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1.
Kalata B1 is a plant protein with remarkable thermal, chemical and enzymatic stability. Its potential applications could be centered on the possibility of using its cyclic structure and cystine knot motif as a scaffold for the design of stable pharmaceuticals. To discover potent dengue NS2B–NS3 protease inhibitors, we have prepared various kalata B1 analogues by varying the amino acid sequence. Mass spectrometric and biochemical investigations of these analogues revealed a cyclopeptide whose two fully oxidized forms are substrate-competitive inhibitors of the dengue viral NS2B–NS3 protease. Both oxidized forms showed potent inhibition with Ki of 1.39 ± 0.35 and 3.03 ± 0.75 μM, respectively. 相似文献
2.
Adam J. Davenport Christopher C. Stimson Massimo Corsi Darshan Vaidya Edward Glenn Timothy D. Jones Sarah Bailey Mark J. Gemkow Ulrike Fritz David J. Hallett 《Bioorganic & medicinal chemistry letters》2010,20(17):5165-5169
A series of potent and subtype selective H3 receptor antagonists containing a novel tetrazole core and diamine motif is reported. A one-pot multi-component Ugi reaction was utilised to rapidly develop the structure–activity relationships (SAR) of these compounds. Optimisation for liver microsome stability (t1/2 >60 min), minimal CYP inhibition (IC50 >50 μM) and high cell permeability (Caco-2 Papp >20 × 10?6 cm/s) identified several compounds with drug-like properties. 相似文献
3.
《Process Biochemistry》2010,45(6):892-896
A novel β-glucosidase capable of hydrolyzing indican to indigo was mined and isolated from Sinorhizobium meliloti using a systematic approach. The corresponding gene was amplified by PCR and overexpressed in the soluble fraction as an MBP fusion protein. The resulting enzyme easily purified to apparent homogeneity via a consecutive step in the affinity column. The recombinant enzyme was determined to be a monomer with a calculated molecular mass of 52 kDa and showed the maximum activity for indican at pH 7.0 and 45 °C. The kinetic parameters for indican, KM and Vmax, were determined to be 0.97 mM and 355.6 μM/min/mg protein, respectively, at pH 7.0 and 35 °C. Additionally, this enzyme hydrolyzed both the β-(1-4)- and β-(1-6)-glucosidic bonds and revealed a minor activity against α-d-glucosides. Furthermore, the enzyme was severely inhibited by DTT, indicating a possibility that the oxidation of amino acids could play a crucial role in the activity of the enzyme. 相似文献
4.
《Bioorganic & medicinal chemistry》2016,24(16):3618-3623
In vivo imaging of β-amyloid (Aβ) aggregates consisting of Aβ(1–40) and Aβ(1–42) peptides by positron emission tomography (PET) contributes to the diagnosis and therapy for Alzheimer’s disease (AD). Because 64Cu (t1/2 = 12.7 h) is a radionuclide for PET with a longer physical half-life than 11C (t1/2 = 20 min) and 18F (t1/2 = 110 min), it is an attractive radionuclide for the development of Aβ imaging probes that are suitable for routine use. In the present study, we designed and synthesized two novel 64Cu labeled benzofuran derivatives and evaluated their utility as PET imaging probes for Aβ aggregates. In an in vitro binding assay, 6 and 8 showed binding affinity for Aβ(1–42) aggregates with a Ki value of 33 and 243 nM, respectively. In addition, these probes bound to Aβ plaques deposited in the brain of an AD model mouse in vitro. In a biodistribution experiment using normal mice, these probes showed low brain uptake (0.33% and 0.36% ID/g) at 2 min post-injection. Although refinement to enhance brain uptake is needed, [64Cu]6 and [64Cu]8 demonstrated the feasibility of developing novel PET probes for imaging Aβ aggregates. 相似文献
5.
Yuan Zhang Guiling Zheng Jianxin Tan Changyou Li Linyou Cheng 《Microbiological research》2013,168(8):512-517
Isolation of Bacillus thuringiensis (Bt) strain or its cry gene encoding insecticidal crystal protein (ICP) with specific toxicity is of great importance to biological control of insect pests. In this study, by screening 66 strains of Bt isolated from soil samples collected in Shandong Province, China, a new cry8-type gene from Bt strain B-JJX was identified via PCR-RFLP method. This novel gene, cry8Ab1, was cloned from the Bt strain B-JJX and expressed in an acrystalliferous mutant strain HD-73?. The open reading frame of the cry8Ab1 gene consists of 3543 bp with a G + C content of 37.99% and encodes a protein of 1180 amino acids with a putative MW of 133.3 kDa which was confirmed by SDS-PAGE analysis. The Cry8Ab1 protein was expressed and released as spherical parasporal crystals from Bt acrystalliferous mutant strain HD-73? along with the presence of spores. In bioassays, this protein was toxic to 3-day-old larvae of the scarabaeid pests, Holotrichia oblita and H. parallela, with an LC50 of 5.72 and 2.00 μg toxin g?1 soil, respectively. The results are in accordance with the insecticidal activities of the original Bt strain B-JJX, which had an LC50 of 1.72 and 0.96 μg toxin g?1 soil against H. oblita and H. parallela, respectively. 相似文献
6.
《Animal Feed Science and Technology》1999,76(3-4):265-274
A study was conducted to determine the effects of moist heat treatment (127°C, 117 kPa steam pressure) for 10 min on protein fractions and in-vitro crude protein (CP) degradability of mustard meal. Rumen undegraded protein (RUP) and amino acid disappearance of unheated, and heated, mustard meal were measured following 12 h of rumen incubation using two ruminally fistulated cows. Intestinal availability of RUP was estimated using an enzymatic (pepsin–pancreatin) procedure. Heat treatment reduced (p<0.05) protein solubility and increased (p<0.05) neutral detergent insoluble CP without affecting acid detergent insoluble CP of mustard meal. Relative to the control, heated mustard meal had a lower (p<0.05) effective in-vitro CP degradability (445.2 vs. 746.8 g kg−1 of CP) and a higher (p<0.05) ruminal escape CP (615.1 vs. 120.2 g kg−1 of CP) value. Amino acid composition was not affected by heat treatment except for the concentration of arginine and lysine which was lower (p<0.05) in heated than in unheated mustard meal. Disappearance of all amino acids following 12 h of rumen incubation was lower (p<0.05) in unheated than in heated mustard meal. Heat treatment increased (p<0.05) the amount of protein available for digestion in the small intestine from 75.7 to 518.1 g kg−1 of CP. It was concluded that moist heating of mustard meal for 10 min will reduce ruminal CP and amino acid degradability without compromising the intestinal availability of ruminal undegraded protein. 相似文献
7.
Hye-Nam Son Ju-Ock Nam Soyoun Kim In-San Kim 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2013,1833(10):2378-2388
TGFBI, a transforming growth factor β-induced extracellular matrix protein, circulates at a level of ~ 300 ng/ml in humans and modulates several integrin-mediated cellular functions. The protein contains an N-terminal EMI domain, four consecutive FAS1 domains, and the RGD motif. Each FAS1 domain and the RGD motif have been known to interact with avb3 integrin. Here, we found that the binding affinity (Kd) of TGFBI for αvβ3 integrin was approximately 3.8 × 10? 8 M, a value ~ 2300-fold higher than that of a single FAS1 domain, and demonstrated that this greater affinity was due to the cooperative action of the four FAS1 domains and the RGD motif. Moreover, TGFBI exhibited more potent anti-angiogenic and anti-tumorigenic activities, even at a 100-fold lower molar dose than the reported effective dose of the FAS1 domain. Finally, our data showed that TGFBI specifically targeted the tumor vasculature and accumulated at the tumor site. Collectively, our results support the theory that TGFBI acts as a potent endogenous anti-tumor and anti-angiogenic molecule by targeting αvβ3 integrin, and highlights the importance of physiological circulating TGFBI levels in inhibiting tumor growth. 相似文献
8.
A gene encoding acidic, thermostable and raw starch hydrolysing α-amylase was cloned from an extreme thermophile Geobacillus thermoleovorans and expressed. The ORF of 1650 bp encodes a 515 amino acid protein (Gt-amy) with a signal peptide of 34 amino acids at the N-terminus. Seven conserved sequences of GH-13 family have been found in its sequence. The specific enzyme activity of recombinant Gt-amy is 1723 U mg−1 protein with a molecular mass of 59 kDa. It is optimally active at pH 5.0 and 80 °C with t1/2 values of 283, 184 and 56 min at 70, 80 and 90 °C, respectively. The activation energy required for its temperature deactivation is 84.96 kJ mol−1. Ca2+ strongly inhibits Gt-amy at 10 mM concentration, and inhibition kinetics with Ca2+ reveals that inhibition occurs as a result of binding to a lower affinity secondary Ca2+ binding site in the active centre in a mixed-type inhibition manner. The Km and kcat of the Gt-amy are 0.315 mg mL−1 and 2.62 × 103 s−1, respectively. Gt-amy is Ca2+-independent at the concentration used in industrial starch saccharification, and hydrolyses raw corn and wheat starches efficiently, and thus, is applicable in starch saccharification at the industrial sub-gelatinization temperatures. 相似文献
9.
Spider silks have great potential as biomaterials with extraordinary properties. Here, we report the cloning and characterization of the major ampullate silk protein gene from the spider Araneus ventricosus. A cDNA encoding the partial major ampullate silk protein (AvMaSp) was cloned from A. ventricosus. An analysis of the cDNA sequence shows that AvMaSp consists of a 240 amino acid repetitive region and a 99 amino acid C-terminal non-repetitive domain. The peptide motifs that were found in the spider major ampullate silk proteins, (A)n, (GA)n, and (GGX)n, were conserved in the repetitive region of AvMaSp. Phylogenetic analysis further confirmed that AvMaSp belongs to the spider major ampullate spidroin family of proteins. The AvMaSp-R cDNA, which encodes the 240 amino acid repetitive domain, was expressed as a soluble 22 kDa polypeptide in baculovirus-infected insect cells. Recombinant AvMaSp-R was degraded abruptly by trypsin. However, AvMaSp-R was stable at 100 °C for at least 30 min. Additionally, the AvMaSp-R was stable at pH values from 2 to 12 for at least 1 h. Taken together, our findings describe the molecular structure and biochemical properties of the A. ventricosus major ampullate silk protein and demonstrate its potential as a biomaterial. 相似文献
10.
Erika Cione Attilio Pingitore Francesco Genchi Giuseppe Genchi 《The international journal of biochemistry & cell biology》2010,42(1):106-112
The adenine nucleotide translocator (ANT) accomplishes the exchange of ATP from the mitochondrial matrix with cytoplasmic ADP. While investigating the biochemical mechanism of retinoic acid (RA) on the ANT via retinoylation, we have found and subsequently demonstrated a positive influence of Coenzyme A (CoA) on the transport of ATP across the membranes of rat liver mitochondria. CoA enhances ANT activity in a dose-dependent manner modifying the Vmax (673.3 ± 20.7 nmol ATP/mg protein/min versus 155.0 ± 1.9 nmol ATP/mg protein/min), the IC50 for the specific inhibitor carboxyatractyloside (CATR) (0.142 ± 0.012 μM versus 0.198 ± 0.011 μM) but not the Km (22.50 ± 0.52 μM versus 22.19 ± 0.98 μM). Data suggest a likely enzymatic involvement in the interaction between ANT and CoA. The effect of CoA is observed in mitochondria from several different tissues. 相似文献
11.
S.V.N. Vijayendra Yutaka KASHIWAGI 《International journal of biological macromolecules》2009,44(1):92-97
A new acid stable exo-β-1,3-glucanase of Rhizoctonia solani purified from a commercial source ‘Kitarase-M’, by a combination of ammonium sulfate precipitation, ion-exchange and gel filtration methods, had specific activity of 0.26 U/mg protein, Km and Vmax values of 0.78 mg/ml and 0.27 mM/min/mg protein, respectively. It had molecular weight of 62 kDa with optimum activity at 40 °C temperature and pH 5.0, with high stability at pH of 3–7. Unique amino acid sequence was found at N-terminal end. The substrate specificity studies confirmed that it is an exo-β-1,3-glucanase. It could hydrolyze curdlan powder to release glucose. 相似文献
12.
Stability of phycocyanin extracted from Spirulina sp.: Influence of temperature,pH and preservatives
Temperature and pH play an important role in the stability of phycocyanin, a natural blue colorant. Systematic investigations showed the maximum stability of phycocyanin was in the pH range 5.5–6.0. Incubation at temperatures between 47 and 64 °C caused the concentration (CR) and half-life of phycocyanin in solution to decrease rapidly. The CR value remained at approximately 50% after incubating for 30 min at 59 °C. After heating at 60 °C for 15 min, the CR value of phycocyanin at pH 7.0 was maintained at around 62–70% when 20–40% glucose or sucrose was added, and the half-life increased from 19 min to 30–44 min. 2.5% sodium chloride was found to be an effective preservative for phycocyanin at pH 7.0 as a CR value of 76% was maintained and the half-life of 67 min was increased. 相似文献
13.
《Process Biochemistry》2014,49(3):490-495
Plant transient expression provides a rapid production platform for recombinant proteins but is linked with low protein yields. To test if plant-specific hydroxyproline (Hyp)-O-glycosylated peptide tags attached to a target protein can improve overall yields of recombinant protein transiently expressed in Nicotiana benthamiana, enhanced green fluorescence protein (EGFP) was expressed as a fusion with 5 or 32 tandem repeats of a serine–proline motif, designated (SP)5 or (SP)32, which is known to direct extensive Hyp-O-glycosylation in plants. EGFP containing the (SP)n motif showed enhanced yields in the order as follows: EGFP < EGFP-(SP)5 ≪ (SP)5-EGFP < (SP)32-EGFP. The EGFP equivalent yield of (SP)32-EGFP was up to 16-fold greater than that of the EGFP control. In addition, both fully glycosylated (SP)32-EGFP (∼115 kDa) and partially glycosylated (SP)32-EGFP (∼40 kDa) were detected in protein extracts of N. benthamiana. These two types of glycoforms were completely segregated between media and cells in tobacco BY-2 cell cultures. 相似文献
14.
Nitrilases have long been considered as an attractive alternative to chemical catalyst in carboxylic acids biosynthesis due to their green characteristics and the catalytic potential in nitrile hydrolysis. A novel nitrilase from Pseudomonas putida CGMCC3830 was purified to homogeneity. pI value was estimated to be 5.2 through two-dimensional electrophoresis. The amino acid sequence of NH2 terminus was determined. Nitrilase gene was cloned through CODEHOP PCR, Degenerate PCR and TAIL-PCR. The open reading frame consisted of 1113 bp encoding a protein of 370 amino acids. The predicted amino acid sequence showed the highest identity (61.6%) to nitrilase from Rhodococcus rhodochrous J1. The enzyme was highly specific toward aromatic nitriles such as 3-cyanopyridine, 4-cyanopyridine, and 2-chloro-4-cyanopyridine. It was classified as aromatic nitrilase. The nitrilase activity could reach up to 71.8 U/mg with 3-cyanopyridine as substrate, which was a prominent level among identified cyanopyridine converting enzymes. The kinetic parameters Km and Vmax for 3-cyanopyridine were 27.9 mM and 84.0 U/mg, respectively. These data would warrant it as a novel and potential candidate for creating effective nitrilases in catalytic applications of carboxylic acids synthesis through further protein engineering. 相似文献
15.
《Process Biochemistry》2010,45(5):757-764
Aminopeptidase from a solvent tolerant strain Pseudomonas aeruginosa PseA was purified and studied for its biochemical and molecular characteristics. Ion-exchange chromatography resulted in 11.9-fold purification and 38% recovery of the 56 kDa enzyme. The enzyme was found to be stable over a pH range of 6.0–8.0 and appreciably thermostable up to 70 °C. PseA aminopeptidase exhibited Km of 3.02 mM and Vmax of 6.71 μmol/mg/min towards l-Leu-p-nitroanilide. Remarkable stability in both hydrophilic and hydrophobic solvents makes PseA aminopeptidase unique. Partial N-terminal sequence of enzyme showed exact match with probable aminopeptidase of P. aeruginosa PAO1, coded by gene pepB. Polymerase chain reaction amplified the 1611-bp open reading frame encoding a 57.51 kDa, 536 amino acid PseA PepB polypeptide. The deduced PseA PepB protein sequence contained a 24-residue signal peptide (2.57 kDa) followed by a 1.28 kDa propeptide and a mature product of 500 residues. Search for conserved domain in PseA aminopeptidase explored its place in zinc-metallopeptidase family. Primary sequence analysis showed the hydrophobic inclination of the protein; and the 3D structure modeling elucidated the presence of a high content of hydrophobic residues on its surface probably imparting solvent stability to it. The enzyme might find potential applications in non-aqueous enzymology due to its marked thermostability and striking solvent stability. 相似文献
16.
P. Dilkin G. Direito M.M.S. Simas C.A. Mallmann B. Corrêa 《Chemico-biological interactions》2010,185(3):157-162
Toxicokinetics and the toxicological effects of culture material containing fumonisin B1 (FB1) were studied in male weaned piglets by clinical, pathological, biochemical and sphingolipid analyses. The animals received a single oral dose of 5 mg FB1/kg of body weight, obtained from Fusarium verticillioides culture material. FB1 was detected by HPLC in plasma collected at 1-h intervals up to 6 h and at 12-h intervals up to 96 h. FB1 eliminated in feces and urine was quantified over a 96-h period and in liver samples collected 96 h post-intoxication. Blood samples were obtained at the beginning and end of the experiment to determine serum enzyme activity, total bilirubin, cholesterol, sphinganine (Sa), sphingosine (So) and the Sa/So ratio. FB1 was detected in plasma between 30 min and 36 h after administration. The highest concentration of FB1 was observed after 2 h, with a mean concentration of 282 μg/ml. Only 0.93% of the total FB1 was detected in urine between 75 min and 41 h after administration, the highest mean concentration (561 μg/ml) was observed during the interval after 8 at 24 h. Approximately 76.5% of FB1 was detected in feces eliminated between 8 and 84 h after administration, with the highest levels observed between 8 and 24 h. Considering the biochemical parameters, a significant increase only occurred in cholesterol, alkaline phosphatase and aspartate aminotransferase activities. In plasma and urine, the highest Sa and Sa/So ratios were obtained at 12 and 48 h, respectively. 相似文献
17.
A series of hydroxy substituted amino chalcone compounds have been synthesized. These compounds were then evaluated for their inhibitory activities on tyrosinase and melanogenesis in murine B16F10 melanoma cell lines. The structures of the compounds synthesized were confirmed by 1H NMR, 13C NMR, FTIR and HRMS. Two novel amino chalcone compounds exhibited higher tyrosinase inhibitory activities (IC50 values of 9.75 μM and 7.82 μM respectively) than the control kojic acid (IC50: 22.83 μM). Kinetic studies revealed them to act as competitive tyrosinase inhibitors with their Ki values of 4.82 μM and 1.89 μM respectively. Both the compounds inhibited melanin production and tyrosinase activity in B16 cells. Docking results confirm that the active inhibitors strongly interact with mushroom tyrosinase residues. This study suggests that the depigmenting effect of novel amino chalcone compounds might be attributable to inhibition of tyrosinase activity, suggesting amino chalcones to be a promising candidate for use as depigmentation agents or as anti-browning food additives. 相似文献
18.
Farsenyl pyrophosphate synthase is a potential molecular drug target of risedronate in Babesia bovis
Akio Ueno Mohamad Alaa Terkawi Miki Yokoyama Shinuo Cao Gabriel Aboge Mahmoud Aboulaila Yoshifumi Nishikawa Xuenan Xuan Naoaki Yokoyama Ikuo Igarashi 《Parasitology international》2013,62(2):189-192
A cDNA encoding farnesyl pyrophosphate synthase of Babesia bovis (BbFPPS) has been isolated, cloned and characterized as molecular drug target. Sequence analysis revealed that BbFPPS contains an open reading frame of 1011 bp with predicted 336 amino acids and molecular mass of 38 kDa. Antiserum raised in mice against recombinant BbFPPS expressed in Escherichia coli specifically reacted with native protein of B. bovis parasites by Western blot analysis and indirect immunofluorescent test. Enzymatic assay using recombinant BbFPPS revealed that the Km value of the enzyme for isopentenyl pyrophosphate and dimethylallyl pyrophosphate was 2.494 ± 1.536 μM. Risedronate inhibited the activity of BbFPPS yielding IC50 value of 8.4 ± 1.2 nM. Furthermore, the in vitro growth of B. bovis was significantly inhibited in the presence of a micromolar concentration of risedronate (IC50 = 4.02 ± 0.91 μM). No regrowth of B. bovis was observed at 10 μM of risedronate in the subsequent viability test. These results demonstrate that BbFPPS is the molecular target of risedronate, which could inhibit the in vitro growth of B. bovis. 相似文献
19.
Haofan Wang Youngjoo Byun Cyril Barinka Mrudula Pullambhatla Hyo-eun C. Bhang James J. Fox Jacek Lubkowski Ronnie C. Mease Martin G. Pomper 《Bioorganic & medicinal chemistry letters》2010,20(1):392-397
We report a strategy based on bioisosterism to improve the physicochemical properties of existing hydrophilic, urea-based GCPII inhibitors. Comprehensive structure–activity relationship studies of the P1′ site of ZJ-43- and DCIBzL-based compounds identified several glutamate-free inhibitors with Ki values below 20 nM. Among them, compound 32d (Ki = 11 nM) exhibited selective uptake in GCPII-expressing tumors by SPECT-CT imaging in mice. A novel conformational change of amino acids in the S1′ pharmacophore pocket was observed in the X-ray crystal structure of GCPII complexed with 32d. 相似文献
20.
《Small Ruminant Research》2009,82(2-3):174-177
The effect of orally administered l-carnitine on biochemical parameters was examined in lactating Tuj-ewes. Ewes were orally given 500 mg of l-carnitine daily for 3 weeks. To evaluate the changes on selected blood indicators (total protein, albumin, glucose, triglyceride, cholesterol, urea, aspartate amino transferase, alanine amino transferase, lipase, triiodothyronine and thyroxine), blood samples were collected at the beginning of the study, and at the end of 1st, 2nd and 3rd week of study. Oral administration of supplemental carnitine significantly decreased serum triglyceride (P < 0.05), glucose (P < 0.05), cholesterol (P < 0.05) and triiodothyronine (P < 0.05) concentrations. In addition, serum thyroxine (P < 0.001) and albumin (P < 0.01) concentrations were significantly elevated as a result of oral carnitine treatment. These results suggest that supplemental l-carnitine improves selected biochemical indicators in Tuj-ewes. 相似文献