Human kallikrein 8 (hK8), whose gene was originally cloned as the human ortholog of a mouse brain protease, is known to be associated with diseases such as ovarian cancer and Alzheimer's disease. Recombinant human pro-kallikrein 8 was activated with lysyl endopeptidase-conjugated beads. Amino-terminal sequencing of the activated enzyme demonstrated the cleavage of a 9-aa propeptide from the pro-enzyme. The substrate specificity of activated hK8 was characterized using synthetic fluorescent substrates. hK8 showed trypsin-like specificity, as predicted from sequence analysis and enzymatic characterization of the mouse ortholog. All synthetic substrates tested containing either arginine or lysine at P1 position were cleaved by hK8. The highest kcat/Km value of 20x10(3)M-1 s-1 was observed with Boc-Val-Pro-Arg-7-amido-4-methylcoumarin. The activity of hK8 was inhibited by antipain, chymostatin, and leupeptin. The concentration for 50% inhibition by the best inhibitor, antipain, was 0.46 microM. The effect of different metal ions on the enzyme activity was analyzed. Whereas Na+ had no effect on hK8 activity, Ni2+ and Zn2+ decreased the activity and Ca2+, Mg2+, and K+ had a stimulatory effect. Ca2+ was the best activator, with an optimal concentration of approximately 10 microM.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1515/BC.2006.091 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Drug repositioning of mesalamine via supramolecular nanoassembly for the treatment of drug-induced acute liver failure.
Theranostics
January 2025
Medicinal Materials Research Center, Biomedical Research Division, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.
Acute liver failure (ALF) is characterized by rapid hepatic dysfunction, primarily caused by drug-induced hepatotoxicity. Due to the lack of satisfactory treatment options, ALF remains a fatal clinical disease, representing a grand challenge in global health. For the drug repositioning to ALF of mesalamine, which is clinically approved for the treatment of inflammatory bowel disease (IBD), we propose a supramolecular prodrug nanoassembly (SPNs).
View Article and Find Full Text PDFDetectEV: A functional enzymatic assay to assess integrity and bioactivity of extracellular vesicles.
J Extracell Vesicles
January 2025
Cell-Tech HUB and Institute for Research and Biomedical Innovation (IRIB), National Research Council of Italy (CNR), Palermo, Italy.
The application of extracellular vesicles (EVs) as therapeutics or nanocarriers in cell-free therapies necessitates meticulous evaluations of different features, including their identity, bioactivity, batch-to-batch reproducibility, and stability. Given the inherent heterogeneity in EV preparations, this assessment demands sensitive functional assays to provide key quality control metrics, complementing established methods to ensure that EV preparations meet the required functionality and quality standards. Here, we introduce the detectEV assay, an enzymatic-based approach for assessing EV luminal cargo bioactivity and membrane integrity.
View Article and Find Full Text PDFSimultaneous Quantification of Total Antibody and Conjugated Payload for DS001 in Rat Serum Using a Hybrid Immuno-Capture LC-MS/MS.
AAPS J
January 2025
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201210, China.
Antibody-drug conjugates (ADCs) are intricate compounds that pose significant challenges in bioanalytical characterization. Therefore, multiple bioanalytical methods are required to comprehensively elucidate their pharmacokinetic (PK) profiles. In this study, we investigated DS001, an ADC consisting of a humanized monoclonal antibody (hRS7), a cleavable chemical linker, and the microtubule inhibitor monomethyl auristatin E (MMAE), with a drug-to-antibody ratio (DAR) of 8.
View Article and Find Full Text PDFStructural characterization of pyruvic oxime dioxygenase, a key enzyme in heterotrophic nitrification.
J Bacteriol
January 2025
Department of Environment and Energy Systems, Graduate School of Science and Technology, Shizuoka University, Shizuoka, Japan.
Nitrification by heterotrophic microorganisms is an important part of the nitrogen cycle in the environment. The enzyme responsible for the core function of heterotrophic nitrification is pyruvic oxime dioxygenase (POD). POD is a non-heme, Fe(II)-dependent enzyme that catalyzes the dioxygenation of pyruvic oxime to produce pyruvate and nitrite.
View Article and Find Full Text PDFExpression and Characterization of Alkaline Phosphatase from KMM 296 in Transiently Transformed Tobacco Leaves and Transgenic Calli.
Plants (Basel)
December 2024
School of Advanced Engineering Studies, Institute of Biotechnology, Bioengineering and Food Systems, FEFU, 10 Ajax Bay, 690922 Vladivostok, Russia.
Alkaline phosphatase (ALP) of the PhoA family is an important enzyme in mammals, microalgae, and certain marine bacteria. It plays a crucial role in the dephosphorylation of lipopolysaccharides (LPS) and nucleotides, which overstimulate cell signaling pathways and cause tissue inflammation in animals and humans. Insufficient ALP activity and expression levels have been linked to various disorders.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!