Kallikrein and kallikrein-like proteinases: purification and determination by chromatographic and electrophoretic methods

Raspi, Giorgio

doi:10.1016/0378-4347(96)00144-2

Cited by 15 publications

(11 citation statements)

References 176 publications

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“…TK activity eluted between 1.6 and 1.8 M NaCl with a minor peak observed at 0.3 M NaCl ( Figure 5). This finding differed from reports of pancreatic, urinary, and salivary kallikreins that eluted between 0.3 and 0.6 M NaCl under similar conditions (13,39,40). Similar tight binding was observed using another anion exchange column (Hitrap Q Pharmacia; data not shown).…”

Section: Bronchial Tk Is Associated With Hyaluronic Acidcontrasting

confidence: 96%

“…TK activity eluted as a single peak at about 0.35 M NaCl (Figure 7). This was consistent with the elution profile of other tissue kallikreins (40), and confirmed the observation that hyaluronidase treatment alters the molecular nature of bronchial TK secreted by serousgland cells.…”

Section: Bronchial Tk Is Associated With Hyaluronic Acidsupporting

confidence: 88%

“…To characterize the TK activity, we used anion exchange chromatography as a first-step purification. Kallikreins have been purified from different exocrine tissues in various species using this approach, and typically showed an elution profile of simple or double peaks, between 0.3 and 0.6 M NaCl (13,39,40). Our initial experiments showed a different profile, in which the main peak of TK activity eluted at high salt concentrations, suggesting that the enzyme had a much tighter binding with the anion exchange beads under these conditions.…”

Section: Discussionmentioning

confidence: 92%

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Bronchial Tissue Kallikrein Activity Is Regulated by Hyaluronic Acid Binding

Forteza

Lauredo²,

Abraham³

et al. 1999

Am J Respir Cell Mol Biol

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Tissue kallikrein (TK) is secreted by serous cells of tracheobronchial submucosal glands and plays a role in allergic airway responses. To better understand the regulation of TK, we used primary cultures of submucosal gland cells that release TK upon stimulation. Media from cultures stimulated with chymase (10(-7) M) showed increased TK activity (0.50 +/- 0.22 mU/ml mean +/- standard error) in comparison with the control group (0.08 +/- 0.02 mU/ml). The increased TK activity was significantly correlated with increases in the levels of the serous cell marker, secretory leukoprotease inhibitor. Anion exchange chromatography of the conditioned culture media showed that TK activity eluted as a broad peak between 1.6 and 1.8 M NaCl, unlike the reported elution (0.3 to 0.6 M NaCl) of kallikreins from other tissues, suggesting that secreted bronchial TK was bound to a negatively charged molecule. Hyaluronidase digestion increased TK activity in both pre- and post-chymase-stimulated culture media, whereas no such change was seen after samples were digested with heparinase or chondroitinase ABC. Further, after hyaluronidase digestion of media, TK eluted from an anion exchange column between 0.3 and 0.6 M NaCl. Enzymatic detection of TK after nondenaturing gel electrophoresis showed that hyaluronidase digestion also reduced the electrophoretic heterogeneity of TK to a single band, whereas adding back hyaluronic acid (HA) to hyaluronidase-digested samples restored the original heterogeneity. Finally, TK activity bound to HA-Sepharose and could be eluted with HA. These studies show that primary cultures of ovine submucosal gland cells secrete TK in a regulated fashion, and that secreted TK binds to HA. This binding reduces TK enzymatic activity; therefore, factors that affect HA turnover could modify the TK activity in the airway lumen. These events could be important in the regulation of kinin-mediated airway inflammation.

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Section: Bronchial Tk Is Associated With Hyaluronic Acidcontrasting

confidence: 96%

Section: Bronchial Tk Is Associated With Hyaluronic Acidsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 92%

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Bronchial Tissue Kallikrein Activity Is Regulated by Hyaluronic Acid Binding

Forteza

Lauredo²,

Abraham³

et al. 1999

Am J Respir Cell Mol Biol

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“…After early studies in the 1930s, when Frey, Kraut, and Werle characterized the first human kallikrein and derived a name from the Greek word for pancreas, καλλικρϵασ (reviewed in Refs. 1 and 2 ), the gene for this kallikrein (KLK1) was not discovered until 1985 ( 3 , 4 ). During the late 1980s, two further genes with high structural similarity to KLK1 , designated KLK2 (glandular kallikrein) ( 5 ) and the most noted member KLK3 (prostate-specific antigen) ( 6 ), were cloned and found to colocalize with KLK1 to the same chromosomal region (19q13.4) ( 7 ).…”

Section: Introductionmentioning

confidence: 99%

A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity

Guo

Skala

Magdolen³

et al. 2016

Journal of Biological Chemistry

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Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology.

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“…Affinity schemes based on heparin for the purification of lipases, phospholipases, and kinases were reviewed by Farooqui et al 448 Focus was also placed on techniques for attaching heparin to supports and detecting its presence after immobilization. Raspi 792 presented a review of sample preparation, purification, and assays for isolating and determining kallikrein proteinases from tissues and body fluids. Knudsen et al 793 reviewed chromatographic and electrophoretic separation strategies for eukaryotic DNA topoisomerases isoforms, and the techniques used for determining their catalytic activity.…”

Section: Enzymesmentioning

confidence: 99%

High‐Performance Liquid Chromatography of Biological Macromolecules

Irgum¹

2000

Encyclopedia of Analytical Chemistry

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Kallikrein and kallikrein-like proteinases: purification and determination by chromatographic and electrophoretic methods

Cited by 15 publications

References 176 publications

Bronchial Tissue Kallikrein Activity Is Regulated by Hyaluronic Acid Binding

Bronchial Tissue Kallikrein Activity Is Regulated by Hyaluronic Acid Binding

A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity

High‐Performance Liquid Chromatography of Biological Macromolecules

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