scholarly journals Protein disulphide-isomerase activity in chick-embryo tissues. Correlation with the biosynthesis of procollagen

1980 ◽  
Vol 191 (3) ◽  
pp. 873-876 ◽  
Author(s):  
B E Brockway ◽  
S J Forster ◽  
R B Freedman
Keyword(s):  
Chick Embryo ◽  
Developmental Stage ◽  
Protein Disulphide Isomerase ◽  
Isomerase Activity

Protein disulphide-isomerase activity was determined in homogenates of chick-embryo tissues. Activities were highest in tissues active in procollagen synthesis and were maximal at the developmental stage of maximal procollagen synthesis. These variations in protein disulphide-isomerase activity correlate closely with those previously observed for protocollagen hydroxylase activities.

scholarly journals The binding of monoribosomes, oligoribosomes and polyribosomes to reticular membranes from rat liver

1978 ◽  
Vol 172 (1) ◽  
pp. 109-114 ◽  
Author(s):  
J A Fielder ◽  
H M Dani ◽  
D Ridge ◽  
B R Rabin
Keyword(s):  
Rat Liver ◽  
Male Rats ◽  
Centrifugal Forces ◽  
Enzyme Protein ◽  
Protein Disulphide Isomerase ◽  
Isomerase Activity ◽  
Wash Solution

1. The activity of the enzyme protein disulphide-isomerase (EC 5.3.4.1) was used to measure the binding of ribosomal fractions to reticular membranes from the liver of unstarved male rats. 2. Membranes degranulated by treatment with KCl plus puromycin and washed by centrifugation through 0.5 M-KCl bound oligoribosomes but not monoribosomes. 3. Substitution of 0.25 M-KCl for 0.5 M-KCl in the wash solution produced membranes that bound monoribosomes and 60 S subunits in addition to oligoribosomes. 4. The binding of all classes of ribosomes to smooth and ‘lightly granulated’ rough membranes was activated by oestradiol. The hormone activation was much greater with polyribosomes from fed rats than for monoribosomes or oligoribosomes from starved rats. 5. Exposure of rough membranes to centrifugal forces caused them to undergo partial degranulation, as demonstrated by unmasking of protein disulphide-isomerase activity.

scholarly journals A direct, continuous, sensitive assay for protein disulphide-isomerase based on fluorescence self-quenching

2005 ◽  
Vol 391 (2) ◽  
pp. 351-357 ◽  
Author(s):  
Arun Raturi ◽  
Panayiotis O. Vacratsis ◽  
Dana Seslija ◽  
Lana Lee ◽  
Bulent Mutus
Keyword(s):  
Platelet Surface ◽  
Protein Disulphide Isomerase ◽  
Isomerase Activity ◽  
Single Turnover ◽  
Reduction Activity ◽  
Crude Sample ◽  
Terminal Amino ◽  
Uv Visible ◽  
Continuous Determination

PDI (protein disulphide-isomerase) activity is generally monitored by insulin turbidity assay or scrambled RNase assay, both of which are performed by UV–visible spectroscopy. In this paper, we present a sensitive fluorimetric assay for continuous determination of disulphide reduction activity of PDI. This assay utilizes the pseudo-substrate diabz-GSSG [where diabz stands for di-(o-aminobenzoyl)], which is formed by the reaction of isatoic anhydride with the two free N-terminal amino groups of GSSG. The proximity of two benzoyl groups leads to quenching of the diabz-GSSG fluorescence by approx. 50% in comparison with its non-disulphide-linked form, abz-GSH (where abz stands for o-aminobenzoyl). Therefore the PDI-dependent disulphide reduction can be monitored by the increase in fluorescence accompanying the loss of proximity-quenching upon conversion of diabz-GSSG into abz-GSH. The apparent Km of PDI for diabz-GSSG was estimated to be approx. 15 μM. Unlike the insulin turbidity assay and scrambled RNase assay, the diabz-GSSG-based assay was shown to be effective in determining a single turnover of enzyme in the absence of reducing agents with no appreciable blank rates. The assay is simple to perform and very sensitive, with an estimated detection limit of approx. 2.5 nM PDI, enabling its use for the determination of platelet surface PDI activity in crude sample preparations.

Detection of protein disulphide-isomerase in sheep pancreas fractions

1982 ◽  
Vol 2 (5) ◽  
pp. 343-349 ◽  
Author(s):  
David A. Hillson ◽  
Jacqueline Anderson
Keyword(s):  
Specific Activity ◽  
Protein Biosynthesis ◽  
Major Site ◽  
General Role ◽  
Protein Disulphide Isomerase ◽  
Isomerase Activity ◽  
Liver Homogenates ◽  
Specific Activities

Conclusions The use of diethylpyrocarbonate to inhibit endogenous ribonuclease in sheep pancreas allows the detection of protein-disulphide-isomerase activity in homogenates, at specific activities of up to 4 units/g. This is higher than the specific activity in sheep liver homogenates (about 2 units/g) or in homogenates of other sheep tissues (16). It is thus evident that high levels of protein-disulphide-isomerase activity are present in sheep pancreas. This is consistent both with the postulated general role of protein disulphide-isomerase in protein biosynthesis (10,11) and with the in vitro action of the enzyme on its conventional substrate scrambled ribonuclease, since pancreas is the major site of ribonuclease synthesis.

scholarly journals The role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity

1996 ◽  
Vol 315 (2) ◽  
pp. 533-536 ◽  
Author(s):  
Arja LAMBERG ◽  
Matti JAUHIAINEN ◽  
Jari METSO ◽  
Christian EHNHOLM ◽  
Carol SHOULDERS ◽  
...  
Keyword(s):  
Β Subunit ◽  
Lipid Transfer ◽  
Α Subunit ◽  
Protein Disulphide Isomerase ◽  
Isomerase Activity ◽  
Transfer Protein ◽  
Transfer Activity ◽  
Disulphide Bond Formation ◽  
Catalytically Active

The microsomal triacylglycerol transfer protein (MTP), an αβ dimer, is obligatory for the assembly of apoB-containing lipoproteins in liver and intestinal cells. The β subunit is identical with protein disulphide isomerase, a 58 kDa endoplasmic reticulum luminal protein involved in ensuring correct disulphide bond formation of newly synthesized proteins. We report here the expression of the human MTP subunits in Spodoptera frugiperda cells. When the α subunit was expressed alone, the polypeptide formed insoluble aggregates that were devoid of triacylglycerol transfer activity. In contrast, when the α and β subunits were co-expressed, soluble αβ dimers were formed with significant triacylglycerol transfer activity. Expression of the α subunit with a mutant protein disulphide isomerase polypeptide in which both -CGHC- catalytic sites had been inactivated also yielded αβ dimers that had comparable levels of lipid transfer activity relative to wild-type dimers. The results indicate that the role of the β subunit in MTP seems to be to keep the α subunit in a catalytically active, non-aggregated conformation and that disulphide isomerase activity of the β subunit is not required for this function.

Protein disulphide-isomerase activity in bacterial osmotic shock preparations

1988 ◽  
Vol 16 (1) ◽  
pp. 57-57 ◽  
Author(s):  
PETER T. BARTH ◽  
CAROLINE BUST ◽  
HILARY C. HAWKINS ◽  
ROBERT B. FREEDMAN
Keyword(s):  
Osmotic Shock ◽  
Protein Disulphide Isomerase ◽  
Isomerase Activity

scholarly journals The catalytic mechanism of the hydroxylation reaction of peptidyl proline and lysine does not require protein disulphide-isomerase activity

1989 ◽  
Vol 263 (2) ◽  
pp. 609-611 ◽  
Author(s):  
R Myllylä ◽  
D D Kaska ◽  
K I Kivirikko
Keyword(s):  
Reaction Mechanisms ◽  
Catalytic Mechanism ◽  
Beta Subunit ◽  
Enzyme Protein ◽  
Protein Disulphide Isomerase ◽  
Lysyl Hydroxylase ◽  
Isomerase Activity ◽  
Hydroxylation Reaction ◽  
Beta 2

Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens. The beta subunit is known to be identical with the enzyme protein disulphide-isomerase and to possess disulphide-isomerase activity even when present in the prolyl 4-hydroxylase tetramer. We here report that lysyl hydroxylase, a homodimer, and algal prolyl 4-hydroxylase, a monomer, do not contain detectable protein disulphide-isomerase activity. Since the hydroxylase reaction mechanisms are similar, the data suggest that the protein disulphide-isomerase activity of the vertebrate prolyl 4-hydroxylase beta subunit is unlikely to be involved in the catalytic mechanism of the hydroxylation reaction.

scholarly journals Protein disulphide-isomerase of chick-embryo tendon

1984 ◽  
Vol 219 (1) ◽  
pp. 51-59 ◽  
Author(s):  
B E Brockway ◽  
R B Freedman
Keyword(s):  
Chick Embryo ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
High Speed ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Supernatant Fraction ◽  
Marker Enzyme ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase can be partially purified from the high-speed-supernatant fraction of extensively disrupted chick-embryo tendon tissue. The catalytic properties of the preparation resemble those of the enzyme from mammalian liver. Gel electrophoresis and isoelectric focusing show the enzyme to be very acidic, with pI 4.4 +/- 0.3. Gel filtration indicates an Mr for the active enzyme of 140 000. The enzyme can be partially purified by preparative gel filtration or isoelectric focusing, but its limited stability has prevented purification to homogeneity; active fractions from both gel filtration and isoelectric focusing show two major polypeptide components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The major polypeptides present in partially purified preparations have Mr 45 000 and 55 000; the latter band co-distributes with the enzyme activity in fractionations by both gel filtration and isoelectric focusing. The subcellular location of the enzyme cannot be established from work on homogenates of whole tissue, which are extensively disrupted. In homogenates from isolated tendon cells, the enzyme is located in a vesicle fraction that is excluded from Sepharose 2B but is of low density and can only be sedimented at very high speeds. This fraction is identified as deriving from the endoplasmic reticulum on the grounds of marker-enzyme studies and electron microscopy.

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