scholarly journals COLCEMID INHIBITION OF CELL GROWTH AND THE CHARACTERIZATION OF A COLCEMID-BINDING ACTIVITY IN SACCHAROMYCES CEREVISIAE

1972 ◽  
Vol 55 (2) ◽  
pp. 355-367 ◽  
Author(s):  
James E. Haber ◽  
John G. Peloquin ◽  
Harlyn O. Halvorson ◽  
Gary G. Borisy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Filtration ◽  
Culture Conditions ◽  
Binding Activity ◽  
Affinity Constant ◽  
Binding Substance ◽  
Related Drug ◽  
Antimitotic Drug ◽  
The Difference

Under restricted culture conditions, the growth and division of Saccharomyces cerevisiae was inhibited by the antimitotic drug Colcemid; in contrast, the related drug colchicine had no effect. The difference in the sensitivity of yeast to these two agents was not dependent on their ability to permeate the cell but rather reflected an inherent difference in the affinity of the two drugs for a cellular-binding site. The binding moiety was characterized by gel filtration as a macromolecule of approximately 110,000 mol wt with an affinity constant for Colcemid of 0.5 x 104 liters per mole; in addition, this macromolecule was retained by diethylaminoethyl (DEAE) ion exchangers. On the basis of these properties, the Colcemid-binding substance in S. cerevisiae cells was provisionally identified as microtubule subunits.

scholarly journals Characterization of a Gi-protein from Trypanosoma cruzi epimastigote membranes

1992 ◽  
Vol 287 (2) ◽  
pp. 443-446 ◽  
Author(s):  
O A Coso ◽  
A Díaz Añel ◽  
H Martinetto ◽  
J P Muschietti ◽  
M Kazanietz ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Pertussis Toxin ◽  
Polyclonal Antibodies ◽  
Gel Filtration ◽  
Binding Activity ◽  
Gi Protein ◽  
Liver Membranes ◽  
Blocking Capacity ◽  
Polypeptide Band

A guanosine 5′-[gamma-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43,000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-alpha common, AS/7 anti-alpha t, anti-alpha i1 and anti-alpha i2 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-beta antibody, of about 30 kDa, was also detected in the membrane fraction.

scholarly journals Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
María-Efigenia Álvarez-Cao ◽  
María-Esperanza Cerdán ◽  
María-Isabel González-Siso ◽  
Manuel Becerra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Food Industry ◽  
Culture Conditions ◽  
Raffinose Family Oligosaccharides ◽  
Recombinant Production ◽  
Gene Coding ◽  
Productive Process ◽  
Hydrolysis Of ◽  
Degree Of Purification

Abstract Background α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. Results The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-d-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. Conclusions ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.

scholarly journals Molecular Characterization of Saccharomyces cerevisiae TFIID

2002 ◽  
Vol 22 (16) ◽  
pp. 6000-6013 ◽  
Author(s):  
Steven L. Sanders ◽  
Krassimira A. Garbett ◽  
P. Anthony Weil
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Molecular Mass ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Calculated Molecular Mass ◽  
General Transcription Factor

ABSTRACT We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.

scholarly journals Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Alexsandro Sobreira Galdino ◽  
Roberto Nascimento Silva ◽  
Muriele Taborda Lottermann ◽  
Alice Cunha Morales Álvares ◽  
Lídia Maria Pepe de Moraes ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Soluble Starch ◽  
Alpha Amylase ◽  
Enzyme Specificity ◽  
Wild Type ◽  
Spectra Analysis ◽  
Exclusion Chromatography ◽  
The Difference ◽  
Thermal Stability Of

An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH4+ and inhibited by Cu+2 and Hg+2. Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to Km values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1w, points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels.

scholarly journals Purification and partial characterization of four proteins from human parotid saliva

1971 ◽  
Vol 123 (3) ◽  
pp. 455-464 ◽  
Author(s):  
Anders Bennick ◽  
George E. Connell
Keyword(s):  
Serum Proteins ◽  
Protein A ◽  
Gel Filtration ◽  
Binding Activity ◽  
Significant Activity ◽  
Parotid Saliva ◽  
Polyacrylamide Electrophoresis ◽  
Ph Range ◽  
Human Parotid Saliva

Four proteins, which have been designated A, B, C and D, have been purified from human parotid saliva. These proteins are the major constituents of parotid saliva which migrate rapidly to the anode in polyacrylamide electrophoresis at pH9.5. Gel filtration and polyacrylamide electrophoresis were employed in the purification procedures. After purification all four preparations were tested for homogeneity by electrophoresis at pH2.8 and 9.5, by isoelectric focusing in the pH range 3–10, by immunodiffusion, and by sedimentation in the analytical ultracentrifuge. None of the proteins showed significant activity in assays for amylase, acid and alkaline phosphatase, protease, lysozyme, ribonuclease, peroxidase, β-glucuronidase, β-galactosidase, iron-binding activity and esterase. No cross-reactions were detected with antisera specific for lactoferrin and 15 serum proteins. All four proteins were rich in glutamic acid, proline and glycine and were lacking completely the sulphur-containing amino acids. Proteins A and C contained no threonine or tyrosine. Carbohydrate could be demonstrated only in protein A at a concentration of 4% of the total protein.

scholarly journals Identification of Sec36p, Sec37p, and Sec38p: Components of Yeast Complex That Contains Sec34p and Sec35p

2002 ◽  
Vol 13 (5) ◽  
pp. 1484-1500 ◽  
Author(s):  
Rachna J. Ram ◽  
Baojie Li ◽  
Chris A. Kaiser
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Affinity Purification ◽  
Gel Filtration ◽  
Missense Mutations ◽  
Functional Connection ◽  
Copi Vesicle

The Saccharomyces cerevisiae proteins Sec34p and Sec35p are components of a large cytosolic complex involved in protein transport through the secretory pathway. Characterization of a new secretion mutant led us to identify SEC36, which encodes a new component of this complex. Sec36p binds to Sec34p and Sec35p, and mutation of SEC36 disrupts the complex, as determined by gel filtration. Missense mutations of SEC36 are lethal with mutations in COPI subunits, indicating a functional connection between the Sec34p/sec35p complex and the COPI vesicle coat. Affinity purification of proteins that bind to Sec35p-myc allowed identification of two additional proteins in the complex. We call these two conserved proteins Sec37p and Sec38p. Disruption of either SEC37or SEC38 affects the size of the complex that contains Sec34p and Sec35p. We also examined COD4,COD5, and DOR1, three genes recently reported to encode proteins that bind to Sec35p. Each of the eight genes that encode components of the Sec34p/sec35p complex was tested for its contribution to cell growth, protein transport, and the integrity of the complex. These tests indicate two general types of subunits: Sec34p, Sec35p, Sec36p, and Sec38p seem to form the essential core of a complex to which Sec37p, Cod4p, Cod5p, and Dor1p seem to be peripherally attached.

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.

The Immunological Characterization of Human Antibodies to Factor VIII Isolated by Immuno-Affinity Chromatography

1981 ◽  
Vol 45 (01) ◽  
pp. 060-064 ◽  
Author(s):  
M L Kavanagh ◽  
C N Wood ◽  
J F Davidson
Keyword(s):  
Factor Viii ◽  
Affinity Chromatography ◽  
Gel Filtration ◽  
Immunological Characterization ◽  
Human Antibodies ◽  
Heavy Chains ◽  
Light Chain Type ◽  
Antibody Preparations ◽  
Chain Type

SummaryNine human antibodies to factor VIII were isolated from haemophilic plasmas by affinity chromatography and gel filtration and six were subsequently subjected to immunological characterization. Three partially purified preparations were similarly characterized. Eight of the antibodies were characterized as being exclusively IgG and one preparation was found to contain IgM. Seven of the antibodies contained only a single light chain type, four being of type lambda and three of type kappa. Two antibody preparations contained both kappa and lambda light chains. In four of the preparations, only a single heavy chain sub-class could be demonstrated, three of IgG3 and one of IgG4. Of the remainder, three were a mixture of IgG3 and IgG4 sub-classes and one contained both IgG2 and IgG4. IgG sub-classification could not be achieved with the IgM-containing preparation. These results demonstrate a restricted heterogeneity of light and heavy chains in human antibodies to factor VIII.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

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