Polypeptide Band Of Molecular Weight Research Articles

Antimicrobial and anticancer potential of low molecular weight polypeptides extracted and characterized from leaves of Azadirachta indica

Low molecular weight antimicrobial polypeptides were extracted and purified from the young fresh leaves of Azadirachta indica (neem). The total protein extracted was precipitated with 15% TCA-Acetone. The total purified proteins yielded from the two extraction methods were 122.33±2.21 and 115.09±1.88mg/g of the total fresh weight. The SDS-PAGE analysis identified the presence of eight low molecular weight polypeptide bands. The antimicrobial activity of the resolved bands was detected by Polyacrylamide gel-Agar overlay diffusion assay (PAG-ADA). Their broad-spectrum bactericidal activity was confirmed using the same technique and found three low molecular weight bands from 11 to 14kDa collectively exhibiting superior bactericidal activities against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermis, Enterococcus faecalis, Pseudomonas aeruginosa and fungicidal activity against Candida tropicalis. The FTIR spectrum of the protein bands depicted the presence of hydroxyl and carbonyl groups in the protein bands. These polypeptides were characterized by MALDI-TOF/TOF analysis. Further, the purified protein extract was found to be active against HELA, BT-549 and Neuro-2a cell lines with IC50 value of 74.03±2.31, 64.82±1.64, 238.32±2.12 and 109.94±2.96, 59.61±0.75 for 24h and 48h, respectively. The results of present study indicate that these polypeptides exhibit broad spectrum antimicrobial and anticancer activity and can therefore be explored for their therapeutic potential.

Purification and Characterization ofζ-Crystallin from the Camel Lens

ζ-crystallin a novel NADPH: quinone oxidoreductase was purified from the cortex of the camel ( Camelus dromedarius) lens to homogeneity by Sepharose CL-6B gel filtration column and 2′, 5′ ADP-Sepharose 4B affinity column chromatography in the presence of dithiothreitol. The purified ζ-crystallin has a molecular weight of 140 kDa, as determined by Superose 12 gel filtration column. SDS-PAGE showed a single polypeptide band of molecular weight 35 kDa, suggesting that the native enzyme is composed of four identical subunits. The isoelectric point of the enzyme was 7.6 on native polyacrylamide gel. The enzyme was purified 20-fold over homogenate with a specific activity of 26.0 Units/mg protein, and an overall recovery of 53%. This enzyme was NADPH specific and followed Michaelis-Menten kinetics. K m values for the reduction of 9,10-phenanthroquinone and oxidation of NADPH were 17 μM and 6.9 μM, respectively, at pH 7.8. The V max values of the enzyme for 9,10 phenanthroquinone and NADPH were 32 μmole min -1, mg -1 protein and 22.7 μmole min -1 mg -1 protein, respectively.

High molecular weight polypeptide bands specific for equine herpesvirus 4

Serum neutralisation (SN) and immunoblotting were used in attempts to distinguish between natural infections with the closely related viruses equine herpesvirus 1 (EHV-1) and equine herpesvirus 4 (EHV-4). Horse sera ( n = 323) collected in 1990 from studs with no experience of EHV-1 abortions as well as 197 sera collected in 1992 from studs with a history of EHV-1 abortions were tested by SN. The two groups differed in the proportion with measurable EHV-1 antibody, the 1992 group being significantly higher. Both groups had high proportions with EHV-4 antibody and no serum had antibody to EHV-1 alone. Pools of positive sera were prepared as probes in immunoblots. High molecular weight (> 200 kDa) polypeptide bands specific for EHV-4 were detected. No bands specific for EHV-1 only were found. The specific EHV-4 polypeptides shared some properties with gp2 of EHV-1.

Physiological and biochemical analysis of the glutamine synthetase-impaired mutants of the nitrogen-fixing cyanobacteriumNostoc muscorum

Three types of glutamine synthetase (GS)-impaired mutants (gln) ofNostoc muscorum were isolated as ethylenediamine (EDA)-resistant phenotypes and characterized with respect to heterocyst development, nitrogen fixation, ammonium metabolism, photosynthetic characteristics, and glutamine synthetase activity. The criterion for categorizing the mutants was the extent of loss of GS activity (both in transferase and biosynthetic assays) compared with wild type; it was 70% in EDA-1, 30% in EDA-2, and more than 90% in EDA-3 strains. The level of nitrogenase activity in mutant strains was proportionate to heterocyst frequency and was found refractory to ammonium and EDA repression. In EDA-resistant strains, development of heterocysts and their spacing pattern remained unaffected and did not respond to treatment of amino acid analogues, drugs, and ammoniacal compounds which otherwise either stimulated or suppressed the number and altered the spacing pattern in wild type. A biphasic pattern of ammonium uptake indicating two transport systems was observed in all the strains except that the Km values for both high- and low-affinity systems were altered in mutant strains. In vivo treatment with MSX or EDA significantly inhibited the GS activity in wild type, whereas mutant strains did not respond to these treatments and were able to liberate NH 4 + continuously into the medium without MSX treatment. During NH 4 + uptake, percentage inhibition of O2 evolution and changes in increase of fluorescence intensity were low in EDA strains compared with wild type. Assessment of GS protein with antibodies against GS and quantitative polyacrylamide gel electrophoresis (PAGE) suggested that loss in specific activity of GS per milligram of extractable protein in EDA mutants was owing to low production of GS-specific protein. SDS-PAGE of purified GS enzyme from all the strains revealed only one polypeptide band of molecular weight of about 51.28 kDa.

Biochemical changes and in‐vitro protein digestibility of the endosperm of germinating dolichos lablab

AbstractA gradual decrease was noted in total protein, soluble protein and protein fractions such as albumin, globulin, prolamine andglutelin in the endosperm of Dolichos lablab L germinated for 8 days. Prolamine showed a maximum decrease of 85% whereas only a 25% decrease was noted in the globulin fraction. Both acid and alkaline protease activities increased during germination, and the acid protease activity was consistently higher than the alkaline protease activity throughout. Electrophoretic studies showed that high molecular weight polypeptide bands disappeared with the appearance of new low molecular weight polypeptide bands in the endosperm proteins of the germinating seeds. Total phenolics and tannin contents and trypsin inhibitory activity also increased during the progressive germination. Germination increased the in‐vitro protein digestibility of the endosperm of Dolichos lablab in the presence of proteolytic enzymes like pepsin, trypsin and pancreatin. It showed a negative but non‐significant correlation with trypsin inhibitory activity, phenolics and tannin contents. Amylase activity also increased gradually up to the sixth day of germination which coincided with the increase in the reducing sugars.

Review on the significance of starch and protein to wheat kernel hardness

AbstractWheat starch contains large lenticular (A‐type) and small spherical (B‐type) granules, with some intermediate (underdeveloped A‐type), composed of two structurally different polysaccharides, amylose (20–30%) and amylopectin (70–80%), plus small amounts of lipids, nitrogen and phosphorus. Vitreous character is related to air spaces between granules, whereas hardness relates to protein‐starch bond strength and protein matrix continuity between granules. Grain hardness is controlled genetically by one or more major genes, modified by one or more minor genes. Environmental factors also exert effects. Most hardness measurements are grouped into categories based on grinding, crushing, abrasion or indentation. Recent methods, viz microscopy, NIR, single kernel cutting, tensile strength and an acoustical technique, seem to have promise.Three basic mechanisms of grain hardness have been postulated: (a) chemically induced adhesion between the protein matrix and starch granule, (b) continuity of the protein matrix, and (c) net charge on the protein. Some recent studies have revealed small amounts of protein as an integral component of the starch granule. The surface granule proteins showed relatively low molecular weight polypeptide bands (5–30 kDa), whereas the integral starch granule proteins had higher molecular weights (59–149 k Da). The 59 kDa protein is postulated to be the enzyme that synthesises amylose. The 15 kDa polypeptide is related to kernel softness, because it is strong in soft wheats, weak or faint in hard wheats, and absent in durum wheats. Reconstituted tablets made from hard wheat starch showed higher tensile strength than those made from soft wheat. Tablets reconstituted from soft wheat starch free from 15 kDa protein exhibited increased tensile strength, suggesting that the 15 kDa protein acts as a ‘non‐sticking’ agent between the starch granules and the protein matrix. Some lipids are associated with arnylose and the starch granule surface but their significance is not clear. Key words. Starch, kernel hardness measurement, hardness mechanism, starch granule proteins.

Isolation, properties and role in progesterone biosynthesis of cytosolic malic enzyme from human term placenta.

Cytosolic malic enzyme (L-malate: NADP oxidoreductase decarboxylating, EC I. I. I.40) has been isolated and purified from postmitochondrial supernatant of human term placenta by ammonium sulphate fractionation, chromatography on diethylaminoethyl- (DEAE-)cellulose, Sepharose 6B, ADP-Sepharose 4B and Ultrogel AcA-34 to apparent homogeneity as judged from polyacrylamide gel electrophoresis (PAGE). The specific activity of the purified enzyme was 24.0 mumol X min-1 X mg-1 protein, which corresponds to about 7500-fold purification. The molecular weight of the native enzyme was determined by gel filtration to be about 250,000. Sodium dodecyl sulphate- (SDS-)PAGE showed one polypeptide band of molecular weight 63,000. It appears that the native protein is a tetramer composed of subunits of identical molecular weight. The isoelectric point of the purified malic enzyme was pH 5.55. The enzyme was shown to carboxylate pyruvate in the presence of high concentrations of pyruvate and bicarbonate at about 80 per cent of the rate of the forward reaction. The optimum pH for the carboxylation reaction was pH 7.3, and that for the decarboxylation reaction varied with malate concentration. The Km values, determined at pH 7.2, for malate, NADP+, Mn2+, and Mg2+ were 81 microM, 10 microM, 2.5 microM and 0.6 mM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 4 mM, 25 microM and 20 mM, respectively. The enzyme converted malate to pyruvate (at pH 6.3) in the presence of 5 mM NAD+ at approximately 80 per cent of the maximum rate with NADP+. It exhibited oxaloacetate decarboxylase activity at about 10 per cent of the rate of oxidative decarboxylation of malate (with NADP+ as coenzyme) and pyruvate reductase activity at about 4 per cent of the rate of oxidative decarboxylation of malate with NADP+. The oxidative decarboxylation of malate was inhibited by malate at lower values of pH of incubation medium. This inhibition gradually decreased as the pH of the incubation medium increased. No inhibition was observed at pH 8.2. The addition of purified cytoplasmic malic enzyme, pyruvate, bicarbonate and NADPH generating system (consisting of NADP+, glucose 6-phosphate, glucose 6-phosphate dehydrogenase) stimulated about twofold progesterone biosynthesis by the isolated human placental mitochondria. This stimulation was abolished by arsenite and fluorocitrate. A possible role for the cytosolic malic enzyme in the regulation of progesterone biosynthesis in human placenta is discussed.

Purification and Molecular and Catalytic Properties of Phosphoribulokinase from the Cyanobacterium Chlorogloeopsis fritschii

Summary: Phosphoribulokinase (ATP:d-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) was purified from the cyanobacterium Chlorogloeopsis fritschii. The enzyme had a molecular weight of 230000, as determined by gel filtration, and a pH optimum of 8·6. Divalent cations were essential for activity, maximal activity being supported by Mg2+, while Mn2+, Ca2+ and Co2+ were less effective. AMP, ADP, phosphoenolpyruvate, aspartate and malate inhibited enzyme activity completely at 1 mm. No effects on phosphoribulokinase activity were observed with NAD, NADH, NADP or NADPH at up to 10 mm or with glyoxylate at up to 20 mm. The enzyme was activated in semi-purified extracts by the addition of dithiothreitol and reduced glutathione. SDS-PAGE of SDS-dissociated enzyme revealed only one polypeptide band of molecular weight 40000. This suggests that C. fritschii phosphoribulokinase is a hexamer consisting of six subunits of identical size.

Isolation of the cytoskeleton from Giardia. Tubulin and a low-molecular-weight protein associated with microribbon structures.

The sucking disk of Giardia is supported by a large, plate-like organelle: the ventral disk cytoskeleton. Extraction by Triton-X 100 of Giardia trophozoites from the mouse gut, or of G. duodenalis or G. lamblia grown from cultures, yields cell-free disk cytoskeletons. Up to 8 flagellar axonemes may be attached to an isolated disk. Disks are seen in the electron microscope to be composed of concentrically coiled microtubules bonded to microribbons. Microribbons are large, laminated structures, linked by dense networks of crossbridges. They are made up of regularly arranged subunits. Microtubules and microribbons are preserved in Triton for long periods, but crossbridges are slowly dissolved. Whereas the addition of ATP causes axonemes to resume bending, active movements were not detected in disks. It seems more likely that a disk is a passive effector, which may be acted upon by other contractile structures of the cytoplasm. It is highly specialized and quintessential cytoskeleton. Disks and axonemes will dissolve in sodium dodecyl sulphate (SDS), and after SDS-gel electrophoresis 2 prominent bands are apparent. One, corresponding to tubulin, migrates in low ionic strength, high pH buffers as 2 closely spaced bands of equal staining density. The other, a smaller protein, is a complex polypeptide band of molecular weight 30 000 Daltons. Allowing for staining differences, the 2 proteins are probably present in cytoskeletons in roughly equal amounts. Because of their size microribbons account for at least 75% of the structured material stained by electron stains in pellets of cytoskeletons. Disk and axoneme microtubules comprise the minority fraction. This result suggests that, like microtubules, microribbons are a source of structural tubulin and probably also contain the 30 000 mol. wt protein.

Purification and some properties of extramitochondrial malic enzyme from rat skeletal muscle

Extramitochondrial malic enzyme ( l-malate:NADP + oxidoreductase (oxalo-acetate-decarboxylating), EC 1.1.1.40) has been isolated from postmitochondrial supernatant of rat skeletal muscle, by (NH 4) 2SO 4 fractionation, chromatography on DEAE-cellulose, Sepharose 6B, ADP-Sepharose and Ultrogel AcA-34 to apparent homogeneity as judged from polyacrylamide gel electrophoresis. Specific activity of purified enzyme was 20 μmol · min −1 per mg protein, which corresponds to about 3000-fold purification. The molecular weight of the native enzyme was determined by gel filtration to be 264 000. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis showed one polypeptide band of molecular weight 63 000. Thus, it appears that the native protein is a tetramer composed of identical molecular weight subunits. The isoelectric point of the isolated enzyme was at pH 6.15. The enzyme was shown to carboxylate pyruvate in the presence of high concentrations of bicarbonate and pyruvate at about 80% of the rate of the forward reaction. The K m values, determined at pH 7.2 for malate and NADP, were 0.125 mM and 11 μM, respectively. The K m values for pyruvate, NADPH and bicarbonate were 4.0 mM, 6.6 μM and 24 mM, respectively. The optimum pH for carboxylation reaction was at pH 7.1. The optimum pH for decarboxylation reaction varied with the malate concentration. The purified malic enzyme catalyzed the decarboxylation of oxaloacetate at pH 4.5. In a system consisting of isolated rat skeletal muscle mitochondria, pyruvate, bicarbonate and NADPH, cytoplasmic malic enzyme is able to replace added malate in stimulating oxidation of acetyl-CoA formed by oxidative decarboxylation of pyruvate. It is suggested that extramitochondrial malic enzyme might be one of the enzymes involved in the anaplerotic supply of Krebs cycle intermediates in skeletal muscle.

Structural chemistry of outer dense fibers of rat sperm.

Structural chemistry of outer dense fibers of rat sperm.