Isozymes of α-galactosidase from Bacillus stearothermophilus

1980 ◽  
Vol 26 (8) ◽  
pp. 978-984 ◽  
Author(s):  
Dennis M. Pederson ◽  
Richard E. Goodman
Keyword(s):  
Gel Electrophoresis ◽  
Half Life ◽  
Protein Concentration ◽  
Mixed Type ◽  
Bacillus Stearothermophilus ◽  
Single Band ◽  
Molecular Forms ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Low Protein

Two molecular forms of α-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. α-Galactosidase I (with the substrate p-nitrophenyl α-D-galactopyranoside (PNPG)) has a pH optimum of 6 and a half-life at 65 °C of > 2 h at low protein concentration. α-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 °C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the β-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are α-galactosidase I, 280 000 ± 30 000 and α-galactosidase II, 325 000 ± 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 + 500 for α-galactosidase I and 84 000 ± 500 for α-galactosidase II, suggest that both enzymes consist of four subunits.

β-Galactosidase from Bacillus stearothermophilus

1976 ◽  
Vol 22 (6) ◽  
pp. 817-825 ◽  
Author(s):  
Richard E. Goodman ◽  
Dennis M. Pederson
Keyword(s):  
Activation Energy ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Bacillus Stearothermophilus ◽  
Thermal Inactivation ◽  
Ph Dependence ◽  
Temperature Sensitive ◽  
Optimum Temperature ◽  
Arrhenius Activation Energy ◽  
Ph Optimum

Several strains of thermophilic aerobic spore-forming bacilli synthesize β-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The β-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 °C and the Arrhenius activation energy is about 24 kcal/mol below 47 °C and 16 kcal/mol above that temperature. At 55 °C the Km is 0.11 M for lactose and 9.8 × 10−3 M for o-nitrophenyl-β-D-galactopyranoside. The enzyme is strongly product-inhibited by galactose (Ki = 2.5 × 10−3 M). It is relatively stable at 50 °C, losing only half of its activity after 20 days at this temperature. At 60 °C more than 60% of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein, and in the presence of 4 mg/ml of bovine serum albumin the enzyme is only 18% inactivated in 10 min at 60 °C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000.

Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. Purification to homogeneity and some properties

1980 ◽  
Vol 58 (1) ◽  
pp. 40-48 ◽  
Author(s):  
E. B. Waygood ◽  
T. Steeves
Keyword(s):  
Escherichia Coli ◽  
Protein Concentration ◽  
Phosphotransferase System ◽  
Temperature Dependent ◽  
Extra Copy ◽  
Ph Optimum ◽  
Low Protein ◽  
Kinetic Activity ◽  
Ping Pong ◽  
Enzyme I

Enzyme I of the phosphoenolpyruvate – sugar phosphotransferase system (PTS) has been purified to homogeneity from Escherichia coli. A merodiploid strain P650 which had an extra copy of the gene for enzyme I resulting in a twofold increase in the amount of activity was used. The enzyme is a dimer of 67 000 ± 5000 molecular weight subunits. At low protein concentration and 4 °C the monomer predominates, while at room temperature the dimer predominates. At higher protein concentrations (2 to 10 mg) this reversible temperature-dependent association-dissociation is not found. Enzyme I has a pH optimum of pH 7.2, a Km for HPr of 9 ± 3 μM, a Km for phosphoenolpyruvate of 0.18 ± 0.04 mM, and kinetics that are consistent with a bi bi Ping-Pong mechanism. No allosteric regulation of kinetic activity has been found. The amino acid composition has been determined and the ε1%280 nm is 4.4. Evidence suggests that the phosphorylated form of enzyme I is more stable.

Investigation by HPLC of the Catabolism of Recombinant Tissue Plasminogen Activator in the Rat

1988 ◽  
Vol 60 (01) ◽  
pp. 107-112 ◽  
Author(s):  
Roy Harris ◽  
Louis Garcia Frade ◽  
Lesley J Creighton ◽  
Paul S Gascoine ◽  
Maher M Alexandroni ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Half Life ◽  
Recombinant Tissue Plasminogen Activator ◽  
Rat Plasma ◽  
High Molecular Weight Complex ◽  
Molecular Weights ◽  
Major Activity ◽  
Tissue Plasminogen

SummaryThe catabolism of recombinant tissue plasminogen activator (rt-PA) was investigated after injection of radiolabelled material into rats. Both Iodogen and Chloramine T iodination procedures yielded similar biological activity loss in the resultant labelled rt-PA and had half lives in the rat circulation of 1 and 3 min respectively. Complex formation of rt-PA was investigated by HPLC gel exclusion (TSK G3000 SW) fractionation of rat plasma samples taken 1-2 min after 125I-rt-PA injection. A series of radiolabelled complexes of varying molecular weights were found. However, 60% of the counts were associated with a single large molecular weight complex (350–500 kDa) which was undetectable by immunologically based assays (ELISA and BIA) and showed only low activity with a functional promoter-type t-PA assay. Two major activity peaks in the HPLC fractions were associated with Tree t-PA and a complex having a molecular weight of ̴ 180 kDa. HPLC fractionation to produce these three peaks at various timed intervals after injection of 125I-rt-PA showed each to have a similar initial rate half life in the rat circulation of 4-5 min. The function of these complexes as yet is unclear but since a high proportion of rt-PA is associated with a high molecular weight complex with a short half life in the rat, we suggest that the formation of this complex may be a mechanism by which t-PA activity is initially regulated and finally cleared from the rat circulation.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Does Low Protein Concentration of Tissue-type Plasminogen Activator Predict a Low Risk of Spontaneous Deep Vein Thrombosis?

1995 ◽  
Vol 74 (02) ◽  
pp. 718-721 ◽  
Author(s):  
Jørgen Gram ◽  
Johannes Sidelmann ◽  
Jørgen Jespersen
Keyword(s):  
Deep Vein Thrombosis ◽  
Confidence Interval ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Protein Concentration ◽  
Tissue Type ◽  
Vein Thrombosis ◽  
Low Protein ◽  
Deep Vein ◽  
Pai 1

SummaryMany reports have demonstrated an abnormal fibrinolysis in a subset of patients with deep vein thrombosis. We have studied systemic global fibrinolytic activity and protein concentrations of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in plasma of 25 young patients with a previous instance of spontaneous deep vein thrombosis documented by phlebography and in 50 healthy controls. The two populations were comparable with respect to a number of base-line variables (age, height, weight, etc.), while the patients had significantly lower fibrinolytic activity (p <0.02), and significantly higher protein concentrations of t-PA (p <0.0001) and PAI-1 (p <0.0006).We used probit scale plots to identify the consequence of different cut-off points to separate patients from controls. Reasonable separation could be obtained for t-PA with a cut-off point of 5.2 ng/ml and for PAI-1 18 ng/ml. The sensitivity and specificity for these cut-off points were for t-PA 73% (95% confidence interval 63%-84%) and for PAI-1 67% (confidence interval 55%-77%). The negative predictive value with a cut-off point t-PA concentration of 5.2 ng/ml was 85% (95% confidence interval 70%-94%). We observed a significantly negative association between concentration of t-PA and fibrinolytic activity (rs = -0.47; p <0.005) and also between PAI-1 and fibrinolytic activity (rs = -0.78; p <0.005).We conclude that a young healthy population is characterized by low protein concentration of t-PA (and PAI-1) compared with young patients with a previous instance of spontaneous vein thrombosis, and we tentatively state that a low protein concentration of t-PA predicts a low risk of spontaneous deep vein thrombosis.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

scholarly journals Quality Attributes of Ultra-High Temperature-Treated Model Beverages Prepared with Faba Bean Protein Concentrates

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1244
Author(s):  
Malik Adil Nawaz ◽  
Tanoj Kumar Singh ◽  
Regine Stockmann ◽  
Hema Jegasothy ◽  
Roman Buckow
Keyword(s):  
Physicochemical Properties ◽  
Faba Bean ◽  
Protein Concentration ◽  
High Concentration ◽  
Molecular Weights ◽  
Oil In Water ◽  
Sds Page ◽  
Lower Protein ◽  
7S Globulin ◽  
Bean Protein

The objective of this research was to develop a model faba bean drink with a high concentration of protein (>4% w/w). The protein molecular weights and frequency for both faba and soy were assessed using SDS-PAGE. Results showed similarities in the protein molecular weight of both faba and soy (mainly 11S globulin ~Glycinin and 7S globulin ~β-conglycinin). Thus, faba can be considered as a potential soy replica in plant-based milk beverages. Oil-in-water emulsions (5–8% w/w available protein) were prepared using faba bean protein concentrate (FPC), 1% sunflower oil, and 0.2% sunflower lecithin. These emulsions were used as model beverages and were further investigated for UHT processibility, stability, and physicochemical properties. The physicochemical properties of emulsions at various processing stages viz., coarse emulsification, homogenisation, and UHT, were measured. An increase in the protein concentration and thermal treatment resulted in an increased oil droplet size, coalescence and flocculation, and protein aggregation. Lower protein concentrations viz., 5–6%, showed greater negative ζ-potential, and thereby, high dispersibility through enhanced electrostatic repulsions than those of higher concentrations (7–8%). Furthermore, an increase in protein concentration and UHT treatment resulted in an increased creaming index. In total, 21 different volatile compounds were detected and quantified, representing different chemical classes, namely alcohols, aldehydes, ketones, esters, furan, and acids. These volatiles have major consequences for the overall flavour chemistry of the model beverage product. Overall, this study showed the potential for application of faba bean as a protein source in UHT-treated legume-based beverages and identified areas for further development.

STARCH GEL ELECTROPHORESIS OF MYOGEN FROM CHICKEN BREAST MUSCLE IN CONSTANT AND GRADIENT BUFFER SYSTEMS

1963 ◽  
Vol 41 (1) ◽  
pp. 369-387 ◽  
Author(s):  
J. M. Neelin
Keyword(s):  
Gel Electrophoresis ◽  
Protein Concentration ◽  
Breast Muscle ◽  
Chicken Breast ◽  
Starch Gel Electrophoresis ◽  
Two Dimensional ◽  
Sodium Cacodylate ◽  
Gradient Systems ◽  
Optimal Separation ◽  
Starch Gel

By varying conditions of starch gel electrophoresis, factors contributing to the resolution of myogen proteins from chicken breast muscle have been studied. Variables examined included composition of the myogen extractant, protein concentration, ionic strength of electrophoretic media, pH of gel media, plane and direction of electrophoresis, and the nature of cations and anions in gel media and bridge solutions. The significance of anions was more closely studied with constant buffer systems, and gradient systems in which bridge electrolyte differed from, and gradually altered, the gel medium. Optimal separation was obtained in gradient systems with 0.10 M sodium chloride bridge solutions, and gel media of sodium cacodylate, pH 6.9, μ 0.010, which resolved 12 cationic zones, and sodium veronal, pH 7.4, μ 0.010, which resolved 10 anionic zones. These buffers in two-dimensional sequence revealed a total of about 24 components in this myogen.

The effect on the reproductive performance of sows of dietary protein concentration and pattern of feeding in pregnancy

1969 ◽  
Vol 73 (2) ◽  
pp. 301-309 ◽  
Author(s):  
I. H. Pike ◽  
T. G. Boaz
Keyword(s):  
Reproductive Performance ◽  
Crude Protein ◽  
Protein Concentration ◽  
Fish Meal ◽  
Live Weight ◽  
Lactation Performance ◽  
Large White ◽  
Low Protein ◽  
Lower Capacity ◽  
In Pregnancy

SUMMARYIn a factorial experiment the effect of two protein intakes and three patterns of feeding in the second pregnancy of 48 Large White x Wessex Saddleback sows was examined. The high protein (HP) diet (19·5% crude protein) contained 15% white fish meal. The low protein (LP) diet (10·5% crude protein) contained cereal protein only. Nutrient components of the diets differed in protein only. The pattern treatments involved allowances of 1·8 kg (L), 2·7 kg (C) and 3·6 kg (H) per day, the three pregnancy patterns being HL, C and LH with the changeovers made from the 49th to the 63rd day post coitum (p.c). Sows on the three pattern treatments received the same total amount of feed from 0–112 days p.c. and were treated alike at farrowing and during lactation.Fertility and parturition results were similar for all treatments, but the number of piglets alive after birth (when weighed) was least for LP sows on the HL pattern. At 3 weeks of age the size and weight of litters on HP sows were significantly greater than those on LP sows (P < 0·05 and < 0·001 respectively). More piglets were weaned by HP sows than LP sows (P < 0·05). HP sows gained more weight in pregnancy (P < 0·001) which was slightly longer, and lost more weight in lactation (P < 0·05) than LP sows.The HL pattern of feeding was associated with smaller live weight gains in pregnancy than the LH pattern (P < 0·001) and the total birth weight of HL litters was lighter than LH (P < 0·05), mean piglet weights being similar. Lactation performance was unaffected by pattern treatment.The main conclusion is that a low intake, particularly during the latter half of pregnancy, of protein which is of vegetable origin, is associated with decreased viability of the piglets at birth and in early suckling life, and with lower capacity of the sows for milk production.

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