Purification, biochemical and biophysical characterization of an acidic α-galactosidase from the seeds of Annona squamosa (custard apple).

Alpha galactosidase is an exoglycosidase that cleaves α-D-galactose and has numerous applications in medicine, biotechnology, food and pharma industries. In this study, a low molecular weight acidic α-galactosidase was identified from the seeds of custard apple. The purification of α-galactosidase from the crude extract of defatted seeds was achieved by employing ammonium sulphate fractionation, hydrophobic interaction and gel filtration chromatographic techniques. The purified custard apple α-galactosidase (CaG) migrated as a single band in native PAGE corresponding to molecular weight of ~67 kDa and cleaved chromogenic, fluorogenic and natural substrates. CaG was found to be a heterodimer with subunit masses of 40 and 30 kDa. The kinetic parameters such as KM and Vmax were found to be 0.67 mM and 1.5 U/mg respectively with p-nitrophenyl α-D-galactopyranoside. Galactose, methyl α-D-galactopyranoside and D-galacturonic acid inhibited CaG activity in mixed mode. The CD spectral analysis at far UV region showed that purified CaG exists predominantly as helix (35%), beta sheets (16.3%) and random coils (32.3%) in its secondary structure. These biochemical and biophysical properties of CaG provide leads to understand its primary sequence and glycan structures which will eventually define its novel physiological roles in plants and potential industrial applications.

Truncated domains of human serum albumin improves the binding efficiency of uremic toxins: A surface plasmon resonance and computational approach.

Albumin binding is the major cause for the toxicity of protein bound uremic toxins (PBUTs) in uremic patients. Albumin binding property is exploited to address this issue, as some of the extracorporeal dialysis systems use albumin as dialysate. In this line, a detailed study about binding of PBUTs to human serum albumin (HSA) and its domains gives valuable information. The focus of this work emphasizes the mechanism of binding of HSA and its domains with a few selected PBUTs such as hippuric acid (HA), indole acetic acid (IAA) and melatonin. The HSA domains (D2, D3 and D2-3) were expressed in Pichia pastoris and purified by using Albupure matrix. The binding of the expressed domains and HSA, with PBUTs, was measured using surface plasmon resonance and analyzed. All the three domains have significant affinity towards PBUTs, while D3 had greater affinity for all the three selected PBUTs. Docking studies showed that the basic amino acid, lysine, was forming hydrogen bond with PUBTs inorder to stabile these complex. This study would be having therapeutic importance for preparing the extracorporeal dialysis systems, in combination of different domains of HSA to remove the PBUTs.

Role of Herborn (K240E) and Milano Slow (D375H) human serum albumin variants towards binding of phenylbutazone and ibuprofen.

Human serum albumin (HSA) is the binding cargo in blood plasma. The binding of drugs to HSA determines the pharmacokinetics and pharmacodynamics of the drugs. There are 67 natural genetic variants of HSA were reported in literature. Studying the effect of albumin modifications on drug binding helps to treat the patients with proper medication. In the present study, we have aimed to understand the effect of two natural variants of HSA, such as Herborn (K240E) and Milano Slow (D375H) on the binding of phenylbutazone and ibuprofen. For this, we have generated K240E and D375H mutants and also double mutant (K240E/D375H) of HSA using site directed mutagenesis. The recombinant HSA and its variants were expressed in Pichia pastoris. The interaction of HSA and its variants to phenylbutazone and ibuprofen was studied using fluorescence spectroscopy. Our results showed that there is no significant effect of K240E and D375H mutations on phenylbutazone and ibuprofen binding. But the effect is significant when both the mutations were there in a single protein (K240E/D375H). Further, the CD spectroscopy data showed that there is no effect of phenylbutazone and ibuprofen binding on the conformation of protein, except in case of D375H, where there is a conformational change in the binding pocket with the ibuprofen binding.

Changes in the photosynthetic apparatus and lipid droplet formation in Chlamydomonas reinhardtii under iron deficiency.

The unicellular photosynthetic alga Chlamydomonas reinhardtii was propagated in iron deficiency medium and patterns of growth, photosynthetic efficiency, lipid accumulation, as well as the expression of lipid biosynthetic and photosynthesis-related proteins were analysed and compared with iron-sufficient growth conditions. As expected, the photosynthetic rate was reduced (maximally after 4 days of growth) as a result of increased non-photochemical quenching (NPQ). Surprisingly, the stress-response protein LHCSR3 was expressed in conditions of iron deficiency that cause NPQ induction. In addition, the protein contents of both the PSI and PSII reaction centres were gradually reduced during growth in iron deficiency medium. Interestingly, the two generations of Fe deficiency cells could be able to recover the photosynthesis but the second generation cells recovered much slower as these cells were severely in shock. Analysis by flow cytometry with fluorescence-activated cell sorting and thin layer chromatography showed that iron deficiency also induced the accumulation of triacylglycerides (TAG), which resulted in the formation of lipid droplets. This was most significant between 48 and 72 h of growth. Dramatic increases in DGAT2A and PDAT1 levels were caused by iron starvation, which indicated that the biosynthesis of TAG had been increased. Analysis using gas chromatography mass spectrometry showed that levels of 16:0, 18:0, 18:2 and 18:3Δ9,12,15 fatty acids were significantly elevated. The results of this study highlight the genes/enzymes of Chlamydomonas that affect lipid synthesis through their influence on photosynthesis, and these represent potential targets of metabolic engineering to develop strains for biofuel production.

Exploration of binding studies of ß-oxalyldiamino propionic acid (ß-ODAP), a non-protein amino acid with human serum albumin-biophysical and computational approach.

Communicated by Ramaswamy H. Sarma.

Human Naa50 Protein Displays Broad Substrate Specificity for Amino-terminal Acetylation: DETAILED STRUCTURAL AND BIOCHEMICAL ANALYSIS USING TETRAPEPTIDE LIBRARY.

Amino-terminal acetylation is a critical co-translational modification of the newly synthesized proteins in a eukaryotic cell carried out by six amino-terminal acetyltransferases (NATs). All NATs contain at least one catalytic subunit, and some contain one or two additional auxiliary subunits. For example, NatE is a complex of Naa10, Naa50, and Naa15 (auxiliary). In the present study, the crystal structure of human Naa50 suggested the presence of CoA and acetylated tetrapeptide (AcMMXX) that have co-purified with the protein. Biochemical and thermal stability studies on the tetrapeptide library with variations in the first and second positions confirm our results from the crystal structure that a peptide with Met-Met in the first two positions is the best substrate for this enzyme. In addition, Naa50 acetylated all MXAA peptides except for MPAA. Transcriptome analysis of 10 genes that make up six NATs in humans from eight different cell lines suggests that components of NatE are transcribed in all cell lines, whereas others are variable. Because Naa10 is reported to acetylate all amino termini that are devoid of methionine and Naa50 acetylates all other peptides that are followed by methionine, we believe that NatE complex can be a major contributor for amino-terminal acetylation at the ribosome exit tunnel.