scholarly journals Prolyl 4-hydroxylase from Volvox carteri. A low-Mr enzyme antigenically related to the α subunit of the vertebrate enzyme

1988 ◽  
Vol 256 (1) ◽  
pp. 257-263 ◽  
Author(s):  
D D Kaska ◽  
R Myllylä ◽  
V Günzler ◽  
A Gibor ◽  
K I Kivirikko
Keyword(s):  
Green Alga ◽  
Polyacrylamide Gel Electrophoresis ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Affinity Column ◽  
Major Protein ◽  
Volvox Carteri ◽  
Chlamydomonas Reinhardii ◽  
Α Subunit ◽  
Major Protein Band

Prolyl 4-hydroxylase was isolated in a highly purified form from a multi-cellular green alga, Volvox carteri, by a procedure consisting of ion-exchange chromatography and affinity chromatography on poly(L-hydroxyproline) coupled to Sepharose. Two other affinity-column procedures were also developed, one involving 3,4-dihydroxyphenylacetate and the other 3,4-dihydroxyphenylpropionate linked to Sepharose. The Km values of the Volvox enzyme for the co-substrates and the peptide substrate, as well as the inhibition constants for selected 2-oxoglutarate analogues, were similar to those of the enzyme from Chlamydomonas reinhardii, except that the Km for 2-oxoglutarate with the Volvox enzyme was 6-fold greater. The temperature optimum of the Volvox enzyme was also 10 degrees C higher. The apparent Mr of the Volvox enzyme by gel filtration was about 40,000, being similar to that reported for the Chlamydomonas enzyme but markedly lower than that of the vertebrate enzymes. A similar apparent Mr of about 40,000 was also found for prolyl 4-hydroxylase from the green alga Enteromorpha intestinalis, whereas the enzyme from various vascular plants gave an apparent Mr greater than 300,000. SDS/polyacrylamide-gel electrophoresis demonstrated in the highly purified Volvox enzyme the presence of a major protein band doublet with a Mr of about 65,000 and a minor doublet of Mr about 55,000-57,000. A polyclonal antiserum, prepared against the Mr-65,000 doublet, stained in immunoblotting the Mr-65,000 doublet as well as the alpha subunit, but not the beta subunit, of the vertebrate prolyl 4-hydroxylase. An antiserum against the beta subunit of the vertebrate enzyme stained in immunoblotting a Mr-50,000 polypeptide in a partially purified Volvox enzyme preparation, but did not stain either the Mr-65,000 or the Mr-55,000-57,000 doublet of the highly purified enzyme. The data thus suggest that the active Volvox carteri prolyl 4-hydroxylase is an enzyme monomer antigenically related to the alpha subunit of the vertebrate enzyme.

scholarly journals Studies on the α-subunit of bovine brain S-100 protein

1984 ◽  
Vol 218 (3) ◽  
pp. 691-696 ◽  
Author(s):  
H R Masure ◽  
J F Head ◽  
H M Tice
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fluorescence Emission ◽  
Bovine Brain ◽  
Alpha Subunit ◽  
Tryptophan Residue ◽  
Affinity Column ◽  
Apparent Dissociation Constant ◽  
Α Subunit ◽  
S 100

A method is described for the rapid purification of both S-100 protein and calmodulin from crude bovine brain extracts by the use of a fluphenazine-Sepharose affinity column eluted stepwise with decreasing concentrations of free Ca2+. Protein containing only alpha-subunit was purified from preparations of S-100 protein by anion-exchange chromatography. This protein co-migrated with the alpha-subunit of S-100 protein on sodium dodecyl sulphate/urea/polyacrylamide-gel electrophoresis and had an amino acid composition identical with that previously reported for this subunit. The results of u.v.-absorption and fluorescence-emission spectroscopy indicate that the tryptophan residue of the purified alpha-subunit of S-100 protein undergoes a Ca2+-induced change in environment. Measurements of changes in tryptophan fluorescence with increasing Ca2+ concentrations suggest an apparent dissociation constant of the alpha-subunit for Ca2+ of 7 × 10(-5)M in the absence of K+. In the presence of 90mM-K+ this value is increased to 3.4 × 10(-4)M.

Partial purification and characterization of isopenicillin N epimerase activity from Streptomyces clavuligerus

1983 ◽  
Vol 29 (11) ◽  
pp. 1526-1531 ◽  
Author(s):  
Susan E. Jensen ◽  
Donald W. S. Westlake ◽  
Saul Wolfe
Keyword(s):  
Pyridoxal Phosphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Major Protein ◽  
Major Protein Band ◽  
Isopenicillin N

Epimerase activity, which converts isopenicillin N to penicillin N, has been partially purified from cell-free extracts of Streptomyces clavuligerus. No stimulating cofactors of this activity were found, and neither EDTA nor anaerobic incubation caused significant inhibition of activity. Although pyridoxal phosphate did not stimulate epimerase activity, the presence of this cofactor was necessary for the stabilization of enzymic activity during the purification process. Epimerase activity was purified 35.5-fold by a combination of salt precipitation, gel filtration, and ion exchange chromatography. Gel filtration indicated that the epimerase has a molecular weight of 60 000 and sodium dodecyl sulphate – polyacrylamide gel electrophoresis of the 35.5-fold purified epimerase showed a major protein band running near that location. Pyridoxal phosphate antagonists did not uniformly inhibit epimerase activity, but the inhibitory effect of hydroxylamine could be partially reversed by pyridoxal phosphate.

scholarly journals Properties of Peanut (KAC431) Protein Hydrolysates and Their Impact on the Quality of Gluten-Free Rice Bread

Foods ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 942 ◽  
Author(s):  
Suphat Phongthai ◽  
Nuttapon Singsaeng ◽  
Rossarin Nhoo-ied ◽  
Thipubol Suwannatrai ◽  
Regine Schönlechner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Functional Properties ◽  
Positive Impact ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Hydrolysates ◽  
Degree Of Hydrolysis ◽  
Major Protein ◽  
Gluten Free ◽  
Major Protein Band

Protein hydrolysates (PH) with a degree of hydrolysis (DH) of 5%, 10%, and 13% from two varieties of peanut were prepared using two commercial enzymes, Alcalase and Flavourzyme. The content of essential amino acids (30,290 mg/100 g) and hydrophobic amino acids (34,067 mg/100 g) of the peanut variety Kalasin 2 (KAC431) protein was higher than that of a common variety, Kalasin 1 (KAC1) (p < 0.05). The protein molecular weight distributions of the two varieties of peanut detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were similar, ranging from 15 to 75 kDa, with a major protein band at 50–75 kDa. The antioxidant and functional properties of derived PHs were influenced by DH. Although the foaming ability of protein was improved by DH5%, it was obviously decreased upon increasing DH further. The best emulsifying properties were observed in PH with DH5% (p < 0.05). The incorporation of PH with a small DH, especially when produced using Flavourzyme, had a highly positive impact on the specific volume and relative elasticity of gluten-free bread. The effect of PHs on bread quality was highly correlated with their functional properties. This study suggests that partially enzymatically modified proteins are suitable for incorporation in food products such as bread and other gluten-free products.

scholarly journals The adipocyte Goα-immunoreactive polypeptide is different from the α subunit of the brain Go protein

1989 ◽  
Vol 260 (1) ◽  
pp. 307-310 ◽  
Author(s):  
B Rouot ◽  
J Carrette ◽  
M Lafontan ◽  
P Lan Tran ◽  
J A Fehrentz ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Molecular Mass ◽  
G Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mass Range ◽  
Alpha Subunit ◽  
Α Subunit ◽  
Positive Immunoreactivity ◽  
Rat Adipose Tissue ◽  
The Brain

Rat adipose tissue possesses two Bordetella pertussis toxin (PTX) substrates and, in the same 39-41 kDa molecular mass range, positive immunoreactivity has also been reported with antibodies against the alpha subunit of Go, the major brain GTP-binding protein (G-protein). In this study, the presence of the brain Go alpha subunit at 39 kDa in adipocytes was reassessed, since direct correspondence between PTX substrates and Go alpha immunoreactivity has not yet been clearly established. On resolutive SDS/polyacrylamide-gel electrophoresis, the PTX substrates of human adipocytes were compared with the three PTX substrates found in brain. No ADP-ribosylated substrate at the level of the 39 kDa brain Go alpha could be detected in adipocyte membranes. Immunoblotting of human adipocyte membranes stained with our anti-Go alpha antibodies confirmed the presence of a positive immunoreactivity in this tissue, but the apparent molecular mass of the immunoreactive polypeptide in adipocytes was higher than that found in nervous tissues. Taken together, these results indicate that the brain Go alpha subunit is not present in adipose tissue. They also suggest the existence of a G-protein in adipocytes which is immunologically related to Go alpha but having a slightly higher molecular mass.

Extracellular calmodulin and its association with epidermal growth factor in normal human body fluids

1988 ◽  
Vol 118 (3) ◽  
pp. 501-NP ◽  
Author(s):  
S. Mac Neil ◽  
R. A. Dawson ◽  
G. Crocker ◽  
C. H. Barton ◽  
L. Hanford ◽  
...  
Keyword(s):  
Growth Factor ◽  
Breast Milk ◽  
Epidermal Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Biologically Active ◽  
Major Protein ◽  
Major Protein Band ◽  
Normal Human ◽  
Epidermal Growth

ABSTRACT In this study we describe the occurrence of a calmodulin-like protein in normal human biological fluids. Extraction of the calmodulin-like protein from breast milk, saliva, serum and urine provided an extract with enhanced calmodulin immunoreactivity which, in the case of milk and saliva, showed a protein band co-migrating with authentic calmodulin (Mr 17 000) on sodium dodecylsulphate-polyacrylamide gel electrophoresis. However, in milk, saliva and serum a major protein band of Mr 14 000–15 000 was always observed, which we speculate may be related to calmodulin, possibly as a partially degraded form. Estimates of biologically active calmodulin in most normal extracellular fluids were of the order which we have found will stimulate cell division when added to the extracellular medium of cells in culture. Levels ranged from 0·03 nmol/l in urine to 18·6 nmol/l in breast milk, and exhibited a quantitative relationship (r = 0·79, P < 0·01) to epidermal growth factor (EGF) levels in fluids. Where EGF concentrations varied from normal (increased in saliva 24 h after oral surgery and reduced in the urine of patients with renal failure) calmodulin concentrations were similarly affected. The presence of calmodulin in serum may in part be attributable to its release from platelets which are particularly rich in calmodulin. Release of calmodulin from the platelet was associated with that of EGF and other platelet products. J. Endocr. (1988) 118, 501–509

Isolation and Characterization of Sepiapterin Reductase from Drosophila melanogaster

Pteridines ◽  
1993 ◽  
Vol 4 (1) ◽  
pp. 43-50 ◽  
Author(s):  
K.H. Yoon ◽  
K.W. Cha ◽  
S.I. Park ◽  
J.J. Yim
Keyword(s):  
Drosophila Melanogaster ◽  
Carbonyl Compounds ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Major Protein ◽  
Sepiapterin Reductase ◽  
Isolation And Characterization ◽  
Major Protein Band

Summary Sepiapterin reductase, an enzyme that catalyses the synthesis of tetrahydrobiopterin (BH4). was partially purified from Drosophila melanogaster using ammonium sulfate fractionation. Affi-gel blue chromatography and hydroxyapatite chromatography. The molecular weight of the enzyme determined by Ultrogel AcA44 column was 39,000. When the enzyme was subjected to polyacrylamide gel electrophoresis in SDS. a 38.000 MW species was found to be the major protein band. The Km values for sepiapterin and NADPH were determined to he 75.4 µM. and 14 µM, respectively. The optimal temperature and pH for the reaction were 30°C and pH 5.7-6.7. The half-life of the activity was 30 minutes when treated at 48°C The enzyme was markedly inhibited by tri-and tetravalent cations. Fe3+ . Sn4+ and divalent cation. Cd2+ . It was found that pyrimidodiazcpine (a homopterin) and 2.4-diamino-6.7-diisopropyl caused the reduction of sepiapterin reductase activity by about 50% at the concentration of 0.1 mM. Among the neurotransmitters and their precursors tested. 3 mM concentrations of melatonin and N-acetylserotonin inhibited the enzyme activity completely. In addition to sepiapterin. the enzyme uses rather broad spectrum of carbonyl compounds as substrate including menadione. p-nitrohenzaldehyde, and various dicarhonyl compounds

The effect of ageing cooled milk on the composition of the fat globule membrane

1972 ◽  
Vol 39 (1) ◽  
pp. 95-105 ◽  
Author(s):  
M. Anderson ◽  
G. C. Cheeseman ◽  
Dorothy J. Knight ◽  
W. F. Shipe
Keyword(s):  
Gel Electrophoresis ◽  
Milk Fat ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Protein Band ◽  
Major Protein ◽  
Fresh Milk ◽  
Major Protein Band ◽  
Fat Globule

SummaryThe effect of ageing fresh milk for 24 h at 4°C on the composition of 4 fractions of milk fat globule membrane (FGM) – microsomal pellet membrane (MPM), deoxycholate soluble membrane (DOCM), high density membrane (HDM) and low density membrane (LDM) – prepared from washed cream treated with sodium deoxycholate (DOC), was studied for milk of individual cows. Total FGM and its fractions were solubilized by treatment with sodium dodecyl sulphate (SDS), EDTA and β-mercaptoethanol, and the dissociated FGM proteins were separated by polyacrylamide gel electrophoresis in the presence of SDS.Ageing resulted in a greater loss of phospholipid during cream preparation than was found with fresh milk, and also in a reduction in the total amount of FGM isolated. Loss of FGM material was confined to MPM, DOCM and LDM and was accompanied by compositional changes in DOCM and LDM, quantitatively the most significant fractions. Ageing also produced changes in the gel electrophoresis patterns of DOCM, where the band of greatest mobility (mol. wt about 16000) was partially lost, and of LDM, where there was an increase observed in the major protein band.The implication of the results on the proposed models of FGM structure is discussed.

Purification of lactate dehydrogenase isoenzyme-5 from human liver.

1981 ◽  
Vol 27 (1) ◽  
pp. 88-93 ◽  
Author(s):  
S M Pettit ◽  
D A Nealon ◽  
A R Henderson
Keyword(s):  
Enzyme Activity ◽  
Lactate Dehydrogenase ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Major Protein ◽  
Dehydrogenase Isoenzyme ◽  
Major Protein Band

Abstract We present a method for preparing human liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase; EC 1.1.1.27) isoenzyme-5 by sequential ion-exchange chromatography, general-ligand (AMP analog) affinity chromatography, and preparative isoelectric focusing. The yield ws 40%, with a 493-fold purification. The final specific activity was 458 kU per gram of protein. The preparation contained less than 0.2% of lactate dehydrogenase isoenzyme-4, was homogeneous by agarose gel electrophoresis and also by polyacrylamide gel electrophoresis at pH 8.9 and 6.9, and showed one major protein band (containing all the enzyme activity) and one minor anodic contaminant (containing no enzyme activity) by analytical isoelectric focusing. The enzyme had a mean pI value of 9.59 (SD 0.04) (n = 5) at 5 degrees C. By comparison, the pI value of a preparation of rabbit lactate dehydrogenase-5 was 9.16 (5 degrees C).

scholarly journals β-1,3-Glucanase Activity in Peanut Seed (Arachis hypogaea) is Induced by Inoculation with Aspergillus flavus and Copurifies with a Conglutin-Like Protein

2005 ◽  
Vol 95 (5) ◽  
pp. 506-511 ◽  
Author(s):  
X. Q. Liang ◽  
C. C. Holbrook ◽  
R. E. Lynch ◽  
B. Z. Guo
Keyword(s):  
Arachis Hypogaea ◽  
Aspergillus Flavus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Major Protein ◽  
Peanut Seed ◽  
Glucanase Activity ◽  
Infected Seed ◽  
Major Protein Band ◽  
Resistant Genotypes

Infection of peanut (Arachis hypogaea) seed by Aspergillus flavus and A. parasiticus is a serious problem that can result in aflatoxin contamination in the seed. Breeding resistant cultivars would be an effective approach to reduce aflatoxin accumulation. The objective of this study was to investigate the expression of the pathogenesis-related (PR) protein β-1,3-glucanase and the isoform patterns in peanut seed inoculated with A. flavus. Peanut genotypes GT-YY9 and GT-YY20 (both resistant to A. flavus infection) and Georgia Green and A100 (both susceptible to A. flavus infection) were used in this study. The activities of β-1,3-glucanase were similar in the uninfected seed of all genotypes, but increased significantly in the resistant genotypes after inoculation in comparison with the susceptible genotypes. An in-gel (native polyacrylamide gel electrophoresis [PAGE]) enzymatic activity assay of β-1,3-glucanase revealed that there were more protein bands corresponding to β-1,3-glucanase isoforms in the infected seed of resistant genotypes than in the infected seed of susceptible genotypes. Both acidic and basic β-1,3-glucanase isoforms were detected in the isoelectric focusing gels. Thin-layer chromatography analysis of the hydrolytic products from the reaction mixtures of the substrate with the total protein extract or individual band of native PAGE revealed the presence of enzymatic hydrolytic oligomer products. The individual bands corresponding to the bands of β-1,3-glucanase isoforms Glu 1 to 5 were separated on the sodium dodecyl sulfate-PAGE, resulting in two bands of 10 and 13 kDa, respectively. The sequences of fragments of the 13-kDa major protein band showed a high degree of homology to conglutin, a storage protein in peanut seed. Conglutin is reported as a peanut allergen, Ara h2. Our data provide the first evidences for peanut having β-1,3-glucanase activities and the association with the resistance to A. flavus colonization in peanut seed. We have not directly demonstrated that conglutin has β-1,3-glucanase activity.

scholarly journals Antibodies directed against a nonapeptide sequence of the γ-aminobutyrate (GABA)/benzodiazepine receptor α-subunit. Detection of a distinct α-like subunit in pig cerebral cortex but not cerebellum

1988 ◽  
Vol 256 (1) ◽  
pp. 291-294 ◽  
Author(s):  
E F Kirkness ◽  
A J Turner
Keyword(s):  
Cerebral Cortex ◽  
Synthetic Peptide ◽  
Benzodiazepine Receptor ◽  
Polyclonal Antiserum ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Amino Acid Residues ◽  
Regional Patterns ◽  
Α Subunit ◽  
Peptide Antibodies

A synthetic peptide, corresponding to amino acid residues 101-109 of the bovine gamma-aminobutyrate/benzodiazepine receptor alpha-subunit, was used to raise a polyclonal antiserum. The reactivity of this antiserum towards polypeptides of both bovine and pig receptor preparations was established by immunoprecipitation and immunoblotting. Anti-peptide antibodies recognized the alpha-subunit (51 kDa) of receptor prepared from pig cerebellum or cerebral cortex. However, a polypeptide of 57 kDa was additionally recognized in cortical, but not cerebellar, preparations. This alpha-like polypeptide appeared larger than the band of polypeptides labelled irreversibly with [3H]muscimol (beta-subunit, 55-57 kDa) and corresponds to a polypeptide detected only in cortex after silver-staining or irreversible labelling with [3H]flunitrazepam. These results support the idea that the distinct regional patterns of polypeptides labelled irreversibly with [3H]flunitrazepam reflect the existence of heterologous distributions of distinct alpha-like subunits.

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