submandibular glands

Abstract

A new protease has been purified to homogeneity from rat submandibular gland homogenate by using DEAE-Sephadex chromatography, chromatofocusing, aprotinin-Sepharose affinity chromatography, and high-performance liquid chromatography. The enzyme has been named esterase B, since it represents the second major esterolytic peak on DEAE-Sephadex chromatography of submandibular gland homogenate. It is an acidic protein (pI = 4.45) with an apparent molecular weight of 27 000. It is heat-stable and has an optimum pH of 9.5. Esterase B hydrolyzed the synthetic substrates tosyl-L-arginine methyl ester and Val-Leu-Arg-p-nitroanilide (S2266). It also cleaved dog plasma kininogen to produce a kinin, identified as bradykinin on reverse-phase high-performance liquid chromatography. Esterase B, however, is only a weak kininogenase, since it had only 5% of the kininogenase activity of equimolar concentrations of glandular kallikrein and had no effect on rat mean blood pressure or on the isolated rat uterus. Esterase B activated plasminogen and had caseinolytic activity. It was inhibited by aprotinin, soybean trypsin inhibitor, lima bean trypsin inhibitor, phenylmethanesulfonyl fluoride, antipain, leupeptin, and p-tosyl-L-lysine chloromethyl ketone. On double immunodiffusion, when reacted with kallikrein and tonin antisera, esterase B showed partial identity with kallikrein but not with tonin. On immunoelectrophoresis against kallikrein antisera, esterase B formed a precipitin arc at a position different from that of kallikrein. Esterase B appears to be a trypsin-like serine protease having some homology with glandular kallikrein.

Estrogen and Luminal Stimulation of Rat Uterine Kallikrein1

“…The use of two antisera raised in different laboratories stresses the consistency of our findings. The minimal variations produced in the staining after coincubation of the antiserum with rK7, a weak kininogenase [24,25], in the oil-stimulated and unstimulated pseudopregnant horn at Day 7, does not permit us to ascertain the presence or absence of this protein, which should be further studied by mRNA analysis and by in situ hybridization.…”

Growth‐promoting activity in serum‐free medium of kallikreinlike arginylesteropeptidases from rat submaxillary gland

“…This result also demonstrates that one of the submaxillary gland growth factors is a toninlike protease which thus belongs to the kallikrein family. Arginylesteropeptidase activity asso- ciated to fraction Sp2 was unaffected by antitonin immunoglobulin (not shown) and should likely correspond to (anlother kallikrein-related esterase(s) (Brandtzaeg et al, 1976;Khullar et al, 1986). The substrate specificity of the enzymes that were purified from rat submaxillary gland was studied and compared to that of thrombin as well as trypsin and commercially available kallikreins by measuring the relative hydrolysis rate of synthetic p-nitroanilide derivatives.…”

A potent vasoconstrictor in the rat submandibular gland.

“…The rat submandibular gland contains a number of arginine esterases. A few of them have been well characterized, such as glandular kallikrein, 24 tonin, 3 esterase B, 7 and esterase y. 6 Under our experimental conditions, kallikrein and esterase y should be retained by the DEAE-Sephadex A-50 column.…”

“…Both contractile and Pro-Phe-Arg-MCA activity are found in a purified protein (pi=7.3) that is clearly separable from tonin (pi=6.2) as well as from a third esterase, which we have previously named esterase B (pl=5.6). 7 ' 25 Characteristics of other submandibular gland esterases are still unknown. All of them belong to a group of immunologicalry related serine proteases coded by a set of closely associated genes named the kallikrein gene family.…”

“…7 The extract was dialyzed against 0.1 M phosphate buffer (pH 7.0) containing 1 mM Na 2 EDTA and applied to a DEAESephadex A-50 column equilibrated with the same buffer. The column was washed until A^ was below 0.005.…”