Bufadienolides, one kind of steroids, are the major active component secreted by ear-side gland of Bufo species. Preliminary studies on high-throughput transcriptome sequencing about B. bufo gargarizans showed that the expression of 3β-Hydroxysteroid dehydrogenase (3βHSD) in ear-side gland was nearly 20 times higher than that in liver. The enzyme 3βHSD is an essential step in the biosynthesis of steroid such as progesterone, estrogens and androgens in steroidogenic tissues. Accordingly, 3βHSD is probably an important enzyme involved in the biosynthesis of bufadienolides. In this study, Bbg-3βHSD cDNA was cloned from the ear-side gland of B. bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pCOLD-Bbg3βHSD which was introduced into E. coli BL21 for prokaryotic expression. Bbg-3βHSD has an open reading frame (ORF) of 1134 bp and encodes 377 amino acid residues. The speculated protein molecular weight is 42.8 kDa and its theoretical isoelectric point is 8.68. Amino acid sequence homologous analysis showed that Bbg-3βHSD was highly homologous to the 3βHSD protein of other species. Phylogenetic tree showed the highest similarity between Bbg-3βHSD and 3βHSD from Rana rugosa. The optimized expression of recombinant Bbg-3βHSD were achieved by inducing with 0.1 mmol L IPTG at 15 °C for 20 h. Enzymatic activity in vitro shows that pregnenolone and dehydroepiandroesterone could be 3β-oxidized by Bbg-3βHSD when NAD was used as the coenzyme. Enzymatic properties showed that the optimum reaction temperature of recombinant Bbg-3βHSD was 40 °C, the optimum pH was 8.5, and the optimum coenzyme concentration was 1.5 mmol L.

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijbiomac.2018.08.165DOI Listing

Publication Analysis

Top Keywords

bufo gargarizans
12
ear-side gland
12
prokaryotic expression
8
3β-hydroxysteroid dehydrogenase
8
dehydrogenase 3βhsd
8
gland bufo
8
amino acid
8
recombinant bbg-3βhsd
8
bbg-3βhsd
7
3βhsd
6

Similar Publications

Nitrate induced hepatic fibrosis in tadpoles of Bufo gargarizans by mediating alterations in toll-like receptor signaling pathways.

Environ Res

January 2025

Life and Environmental Science College, Wenzhou University, 325003, Wenzhou, China; Zhejiang Provincial Key Laboratory for Subtropical Water Environment and Marine Biological Resources Protection, Wenzhou University, 325003, Wenzhou, China. Electronic address:

The nitrate pollution has become an increasingly serious environmental problem worldwide, and the toxic effects of elevated nitrate levels in the environment on aquatic animals remain to be elucidated. The purpose of the present study was to investigate the mechanisms of liver injury to tadpoles after exposure to nitrate from embryonic to metamorphic climax and to assess the recovery process of liver function after cessation of exposure. In the group with continuous nitrate exposure, the livers and thyroid of tadpoles showed remarkably histological lesions, of this with structural disorganization of the hepatocytes, cellular atrophy, and fibrosis, as well as significant reduction in the follicular and colloidal area of the thyroid.

View Article and Find Full Text PDF

Methimazole disrupted skeletal ossification and muscle fiber transition in Bufo gargarizans larvae.

Ecotoxicol Environ Saf

January 2025

Life and Environmental Science College, Wenzhou University, Wenzhou 325003, China; Zhejiang Provincial Key Laboratory for Subtropical Water Environment and Marine Biological Resources Protection, Wenzhou University, Wenzhou 325003, China. Electronic address:

Methimazole (MMI) is an emerging endocrine disrupting chemical (EDC) due to its increasing use in the treatment of thyrotoxicosis (hyperthyroidism), but its potential impact on amphibian development remains largely unexplored. In the present study, the effects of 8 mg/L MMI and 1 μg/L thyroxine (T4) exposure on skeletal ossification and muscle development in Bufo gargarizans tadpoles were comprehensively investigated by double skeletal staining, histological analysis and RNA sequencing. Our results indicated that MMI treatment down-regulated the expression levels of ossification-related genes (e.

View Article and Find Full Text PDF

Cinobufagin modulates vasculogenic mimicry and tumor-associated macrophages to inhibit ovarian cancer progression.

Eur J Pharmacol

January 2025

Key Laboratory of Xin'an Medicine, Anhui Province Key Laboratory of R&D of Chinese Medicine, Ministry of Education, Anhui University of Traditional Chinese Medicine, 103 Meishan Road, Shushan District, Hefei City, Anhui Province, 230038, China; Anhui Academy of Traditional Chinese Medicine Bozhou Branch, Bozhou City, Anhui Province, 236800, China. Electronic address:

Background: Ovarian cancer is among the most prevalent malignant tumors affecting women. While conventional therapies like surgery do provide some measure of disease control, they are accompanied by evident side effects that may readily result in drug resistance. Cinobufagin (HCS) is a water-soluble active component extracted from the dried skin of the Bufo gargarizans.

View Article and Find Full Text PDF

Deciphering substrate promiscuity and specificity of indolethylamine N-methyltransferase family enzymes from amphibian toads.

Bioorg Chem

December 2024

The Research Center of Chiral Drugs, Innovation Research Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China. Electronic address:

N-methylation is a crucial post-modification process in natural product biosynthesis and also contributes to the metabolism of various physiological substances, such as neurotransmitter, hormone, and trace elements. In this study, we identified seven indolethylamine N-methyltransferase (INMT) family enzymes from the amphibian toad Bufo gargarizan with distinct catalytic properties. Among these enzymes, BNMT 1, BNMT 5, BNMT 6 and BNMT 7 exhibited notable promiscuity, demonstrating the ability to methylate multiple derivatives of indolethylamine, phenylethylamine, phenylethanolamine, and nicotinamide.

View Article and Find Full Text PDF

Structurally diverse bufadienolides from the skins of Bufo bufo gargarizans and their cytotoxicity.

Sci Rep

November 2024

Guizhou Provincial College-based Key Lab for Tumor Prevention and Treatment with Distinctive Medicines, Zunyi Medical University, Zunyi, China.

Natural products, with their extensive chemical diversity, distinctive biological activities, and vast reservoirs, provide a robust foundation for advancing cancer therapeutics. A comprehensive phytochemical investigation of the skins from Bufo bufo gargarizans afforded two new bufadienolide derivatives identified as bufalactamides A and B (1-2), along with six known compounds: argentinogenin (3), desacetylcinobufagin (4), desacetylcinobufaginol (5), cinobufaginol (6), bufalin (7) and gamabufalin (8). The structural elucidation of these compounds was meticulously carried out by analyses of spectroscopic data (1D and 2D NMR, HR-ESIMS), and comparison with the literature data.

View Article and Find Full Text PDF

Want 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!