Archives of biochemistry and biophysics最新文献

{"title":"Impact of oxidized phosphatidylcholine supplementation on the lipidome of RAW264.7 macrophages","authors":"Matilde Santos ,&nbsp;Tatiana Maurício ,&nbsp;Rosário Domingues ,&nbsp;Pedro Domingues","doi":"10.1016/j.abb.2025.110384","DOIUrl":"10.1016/j.abb.2025.110384","url":null,"abstract":"<div><div>Oxidized phospholipids (OxPLs) have emerged as critical damage-associated molecular patterns (DAMPs) and modulators of numerous biological processes, including inflammation, playing a significant role in health and disease. Despite their recognized influence on macrophage polarization, the precise mechanisms by which distinct OxPL species shape macrophage behavior remains poorly understood. The present study investigates the impact of two oxidized phosphatidylcholines (OxPC): omega 3 1-stearoyl-2-docosahexaenoyl-<em>sn</em>-glycero-3-phosphatidylcholine (OxPC22:6), and omega 6 1-stearoyl-2-linoleoyl-<em>sn</em>-glycero-3-phosphatidylcholine (OxPC18:2), on the lipidomic profile of RAW264.7 macrophages, through an LC-MS lipidomic analysis. Our findings demonstrate that the OxPCs under study modulate macrophage lipidome differently, highlighting the significance of the sn-2 acyl chain composition for their biological function. When administered alone, neither of the OxPCs induced a pro-inflammatory phenotype in macrophages. OxPC22:6 appears to induce a preparatory pro-inflammatory state in macrophages, improving their subsequent inflammatory responses, while OxPC18:2 seems to induce a resting state on macrophages. Under LPS stimulation, both OxPCs were found to selectively attenuate certain LPS-driven lipidomic changes (PC.O, PC.P, PI.P, PE.P) while amplifying others (DG, Cer, LPC, PE.O, PI.O, TG, PC, PI) and introducing unique alterations to the macrophage lipidome (SM, PE, LPE). Core lipidomic changes, crucial for macrophages' LPS response, were identified, with sustained elevation of TG, DG, Cer, PC, LPC, and PI.O and reduction of PE.O, PI, and CAR. These observations suggest that, in the presence of LPS, mainly OxPC22:6 amplifies the pro-inflammatory lipidomic signature of macrophages. Further research is needed to clarify whether the observed lipidomic adaptations improve, impair, or inhibit macrophages' inflammatory capacities and response.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"768 ","pages":"Article 110384"},"PeriodicalIF":3.8,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143639382","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Amyloid β fragments that suppress oligomers but not fibrils are cytoprotective","authors":"Maria C. Zabala-Rodriguez ,&nbsp;Ken Teter ,&nbsp;Suren A. Tatulian","doi":"10.1016/j.abb.2025.110386","DOIUrl":"10.1016/j.abb.2025.110386","url":null,"abstract":"<div><div>Neurotoxic aggregates of amyloid beta (Aβ) peptide contribute to the etiology of Alzheimer's disease (AD). In this work, we examined how seven overlapping fragments derived from Aβ_1-42 affect the oligomerization and toxicity of the full-length peptide. Four fragments inhibited the toxicity of oligomeric Aβ_1-42 to various degrees, two others conferred no cellular protection against Aβ_1-42 toxicity, and one fragment enhanced both Aβ_1-42 oligomerization and toxicity. The structural and aggregation propensities of the peptides that support strong inhibition of Aβ_1-42 toxicity have been identified. Data analysis allowed elucidation of the mechanisms of action of each of the seven peptide fragments on Aβ_1-42 cytotoxicity. Our work establishes the potential therapeutic value of four Aβ fragments and supports the notion that agents directed to disruption of Aβ oligomers may be more effective AD drug candidates than those targeting Aβ fibrils.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"768 ","pages":"Article 110386"},"PeriodicalIF":3.8,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143630076","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolic role of a genetically conserved aldehyde dehydrogenase in bacterial assimilation of various primary amines","authors":"Tadashi Nakai ,&nbsp;Naoya Miyagi ,&nbsp;Kota Hisamura ,&nbsp;Shoya Matsuba ,&nbsp;Kanji Nishimoto ,&nbsp;Emi Nakai ,&nbsp;Katsuyuki Tanizawa ,&nbsp;Toshihide Okajima","doi":"10.1016/j.abb.2025.110387","DOIUrl":"10.1016/j.abb.2025.110387","url":null,"abstract":"<div><div>Primary amines such as <em>n</em>-butylamine and 2-phenylethylamine serve as good nitrogen, carbon, and energy sources for bacterial growth. In many Gram-negative bacterial species, these amines are first oxidized by a periplasmic enzyme, quinohemoprotein amine dehydrogenase (QHNDH), encoded in an operon termed ‘<em>qhp</em>’, consisting of eight genes (<em>qhpABCDEFGR</em>). A gene predicted to encode an aldehyde dehydrogenase is also highly conserved in the vicinity of the <em>qhp</em> operon. In this study, we found that a 5′-upstream region of the aldehyde dehydrogenase gene in <em>Paracoccus denitrificans</em> has a high promoter activity that responds to <em>n</em>-butylamine supplementation in the culture medium, indicating co-regulation with the <em>qhp</em> genes by the transcriptional regulator QhpR. Hence, we designate this gene as the ninth member of the <em>qhp</em> operon, <em>qhpH</em>. Disruption of <em>qhpH</em> in <em>P. denitrificans</em> neither affected bacterial growth on primary amines, nor impaired QHNDH activity, suggesting the presence of another constitutive aldehyde dehydrogenase(s) compensating for the defect of <em>qhpH</em>. Nevertheless, heterologous expression of <em>qhpH</em> along with the eight <em>qhp</em> genes in an amine non-assimilating bacterium, <em>Rhodobacter sphaeroides</em>, significantly enhanced the growth on <em>n</em>-butylamine, as compared to the slow growth without <em>qhpH</em>. The recombinant QhpH purified from <em>Escherichia coli</em> cells showed high aldehyde dehydrogenase activities toward various aldehydes. These findings demonstrate that the <em>qhpH</em> gene encodes an aldehyde dehydrogenase with broad substrate specificity and is evolutionarily conserved with the <em>qhp</em> operon to play a role in an efficient metabolism of various primary amines in Gram-negative bacteria.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"768 ","pages":"Article 110387"},"PeriodicalIF":3.8,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143628896","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-altitude chronic hypoxia prevents myocardial dysfunction in experimental model of type 2 diabetes","authors":"Yixuan Wan ,&nbsp;Lisha Zhang ,&nbsp;Lei Wang ,&nbsp;Yu Zhang ,&nbsp;FaBao Gao","doi":"10.1016/j.abb.2025.110371","DOIUrl":"10.1016/j.abb.2025.110371","url":null,"abstract":"<div><h3>Background</h3><div>High-altitude chronic hypoxia (CHH) has a favorable impact on the lower prevalence of diabetes together with the better glucose tolerance. However, whether it prevents diabetic cardiomyopathy remains unclear. This study aimed to investigate the effects of CHH on left ventricular (LV) function in experimental model of type 2 diabetes.</div></div><div><h3>Methods</h3><div>Sprague-Dawley rats were randomly divided into control (altitude 500 m), DM (diabetes mellitus and altitude 500 m), CHH (altitude 4250 m and non-diabetic for 2 weeks), CHH-DM2 (altitude 4250 m and DM for 2 weeks), and CHH-DM8 (altitude 4250 m and DM for 8 weeks) groups. The experimental model of type 2 diabetes was induced by a high-fat diet plus low-dose streptozotocin (35 mg/kg, intraperitoneal) after fasted overnight. Left ventricular cardiac function and global myocardial strain were evaluated at 2, and 8 weeks by 7.0 T cardiovascular magnetic resonance. Subsequently, biochemical indices, histological evaluation, and levels of hypoxia-induced factor (HIF)-1α were assessed.</div></div><div><h3>Results</h3><div>Left ventricular ejection fraction (LVEF), global longitudinal (GLS), circumferential (GCS), and radial (GRS) strains significantly decreased in the DM group compared with the controls. However, these abnormalities in DM rats were significantly prevented in the CHH-DM2 group, and were further improved in CHH-DM8 group. Mechanistically, prolonged CHH at high altitude further reduced cardiac apoptosis, and oxidative stress, and increased autophagy, and the expression of HIF-1α in diabetic myocardial tissue.</div></div><div><h3>Conclusions</h3><div>CHH exerted cardioprotective effects by improving LV function, increasing myocardial strain and attenuating cardiac hypertrophy in type 2 diabetic rats, likely through reducing apoptosis and oxidative stress, activating autophagy and HIF-1α signaling in diabetic rats.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"768 ","pages":"Article 110371"},"PeriodicalIF":3.8,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143630078","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PANoptosis-related gene biomarkers in aortic dissection","authors":"Yuting Pu ,&nbsp;Yang Zhou ,&nbsp;Tuo Guo ,&nbsp;Xiangping Chai ,&nbsp;Guifang Yang","doi":"10.1016/j.abb.2025.110385","DOIUrl":"10.1016/j.abb.2025.110385","url":null,"abstract":"<div><h3>Introduction</h3><div>Programmed cell death of vascular smooth muscle cells (VSMCs) is critical in the pathogenesis of aortic dissection (AD), yet the role of PANoptosis—comprising pyroptosis, apoptosis, and necroptosis—remains unclear.</div></div><div><h3>Methods</h3><div>We utilized the GSE213740 single-cell sequencing dataset to assess PANoptosis levels in VSMCs. Datasets GSE153434 and GSE147026 were employed to identify differentially expressed genes (DEGs) and perform weighted gene co-expression network analysis. PANoptosis gene sets were sourced from the GSEA website, with GSE52093 serving as the validation cohort. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-Protein Interaction analyses were conducted, along with assessments of upstream regulators and immune cell infiltration. Validation was performed on aortic tissues from AD patients and mouse models.</div></div><div><h3>Results</h3><div>The single-cell dataset revealed an increased PANoptosis score in VSMCs in AD. Nineteen PANoptosis-related DEGs (PANDEGs) were identified, contributing to VSMC differentiation, DNA damage response, and apoptosis. KEGG analysis highlighted the P53 and TGF-β pathways, with PANDEGs positively correlating with immune cell infiltration. Key PANDEGs GADD45B, CDKN1A, and SOD2 were validated, showing co-expression with α-SMA.</div></div><div><h3>Conclusion</h3><div>The increased PANoptosis score in VSMCs suggests that GADD45B, CDKN1A, and SOD2 play crucial roles in AD.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"768 ","pages":"Article 110385"},"PeriodicalIF":3.8,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143620514","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"microRNA-34 family: A multifunctional miRNA family","authors":"Chen Wang,&nbsp;Ximei Yuan,&nbsp;Yuejun Fu","doi":"10.1016/j.abb.2025.110382","DOIUrl":"10.1016/j.abb.2025.110382","url":null,"abstract":"<div><div>MicroRNAs are endogenous non-coding small RNAs composed of about 22 nucleotides, which are widely found in eukaryotic cells and regulate gene expression at the post-transcriptional level through complementary pairing with target genes, leading to mRNA degradation or translation inhibition. MiR-34 family is a highly conserved miRNA family during evolution. Recent studies have found that members of the miR-34 family are involved in regulating biological processes such as aging, ciliogenesis, and immunity. To have a more comprehensive understanding of miR-34 family, this paper reviewed the functional evolution of miR-34 family, and provided a reference for further research on the related functions of miR-34.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"768 ","pages":"Article 110382"},"PeriodicalIF":3.8,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143613123","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The R337C mutation in the p53 oligomerization domain affects the regulatory domain and its ability to bind response elements: Evidence based on structural and biophysical studies","authors":"Alankrita Singh ,&nbsp;Lakshay Malhotra ,&nbsp;Abhay Mishra ,&nbsp;Simran Kundral ,&nbsp;Pawan Kumar Tiwari ,&nbsp;Saroj Kumar ,&nbsp;Hariprasad Gururao ,&nbsp;Punit Kaur ,&nbsp;Abdul Samath Ethayathulla","doi":"10.1016/j.abb.2025.110381","DOIUrl":"10.1016/j.abb.2025.110381","url":null,"abstract":"<div><div>The homotetrameric form of p53 is critical for performing essential functions like maintaining genomic stability and preventing uncontrolled cell proliferation. In part, these crucial functions are mediated by the p53 C-terminal region (CTR) containing the tetramerization/oligomerization domain (TD/OD) and regulatory domain (RD), responsible for maintaining the protein's oligomeric state and regulating its function. Mutations in the tetramerization domain reduce the transactivation potential and alter the transactivation specificity of p53. This study investigates the effect of high-frequency tetramerization missense mutation p53R337C on protein stability, oligomeric state, and its ability to bind the DNA response elements. For the first time using CD and FTIR spectroscopy, we have shown that the p53 regulatory domain (residues 363–393) and oligomerization domain (residues 327–355) possess a characteristic alpha helix secondary structure, which is enhanced upon binding to DNA, implicating stabilization of the domain. The mutation R337C in the OD impacts the secondary and tertiary structure of p53 CTR, leading to the loss of secondary structure and the formation of unstable tetramers, as shown by CD and DSC thermal studies. Surprisingly, the secondary structure of mutant p53 CTR partially stabilized upon binding to the DNA sequence. Our data suggests that the unstable p53R337C tetramer exhibits weaker binding to the DNA promoter sequence with decreased transcription activity, consistent with previous cell-based assays. Our study conclude that the loss of salt-bridge interactions between Arg337 and Asp352 in the intra-dimer of p53 leads to the formation of unstable tetramers, and the DNA-binding ability of the regulatory domain.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"768 ","pages":"Article 110381"},"PeriodicalIF":3.8,"publicationDate":"2025-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143595963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dual role of Tropomyosin-R160 in thin filament regulation: Insights into phosphorylation-dependent cardiac relaxation and cardiomyopathy mechanisms","authors":"Meaghan E. Barry ,&nbsp;Michael J. Rynkiewicz ,&nbsp;Jian Wen ,&nbsp;An-Yue Tu ,&nbsp;Michael Regnier ,&nbsp;William Lehman ,&nbsp;Jeffrey R. Moore","doi":"10.1016/j.abb.2025.110380","DOIUrl":"10.1016/j.abb.2025.110380","url":null,"abstract":"<div><div>β-adrenergic stimulation causes cell signaling that targets modulation of calcium levels as well as sarcomeric proteins to increases contractility. PKA phosphorylation of serine residues 23 and 24 of troponin I reduces calcium sensitivity and promotes cardiac relaxation. Our protein-protein docking and molecular dynamics studies revealed that Tpm-R160 is adjacent to these phosphorylation sites. In addition, Tpm-R160 has been identified as a disease-causing mutation site. Here, we investigated Tpm-R160's role in thin filament regulation and its interaction with phosphorylated TnI. Using <em>in</em> <em>vitro</em> motility assays, calcium sensitivity was quantified in reconstituted thin filaments containing various combinations of a phosphomimetic version of troponin I (TnI-S23/24D) and tropomyosin where the charge and potential for electrostatic interaction was removed by mutation of Tpm-R160 to a neutral alanine (Tpm-R160A). As expected, the phosphomimetic TnI (TnI-S23/24D) reduced calcium sensitivity in thin filaments with wild-type tropomyosin. However, the phosphorylation-like effect was diminished in the presence of the Tpm-R160A mutation. Notably, Tpm-R160A alone, when paired with wild-type TnI, also showed reduced calcium sensitivity, indicating that mutation of Tpm-R160 affects thin filament regulation in the absence of phosphorylation. Our findings indicate that Tpm-R160 has a dual role in thin filament regulation: (1)it is crucial for proper interaction between phosphorylated TnI and Tpm-R160 during adrenergic-induced cardiac relaxation, and (2) at the same time, the arginine residue itself is additionally required for maintenance of overall calcium sensitivity. These results provide key insight into the molecular defects underlying cardiomyopathy and a framework for development of therapeutic strategies targeting Tpm-R160 interactions. (249 words)</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"768 ","pages":"Article 110380"},"PeriodicalIF":3.8,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143584471","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phytochemical inhibitors of squalene epoxidase: Integrated In silico and In vitro mechanistic insights for targeting cholesterol biosynthesis","authors":"Emadeldin M. Kamel ,&nbsp;Doaa A. Abdelrheem ,&nbsp;Bashir Salah ,&nbsp;Al Mokhtar Lamsabhi","doi":"10.1016/j.abb.2025.110372","DOIUrl":"10.1016/j.abb.2025.110372","url":null,"abstract":"<div><div>Squalene epoxidase (SQLE) is a critical enzyme in the sterol biosynthesis pathway and a promising therapeutic target for various diseases. This study investigates the inhibitory potential of six phytochemicals, amentoflavone, dihydromyricetin, withaferin A, ursolic acid, paeonol, and maslinic acid, against SQLE through an integrated approach combining <em>in silico</em> predictions and experimental validation. Computational analyses, including molecular docking, molecular dynamics (MD) simulations, Potential energy landscape (PEL), MM/PBSA analysis, and ADMET profiling, identified amentoflavone, dihydromyricetin, and withaferin A as the most promising inhibitors, with high binding affinities and stable interactions within the SQLE binding site. Among these, amentoflavone exhibited the strongest binding affinity (−10.4 kcal/mol), binding free energy (−42.01 ± 2.78 kcal/mol), and stability during a 300 ns MD simulation, supported by favorable MD trajectory and interaction energy profiles. Experimental <em>in vitro</em> assays further validated these findings, showing that all tested compounds exhibited inhibitory activity against SQLE, with amentoflavone demonstrating the lowest IC₅₀ (1.92 ± 0.28 μM), confirming its role as a potent inhibitor. Enzyme kinetics studies revealed that maslinic acid and ursolic acid exhibited noncompetitive inhibition, while withaferin A, paeonol, and amentoflavone acted as competitive inhibitors. Dihydromyricetin demonstrated a mixed inhibition mode due to its dual interaction with the enzyme's active and allosteric sites. The pharmacokinetic analysis indicated that all compounds exhibited drug-like properties, with varying ADMET profiles influencing their potential as therapeutic candidates. This study highlights the therapeutic potential of amentoflavone, withaferin A, and dihydromyricetin as potent SQLE inhibitors, underscoring the value of integrated <em>in silico</em> and <em>in vitro</em> approaches in drug discovery.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"768 ","pages":"Article 110372"},"PeriodicalIF":3.8,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143562682","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Standardization of lipid sample preparation for monitoring phospholipase activity","authors":"Laís Cardoso Cunha ,&nbsp;Verônica Silva Valadares ,&nbsp;Jamil Silvano de Oliveira ,&nbsp;Liza Figueiredo Felicori ,&nbsp;Adolfo Henrique Moraes","doi":"10.1016/j.abb.2025.110373","DOIUrl":"10.1016/j.abb.2025.110373","url":null,"abstract":"<div><div>Phospholipase enzymes, such as A1, A2, B, and D, are found in the venom of venomous animals, including brown spiders. Phospholipase D (PLD) isoforms from brown spider venom can cause dermonecrosis, hemolysis, and nephrotoxicity. New methods to monitor PLD activity are essential for understanding its mechanisms and molecular characteristics. One effective approach is using ³¹P nuclear magnetic resonance (³¹P NMR) spectroscopy to track PLD enzymatic activity by identifying the ³¹P signals of phosphorylated substrates and products. However, sample preparation for ³¹P NMR is challenging, as the lipid substrates’ carbon chain length and unsaturation degree can affect solubilization, oxidation, and enzyme interaction, impacting the reaction kinetics. This study standardizes a phospholipid sample preparation method with fatty acids of different chain lengths for monitoring PLD activity. The addition of CHAPS detergent is essential for solubilizing lipids with long-chain fatty acids, but its concentration needs optimization, as higher amounts can inhibit PLD activity. Storing lipids in ethanol, forming lipid films, and injecting nitrogen into stock solutions improved lipid quantification and assay reproducibility. These standardized conditions can be adapted to other experimental approaches for monitoring phospholipase activity.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"768 ","pages":"Article 110373"},"PeriodicalIF":3.8,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143579956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}