Sepharose Column Research Articles

Molecular Targets of the 5-Amido-Carboxamide Bumped Kinase Inhibitor BKI-1748 in Cryptosporidium parvum and HCT-8 Host Cells.

Cryptosporidium parvum is an apicomplexan parasite causing persistent diarrhea in humans and animals. Issuing from target-based drug development, calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs), with excellent efficacies in vitro and in vivo have been generated. Some BKIs including BKI-1748 share a core structure with similarities to the first-generation antiprotozoal drug quinine, which is known to exert notorious side effects. Unlike quinine, BKI-1748 rapidly interfered with C. parvum proliferation in the human colon tumor (HCT) cell line HCT-8 cells and caused dramatic effects on the parasite ultrastructure. To identify putative BKI targets in C. parvum and in host cells, we performed differential affinity chromatography with cell-free extracts from non-infected and infected HCT-8 cells using BKI-1748 and quinine epoxy-activated sepharose columns followed by mass spectrometry. C. parvum proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from both BKI-1748 and quinine columns. However, no C. parvum proteins could be identified binding exclusively to BKI-1748. In contrast, 25 BKI-1748-specific binding proteins originating from HCT-8 cells were detected. Moreover, 29 C. parvum and 224 host cell proteins were identified in both BKI-1748 as well as in quinine eluates. In both C. parvum and host cells, the largest subset of binding proteins was involved in RNA binding and modification, with a focus on ribosomal proteins and proteins involved in RNA splicing. These findings extend previous results, showing that BKI-1748 interacts with putative targets involved in common, essential pathways such as translation and RNA processing.

Investigate the efficacy of size exclusion chromatography for the isolation of extracellular vesicles from C. elegans

Isolation of Extracellular Vesicles (EVs) has been done extensively in the past using ultracentrifugation, a recent shift has been observed towards precipitation, and exosome isolation kits. These methods often co-elute contaminants of similar size and density which makes their detection and downstream applications quite challenging. As well as the EV yield is also compromised in some methodologies due to aggregate formation. In recent reports, size-exclusion chromatography (SEC) is replacing density gradient-based ultracentrifugation as the gold standard of exosome isolation. It outperforms in yield, purity and does not account for any physical damage to the EVs.We have standardized the methodology for an efficient pure yield of homogenous exosomes of size even smaller than 75 nm in Caenorhabditis elegans homogenate. The paper entails the application and optimization of EV isolation by SEC based on previous studies by optimizing bed size and type of sepharose column employed. We propose that this method is economically feasible in comparison with currently available approaches. A comparative study was conducted to investigate the performance of CL-6B in relation to CL-2B and further, this was combined with ultracentrifugation for higher efficacy. The methodology could be introduced in a clinical setting due to its therapeutic potential and scope. The eluted EVs were studied by flow cytometry, nanotracking and characterized for size and morphology.

High yield expression and purification of Aspergillus flavus uricase cloned in Pichia pink™ expression system.

In this research, for the first time, A. flavus uricase gene was cloned in pPink-UOX plasmid under strong alcohol oxidase promoter of Pichia pink expression system after codon optimization. After selecting the best uricase producing clone with an activity of 0.7U/ml at the Flask level, a 5-L fermenter was used to increase the expression of the enzyme. Within 60h, the fermentation process produced 1500g of biomass from 4L of semi defined culture media and expressed 2.5g/L of the enzyme. The purity of recombinant uricase production using three consecutive DEAE Sepharose, CM Sepharose and Phenyl Sepharose columns was above 99%, which was confirmed by SDS-PAGE and RP-HPLC analyses. Size exclusion chromatography analysis showed that the purified enzyme has comparable heterogeneity to the Rasburicase. The yield of recombinant uricase production in this study was 63% and its specific activity was 24U/mg. The high expression of recombinant uricase in the Pichia pink strain and the increased enzyme activity compared to the standard sample indicate the potential of therapeutic and diagnostic applications of recombinant uricase in the present study.

Biochemical, molecular and anti-tumor characterization of L-methionine gamma lyase produced by local Pseudomonas sp. in Egypt

A soil inhabiting Pseudomonas sp. has been examined for producing L- methionine gamma-lyase enzyme. The identity of the tested bacteria was verified by VITEK2, and MALDI-TOF analysis in addition to molecular confirmation by 16S rDNA sequence and submitted in Genbank under accession number ON993898.1. Production of the targeted enzyme was done using a commercial medium including L-methionine, as the main substrate. This obtained enzyme was precipitated using acetone (1:1v/v) followed by purification with Sephadex G100 and sepharose columns. The specific activity of the purified enzyme (105.8 µmol/ mg/min) increased by 1.89 folds after the purification steps. The peptide fingerprint of the native MGL was verified from the proteomics analysis, with identical conserved active site domains with database-deposited MGLs. The molecular mass of the pure MGL denatured subunit was (>40 kDa) and that of the native enzyme was (>150 kDa) ensuring their homotetrameric identity. The purified enzyme showed absorption spectra at 280 nm and 420 nm for the apo-MGL and PLP coenzyme, respectively. Amino acids suicide analogues analysis by DTNB, hydroxylamine, iodoacetate, MBTH, mercaptoethanol and guanidine thiocyanate reduced the relative activity of purified MGL. From the kinetic properties, the catalytic effectiveness (Kcat/km) of Pseudomonas sp. MGL was 10.8 mM -1 S-1 for methionine and 5.51 mM -1 S-1 for cysteine, respectively. The purified MGL showed highly significant antiproliferative activity towards the liver carcinoma cell line (HEPG-2) and breast carcinoma cell line (MCF-7) with half inhibitory concentration values (IC50) 7.23 U/ml and 21.14 U/ml, respectively. No obvious signs of toxicity on liver and kidney functions in the examined animal models were observed.

Affinity purification, identification, and biochemical characterization of Gamma-glutamyl transpeptidase, a membrane anchored enzyme of Gigantocotyle explanatum.

Gamma-glutamyl transpeptidase is an enzyme that facilitates the transfer of glutamyl groups from glutamyl peptides to other peptides or water. Additionally, it also participates in important processes such as amino acid transport, cellular redox control, drug detoxification, apoptosis, and DNA fragmentation in a various organism. In the present study, GGT activity in Gigantocotyle explanatum was examined in order to characterize the enzyme in the helminth system. GGT is isolated using membrane solubilization and purified through affinity column chromatography (Con-A Sepharose column). Km and Vmax values, as well as the optimal pH, optimal temperature, and incubation period, are also determined using enzyme kinetics. The hetero-dimeric property of the enzyme is demonstrated by the purified GGT, which yielded two subunits of 65.5 and 55kDa. The optimal pH and temperature are found to be 8.0 and 37°C, respectively. While assessing the optimal incubation time of the enzyme, it was observed that the purified GGT not only retained its functional integrity up to 15min but also reflected considerable thermostability at higher temperatures, by retaining 78% and 25% of its initial activities at 50°C and 60°C, respectively. One millimolar concentration of 6-Diazo-5-Oxo Nor-isoleucine (DON), a specific inhibitor of GGT, completely abolished GGT activity. These results suggest that GGT in these worms is a catalytically active enzyme with distinguishing characteristics that can be used for further study to comprehend its function in amphistome biology and in host-parasite relationships, especially since the potential therapeutic candidacy of the GGT enzyme has already been indicated in these groups of organisms.

Transthyretin binds soluble endoglin and increases its uptake by hepatocytes: A possible role for transthyretin in preeclampsia?

BackgroundPreeclampsia is a common but life-threatening condition of pregnancy. It is caused by poor placentation resulting in release of trophoblast material (including soluble endoglin (sEng)) into the maternal circulation leading to maternal vascular dysfunction and to the life-threatening condition of eclampsia. The only cure is early delivery, which can have lifelong consequences for the premature child. The thyroid hormone binding protein transthyretin is dysregulated in preeclampsia, however it is not known if this plays a role in disease pathology. We hypothesised that transthyretin may bind sEng and abrogate its negative effects by removing it from the maternal serum. MethodsThe effect of transthyretin on hepatocyte uptake of Alexa-labelled sEng was measured using live cell imaging. Interactions between transthyretin, and sEng were investigated using molecular modelling, direct binding on CnBr Sepharose columns, confocal imaging, and measurement of fluorescence resonance energy transfer. ResultsTransthyretin directly bound to sEng and increased its uptake by hepatocytes. This uptake was altered in the presence of transforming growth factor-β1 (TGF-β1). Molecular modelling predicted that transthyretin and TGF-β1 bind at the same site in sEng and may compete for binding. Endocytosed transthyretin and endoglin entered cells together and co-localised inside hepatocyte cells. ConclusionTransthyretin can bind sEng and increase its uptake from the extracellular medium. This suggests that increasing transthyretin levels or developing drugs that normalise or mimic transthyretin, may provide treatment options to reduce sEng induced vascular dysfunction.

Purification of Anginex: An Inhibitor of Tumor Metastasis using RdRd tag

Anginex is a cytokine-like β-sheet forming peptide of 33 amino acids with potent anti-angiogenic activity.Anginex is essential in inhibiting abnormal processes caused by angiogenesis, such as tumor growth and blood vessel formation. However, Anginex has limitations, including poor stability, short half-life, complicated synthesis, and low purity. Rubredoxin dimer (RdRd) is used as a protein tag to improve stability and detection of Anginex during purification for the first time. A plasmid was designed to contain the RdRd-Anginex fusion protein. RdRd-Anginex plasmid was transformed and expressed in E. coli BL21star cells. The results show RdRd-Anginex has been purified using a Nickel Sepharose column. Enterokinase, a proteolytic enzyme, was then used to cleave between the RdRd from Anginex. The cleaved sample was placed on a Nickel Sepharose column where the Anginex sample was eluted.

An Efficient Gram-Scale Chemical Synthesis of Purine Locked Nucleic Acid Nucleoside-5'-O-Triphosphates.

This article presents a simple, reliable, straight-forward, general, and efficient chemical method for the gram-scale synthesis of purine locked nucleic acid (LNA) nucleotides, such as LNA guanosine-5'-O-triphosphate (LNA-GTP) and LNA adenosine-5'-O-triphosphate (LNA-ATP), starting from the corresponding nucleoside. The reaction pathway employs an improved protection-free "one-pot, three-step" Ludwig synthetic strategy. The first step involves monophosphorylation of nucleoside with phosphorus oxychloride followed by reaction with tributylammonium pyrophosphate and subsequent hydrolysis of the resulting cyclic intermediate to furnish the corresponding LNA nucleotide in good yields. It is noteworthy that the reaction affords high-purity (>99.5%) LNA nucleotide after diethylaminoethyl Sepharose column purification. © 2022 Wiley Periodicals LLC. Basic Protocol: Synthesis of LNA nucleoside-5'-O-triphosphates.

Functional production, characterization, and immobilization of a cold-adapted cutinase from Antarctic Rhodococcus sp.

A lipolytic enzyme (Rcut) was discovered from the Rhodococcusstrain (RosL12) isolated from the Antarctic Ross Sea. The corresponding gene composed of 651 bases encoding 216 amino acids. It was found to be a cutinase gene through BLAST search. Rcut has a signal sequence consisting of 29 amino acids. An active Rcut was produced after the intact gene containing the signal sequence was transformed into Escherichia coli Rosetta-gami™ 2 (DE3) pLysS. Rcut was purified through a nickel-nitrilotriacetic acid purification system and a carboxymethyl Sepharose column chromatography. Its specific activity was 2190 U/mg. Rcut showed the highest activity at 40 °C and had a low activation energy of 3.16 kcal/mol. This means that it is a typical cold-adapted enzyme. Rcut showed high activity towards medium chain fatty acids (C4–C10). Rcut degraded polycaprolactone and polyethylene terephthalate, suggesting that it could be used for decomposition of synthetic plastics causing environmental pollution. Rcut was immobilized on methacrylate-divinyl benzene bead. This immobilized Rcut (immRcut) showed higher thermal stability than the free enzyme. ImmRcut performed transesterification of various esters and ethanol in a non-polar solvent, suggesting that it could be used for the synthesis of industrially useful ester compounds.

Purification and characterization of L-asparaginase extracted from local Iraqi green beans (Phaseoulus vulgaris)

The most prevalent metabolite for the storage and transport of nitrogen used in protein production is L-asparagine; in earlier studies, L-asparaginase enzyme derived from microorganisms was utilized to treat cancer cells; therefore, this study aimed to purify and describe this enzyme derived from Iraqi green bean seeds rather than microbial sources. The enzymes were partially purified from green beans by short steps, including centrifugation of crude enzyme, dialysis by Diethylaminoethyl Sepharose Column, and equilibrated using 20 mM Tris-HCl buffer, pH 8.0, and then applied to a sephacryl S-200. Coulometric methods measured the enzymatic activity at 450 nm, and the unit of activity was calculated by comparing it to a standard curve. Purification of L-asparaginase yielded a 5 percent yield, a 2.7 fold increase in activity, and a 43 unit/ml activity. The pH of asparaginase was optimal at 8.0. After a one-day incubation period, this enzyme became more stable at pH levels ranging from 7.5 to 9.5. This enzyme had the same optimal temperature and thermal stability at 40°C, but it was more stable at temperatures ranging from 20 to 40°C, allowing it to retain its maximal activity.

Partial Purification of Phytocystatin from Moringa oleifera lam

Background: Phytocystatins are plants cysteine protease inhibitors (CPIs) that are known for their numerous uses in medicine and biotechnology. Methods: Different extraction media which include, Sodium Hydroxide, Hydrochloric acid, Sodium Chloride, Sodium phosphate buffer and distilled Water were used to evaluate flower, leave, root, latex, and stem bark of Moringa oleifera for inhibitory activity against cysteine protease (Papain enzyme). The phosphate buffer extract with higher protease inhibitory activity was concentrated by cold acetone precipitation and further subjected to partial purification using ammonium sulphate fractionation and Hydrophobic chromatography on Phenyl Sepharose column. The molecular weight of partially purified protein was estimated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Results: The extracts of Sodium phosphate buffer from the seeds of Moringa oleifera showed higher CPI activity of 11.23 units/mg protein. Cold acetone precipitated extract gave high yield of 94.6% protein with 60% inhibition of protease. The partially purified protein showed a fifty percent inhibitory concentration (IC50) of 1.22±0.3 mg/ml protein against the enzyme with 63% inhibition. An estimated molecular weight of 13.5kDa was obtained from electrophoretic analysis of partially purified protein. Conclusion: Moringa oleifera seeds could be a good source of low molecular weight protein Inhibitor of Cysteine protease and might be useful in biotechnology of traditional medicinal plants, in transgenic crops to arrest the negative pathogenic over expressions of cysteine proteases

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System.

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Heterologous Expression and Functional Characterization of CAP18 from Oryctolagus cuniculus.

Antimicrobial peptides belong to the innate defence system of creatures. These peptides attach to the bacterial membrane in order to die microorganisms by penetrating them. Hence, biotechnology researchers pay more attention to produce antimicrobial peptides for use in various fields. The studies showed that rabbit tissue with inflammation and skin ulcers would be producing CAP18 peptide, which belongs to the cathelicidin group. In this study, the optimized sequence of the cap18 gene was placed into the pPICZAα plasmid after the alpha-factor signal and transformed into Pichia pastoris (X-33 strain). Purification of the recombinant peptide was done based on its histidine tail at C-terminal, and western blotting method was used to demonstrate the purification of rCAP18. The antibacterial activity of the purified and desalted rCAP18 was investigated at different concentrations against pathogenic bacteria. The maximum expression level of rCAP18 (17.5 kDa) was seen 90 h after induction of alcohol oxidase I (AOX1) promoter with methanol. The concentration of rCAP18 was 33 mg/L after purification with Ni-NTA Sepharose column. The function of rCAP18 (4.3, 5.7, 7 µg/ml) was investigated against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Results showed that %CFU/cm2 reached 28% after P. aeruginosa cells treatment with 7 μg/ml of rCAP18. This study presented the findings related to heterologous expression of cap18 gene, and evaluation of rCAP18 antibacterial effects. Our results showed that rCAP18 plays a significant role in inhibiting bacterial growth, especially Gram-negative bacteria.

Production and purification of glutaminase free L-asparaginase from Lysinibacillus fusiformis and its appraisal in acrylamide mitigation of starchy foods

L-asparaginase (EC 3.5.1.1.) is at the leading edge in microbial catalysis and biotechnology, as it possess range of applications in biomedical and food sectors. The L-asparaginase manifests versatile applications in the treatment of cancers and acrylamide reduction in fried and baked food. However, availability of limited enzyme producers, lower activity and stability reduces the commercial exploitation of asparaginase. L-asparaginase was produced and purified from the newly isolated bacterial strain Lysinibacillus fusiformis NP_MK3. The enzyme was partially purified using the DEAE Sepharose column chromatography and gel electrophoresis revealed production of asparaginase isozymes by L. fusiformis. The enzyme activity was enhanced up to twofold (10.55 Ul−1) with the addition of fructose as a carbon source. L-asparaginase exhibited optimal activity at 35 °C and pH 8. Pretreatment of potato chips with purified L-asparaginase had resulted into considerable decrease in acrylamide content. L. fusiformis asparaginase could potentially be employed as a robust biological pretreatment strategy to lessen the acrylamide content in fried and baked foods and thus in a long term reducing the risks of cancer incidence by consumption of such food products.

A novel assay to assess the effects of estrogen on the cardiac calmodulin binding equilibrium

AimsThe Ca2+-binding protein calmodulin (CaM) modulates numerous target proteins but is produced insufficiently to bind all of them, generating a limiting CaM equilibrium. Menopause increases cardiac morbidity; however, it is unknown if the cardiac CaM equilibrium is affected by estrogen. We devised an assay to assess the effects of ovariectomy and estrogen treatment on the cardiac CaM equilibrium. Materials and methodsSprague-Dawley rats received sham surgery or ovariectomy, followed by 2-week treatment with vehicle or 17β-estradiol. Ca2+-saturated left ventricular (LV) lysates were processed through CaM sepharose columns, which retained CaM-binding proteins unoccupied by endogenous CaM. Eluants therefrom were subjected to a competitive binding assay against purified CaM and a CaM biosensor to assess the amounts of unoccupied CaM-binding sites. LV cellular composition was assessed by immunohistochemistry. Key findingsLV eluants processed from sham animals reduce biosensor response by ~32%, indicating baseline presence of unoccupied CaM-binding sites and a limiting CaM equilibrium. Ovariectomy exacerbates the limiting CaM equilibrium, reducing biosensor response by ~65%. 17β-estradiol treatment equalizes the difference between sham and ovariectomized animals. These changes reflect whole tissue responses and are not mirrored by changes in total surface areas of cardiomyocytes and fibroblasts. Consistently, Ca2+-dependent, but not Ca2+-independent, interaction between CaM and the cardiac inositol trisphosphate receptor (IP3R) is reduced following ovariectomy and is restored by subsequent 17β-estradiol treatment. SignificanceOur assay provides a new parameter to assess tissue CaM equilibrium. The exacerbated limiting CaM equilibrium following estrogen loss may contribute to cardiac morbidity and is prevented by estrogen treatment.

Microbial keratinases as potential therapeutic options for neurodegenerative diseases.

Normal proteins, if misfolded, form insoluble aggregates or fibrils, resistant to proteolytic degradation. Such proteins, named amyloid proteins, share a common structural motif, called the cross β sheet structure. The insoluble β-amyloid fibrils have been implicated in neurodegenerative diseases such as Alzheimer's disease (AD). Prions have structural similarities with β-amyloid fibrils. As microbial keratinases can degrade prions, we investigated the possible capacity of microbial keratinases, purified in our lab, for degrading β- amyloid fibrils. Lysozyme is a model system to study protein misfolding. β-Amyloid fibrils of lysozyme were prepared by treating Hen Egg White Lysozyme (HEWL) with 8M Urea for 48 h at 55°C. The fibril formation was confirmed by formation of a fibrous precipitate, immunoblotting with anti β-amyloid antibody, High Performance Liquid Chromatography (HPLC), and Congo Red Absorption spectroscopy. Using Q Sepharose Column Chromatography, two microbial keratinases, Ker1 and Ker2 were purified from an actinomycete strain, Amycolatopsis sp. MBRL 40, previously isolated in our laboratory (MBRL). β-amyloid fibril degrading activity of these keratinases were investigated both in solution or as reconstituted form on cationic/neutral liposomes. Both soluble and reconstituted Ker1 degraded β-amyloid fibrils as confirmed by loss of fibrous precipitate of β-amyloid fibrils, changes in the spectra as observed in HPLC and Congo Red Absorption spectroscopy and loss of signal when immunoblotted with anti β-amyloid antibody. Details of the findings on degradation of β-amyloid degradation by keratinase (Ker1) of indigenous Amycolatopsis sp. MBRL 40 obtained in our laboratory and its potential as a therapeutic option for neurodegenerative diseases shall be presented in this oral paper. Many drugs for treating Alzheimer's disease (AD) are in various stages of clinical trials. These drugs either inhibit the activity of acetylcholinesterase or inhibit the formation of Aβ plaques and their downstream effects e.g. Ca++ channel antagonists, nonsteroidal anti-inflammatory drugs, antioxidants, iron chelators, hypolipidemic drugs etc. Microbial keratinases are novel molecules in this sense as they haven't been investigated so far and we propose these enzymes as new potential therapeutic interventions for neurodegenerative diseases such as AD.

A Purified Alkaline and Detergent-Tolerant Lipase from Aspergillus fumigatus with Potential Application in Removal of Mustard Oil Stains from Cotton Fabric

Abstract In the present study, the lipase from Aspergillus fumigatus was purified which was found to be stable to commercial detergents and oxidising agents. A purification fold of 6.96 and yield of 11.03% were achieved when the enzyme was purified using Octyl Sepharose column chromatography. In presence of various oxidizing agents, the highest activity of lipase was 15.56 U/mg with hydrogen peroxide. Among various surfactants used, the maximum activity exhibited by lipase was with Tween 80. While studying the effect of various detergents, the highest activity of 9.3 U/mg was achieved with "Vanish" detergent. Wash performance was studied with various detergents out of which "Vanish" showed highest oil removal of 79%. Lipase from Aspergillus fumigatus possessed better stability with various surfactants and oxidizing agents. The results of this study have shown that the lipase from Aspergillus fumigatus along with detergent "Vanish" (0.7%) under optimized conditions (5 μg/ml lipase, 40°C wash temperature and 40 min wash duration) improved oil removal from cotton fabric stained with mustard oil by 84%.

Generation of a Universal Human Complement Source by Large-Scale Depletion of IgG and IgM from Pooled Human Plasma.

Complement is a key component of functional immunological assays used to evaluate vaccine-mediated immunity to a range of bacterial and viral pathogens. However, standardization of these assays is complicated due to the availability of a human complement source that lacks existing antibodies acquired either through vaccination or natural circulation of the pathogen of interest. We have developed a method for depleting both IgG and IgM in 200mL batches from pooled hirudin-derived human plasma by sequential affinity chromatography using a Protein G Sepharose column followed by POROS™ CaptureSelect™ IgM Affinity resin. The production of large IgG- and IgM-depleted batches of human plasma that retains total hemolytic and alternative pathway activities allows for improved assay standardization and comparison of immune responses in large clinical trials.

Disorders of proteolytic homeostasis in the liver of rats with hyperhomocysteinemia

Introduction: The liver is a key organ of the body that is responsible for maintaining homeostasis. It helps regulate almost all biochemical pathways associated with metabolism, nutrition, energy supply, and the formation of immunity. Consequently, impaired liver function can contribute to the progression of hyperhomocysteinemia and the development of associated complications. The present study aims to investigate the level and activity of metal-dependent and serine proteases in the liver of rats with hyperhomocysteinemia. Methods: A total of 60 albino nonlinear male rats were used in this study. Hyperhomocysteinemia was induced by intragastric administration of DL-homocysteine thiolactone. Total proteolytic activity was measured using casein as a substrate. To determine the activity of metal-dependent and serine proteases, ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride were used. The levels of MMP-1, MMP-2, and MMP-3 were studied by enzyme-linked immunosorbent assay. The fraction of serine proteases was isolated by affinity chromatography on the benzamidine sepharose column. The composition of serine protease fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results: The pathogenesis of hyperhomocysteinemia was found to be accompanied by a proteolytic imbalance manifested in the increase in the total proteolytic activity, the activity of serine proteases and especially the activity of metal-dependent enzymes. The increase in the levels of MMP-1, MMP-2, and MMP-3 indicates the involvement of these enzymes in the amplification of proteolysis mediated by metal-dependent enzymes. Despite a significant increase in the content of the serine proteases, their activity did not increase significantly. This may be because of the proteolytic degradation of serine proteases, which was confirmed by changes in their qualitative composition. The accumulation of low-molecular-weight proteins and the decrease in the part of proteins with a molecular weight of greater than 50 kDa in the fraction of serine proteases were revealed. Conclusion: Collectively, our observations indicate that impaired proteolysis in the liver may be an important determinant in the development of hyperhomocysteinemia-associated disorders.

Isolation, purification, and characterization of pectin methylesterase inhibitor and polygalacturonase inhibitor protein from Indian lemon (Citrus limon L.)

Proteins acting as powerful inhibitors of plant pectin methylesterase and polygalacturonase were isolated from whole lemon fruits (Citrus limon L.). Pectin methylesterase inhibitor (PMEI) and polygalacturonase inhibitor protein (PGIP) were purified using DEAE Sepharose column, resulting in fold purity of 89.13 and 81.16 and having a molecular mass of 35 and 38 kDa, respectively as estimated using SDS-PAGE and MALDI-TOF mass spectroscopy. The optimum pH of purified PMEI and PGIP was pH 6 and pH 4.5 while the inhibitors showed good stability in the pH range of 5–8 and 3.5 to 5.5, respectively. Both the inhibitors from C. limon demonstrated an optimum temperature of 55 °C. Thermal inactivation data suggested that purified PGIP was more heat stable than PMEI. The inhibition kinetics of PMEI and PGIP towards C. limon PME and C. limon PG was of a non-competitive type. Both PMEI and PGIP obeyed first-order inactivation kinetics. The PMEI and PGIP exhibited different extent of inhibition towards PME and PG from other fruit sources analyzed in this study. As these inhibitors inhibit PME and PG from other plant sources they can be used in fruit-based products to control undesirable endogenous enzyme activities as an alternative to thermal processing.