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.

Characterization, Development and Localization of "Flavanone Synthase" in Tulip Anthers

1978 ◽  
Vol 33 (11-12) ◽  
pp. 841-846 ◽  
Author(s):  
R. Siitfeld ◽  
B. Kehrel ◽  
R. Wiermann
Keyword(s):  
Enzyme Activity ◽  
Gel Chromatography ◽  
Enzyme Release ◽  
Similar Data ◽  
Reaction Products ◽  
Isomerase Activity ◽  
Malonyl Coa ◽  
Specific Activities

1. “Flavanone synthase” was isolated from anthers of Tulipa cv. “Apeldoorn” and partially purified by (NH4) 2SO4 fractionation, gel chromatography and isoelectric focussing. The enzyme preparation was free of chalcone-flavanone isomerase activity. 2. p-Coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA were found to be efficient substrates of the synthase. The products formed were naringenin (5,7,4′-trihydroxyflavanone), eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) and homoeriodictyol (5,7,4′-trihydroxy-3′-methoxyflavanone), respectively. Addition of thiol reagents at concentrations exceeding 10-3 м caused inhibition of the enzyme. “ Release products” , however, were not detectable. Although exclusively chalcones accumulate in the tulip anther, only flavanones but no chalcones were detectable in our in vitro system. 3. The apparent Km values for p-coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA were 1 .7× 10-6 м, 1.6× 10-6 м and 2 .5 ×10-6 м, respectively. Similar data were observed for malonyl- CoA. 4. No cofactors are required for the synthase reaction. The enzyme is strongly inhibited by the reaction products flavanone and coenzyme A . Maximum enzyme activity was found at pH 8.0 and 30 °. The molecular weight was approx. 55,000. 5. Synthase activity develops in early postmeiotic stages of microsporogenesis. Highest specific activities of the enzyme coincide with a maximum in chalcone accumulation within the anthers. 6. The contents of anthers was separated into two fractions, pollen and tapetum. Highest specific activities were observed with tapetum fractions, while pollen fractions exhibited only very low activities. The high enzyme activity in the tapetum fraction points to the important role of the tapetum in the biosynthesis of flavonoids in the loculus of anthers.

Lethality of a tritiated amino acid in early mouse embryos

Development ◽  
1985 ◽  
Vol 88 (1) ◽  
pp. 209-217
Author(s):  
Janet L. Wiebold ◽  
Gary B. Anderson
Keyword(s):  
Specific Activity ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Radiation Induced ◽  
Specific Activities ◽  
Tritiated Amino Acids ◽  
Early Mouse

2- to 4-cell and morula- to blastocyst-stage mouse embryos were cultured for 1 h in tritiated leucine at two specific activities and their subsequent development followed in vitro and in vivo (after transfer to recipients), respectively. 2- to 4-cell embryos that incorporated an average of 42 d.p.m. per embryo were impaired in their ability to develop to the morula and blastocyst stage. Recipients receiving morulae and blastocysts that had incorporated an average of 384 d.p.m. per embryo failed to produce young. Reduction of the specific activity improved the viability of embryos both in vitro and in vivo but development was still less than that of unlabelled embryos. Protein degradation curves were different for both 2- to 4-cell and morulato blastocyst-stage embryos labelled at the two different specific activities. Most studies using tritiated amino acids have employed higher specific activities than those used here and they may have to be reevaluated due to the possibility of radiation-induced artifacts.

scholarly journals Platelet Alterations Caused by Antiplatelet Antibodies in the Circulation

1977 ◽  
Author(s):  
T. Nagasawa ◽  
B.K. Kim ◽  
M.G. Baldini
Keyword(s):  
Platelet Count ◽  
Specific Activity ◽  
Rabbit Model ◽  
Antiplatelet Antibodies ◽  
Large Numbers ◽  
Severe Reduction ◽  
Series Of Experiments ◽  
Specific Activities

It is known that antiplatelet antibodies cause loss of platelet cytoplasmic and granular contents in vitro. It is, however, unknown whether similar platelet changes occur in vivo, in the circulation, leading to destruction and phagocytosis of platelets in the R.E. system. To study this possibility a rabbit model was devised. Severe and stable thrombocytopenia was first produced in rabbits by one intravenous injection of Adriamycin. Large numbers of allogenic platelets labeled in vitro with 51Cr and 14C-serotonin were then infused to raise the circulating platelet count to 180-250 × 103/mm3. A dilute heteroimmune antiplatelet serum prepared in the guinea pig was infused intravenously and platelet samples were collected four times during the subsequent 30 minutes to 24 hours. Platelet hexokinase and β-glucuronidase, 14C-serotonin and 51Cr were measured. Within the first 60 min the specific activity of 51Cr in platelets decreased by 21%, 14C-serotonin declined by 30%, hexokinase by 5% and β-glucuronidase by 29%. During the subsequent 24 hours only 51Cr and hexokinase registered a mild decrease but 51C-serotonin and β-glucuronidase remained essentially unchanged. In a second series of experiments the effect of platelet alloantibodies was studied in rabbits previously immunized with allogenic platelets. The decline in the specific activities of the enzymes and 14C-serotonin was similar to that observed in animals treated with heteroimmune sera but loss of 51Cr was more severe. These results demonstrate that the platelets remaining in the circulation after the disappearance of the immediate effect of hetero- or alloantibodies were qualitatively altered with a severe reduction of their granular and cytoplasmic contents.

The Incorporation of [4-14C] δ-Aminolaevulic Acid into Urinary Porphyrins in Symptomatic Porphyria

1970 ◽  
Vol 39 (2) ◽  
pp. 159-168 ◽  
Author(s):  
P. Goldswain ◽  
E. Dowdle ◽  
Norma Spong ◽  
L. Eales
Keyword(s):  
Oral Dose ◽  
Control Subject ◽  
Single Oral Dose ◽  
Metabolic Pathways ◽  
Specific Activity ◽  
Specific Activities ◽  
Urinary Porphyrins

1. The specific activities of urinary uroporphyrin and coproporphyrin were measured as functions of time following the administration of a single oral dose of [4-14C] δ-aminolaevulic acid (ALA) to six patients with symptomatic porphyria and one control subject. 2. The peak specific activity of coproporphyrin preceded that of uroporphyrin in all subjects studied and exceeded that of uroporphyrin in the patients with symptomatic porphyria. 3. These results are interpreted as indicating the existence of two distinct metabolic pathways in the liver for the disposal of ALA, rather than as contradicting the generally accepted role of uroporphyrinogen as a precursor of coproporphyrinogen.

Proteinsynthese in grünen Blättern

1964 ◽  
Vol 19 (3) ◽  
pp. 235-248 ◽  
Author(s):  
Benno Parthier
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Subcellular Fractions ◽  
Mechanical Destruction ◽  
Cell Organization ◽  
Specific Activities ◽  
Short Time ◽  
Natural Cell

In the green leaves of Nicotiana rustica, protein synthesis of various subcellular fractions has been investigated in vivo after 14CO2-photosynthesis and also in vitro by incorporation of radioactive amino acids. Following photosynthesis, homogenization of the tissues, and differential centrifugation of the homogenates, the results show that all structural particles of the cell are able to use photosynthetically formed amino acids for the incorporation into their proteins. The proteins with the highest specific activities are found in the mitochondria-rich fractions, and with the lowest in the soluble cytoplasma supernatant. High specific activities are also observed in the ribosomal-rich fraction in short-time experiments, and also in the chloroplasts after exposure of the leaves to light. After an osmotic-mechanical destruction of the isolated 14C-labelled chloroplasts, the specific activities of lamellar proteins exceed the colourless soluble proteins of the chloroplasts. A green fraction, sedimented at 1,000 g, and perhaps mainly consisting of broken and leached chloroplasts, shows the highest specific activity of all chloroplast fractions. Obviously, due to the destruction of the natural cell organization, in vitro experiments give not only drastically decreased specific activities but also another distribution of the incorporated amino acids between the subcellular fractions, compared with experiments in vivo.

scholarly journals Requirement of the C-terminal proline residue for stability of the Ca2+-activated photoprotein aequorin

1993 ◽  
Vol 293 (1) ◽  
pp. 181-185 ◽  
Author(s):  
N J Watkins ◽  
A K Campbell
Keyword(s):  
Light Emission ◽  
Specific Activity ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Proline Residue ◽  
Long Term Stability ◽  
Recombinant Aequorin

cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.

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.

EVIDENCE FOR THE INDUCTION OF URIDINE AND DEOXY-URIDINE PHOSPHORYLASES OF REGENERATING RAT LIVER IN VITRO

1965 ◽  
Vol 43 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Esther W. Yamada
Keyword(s):  
Amino Acids ◽  
Tissue Culture ◽  
Rat Liver ◽  
Culture Medium ◽  
Specific Activity ◽  
Tissue Culture Medium ◽  
Uridine Phosphorylase ◽  
Regenerating Rat Liver ◽  
Specific Activities

Increases in the specific activities of undine and deoxyuridine phosphorylases of slices of regenerating rat liver were found 4 hours after incubation in tissue-culture medium containing uridine or 6-azauridine. These increases were not found when the tissue-culture medium contained either 8-azaguanine or puromycin, or when it lacked amino acids. Although both uridine and 6-azauridine were more effective in increasing the specific activity of uridine phosphorylase than that of deoxyuridine phosphorylase, azauridine was more effective than uridine in increasing the specific activities of both enzymes.In time studies, in which slices of regenerating rat liver were incubated in tissue-culture medium containing optimal concentrations of uridine, the specific activities of the two enzymes reached maximum levels at 3–4 hours. Puromycin prevented these increases.

THE RELATIONSHIP BETWEEN CORTICOSTERONE SYNTHESIS FROM ENDOGENOUS PRECURSORS AND FROM ADDED RADIOACTIVE PRECURSORS BY RAT ADRENAL TISSUE IN VITRO

1966 ◽  
Vol 36 (3) ◽  
pp. 231-238 ◽  
Author(s):  
G. P. VINSON
Keyword(s):  
Specific Activity ◽  
Single Point ◽  
Biosynthetic Pathways ◽  
Activity Change ◽  
Adrenal Tissue ◽  
Specific Activities ◽  
The Relationship ◽  
Synthesis Of Hormones ◽  
Venous Plasma

SUMMARY A kinetic study of corticosterone recovered during incubation of rat adrenal tissue with [4-14C]progesterone and [16-3H]pregnenolone shows that both its 3H: 14C ratio and its specific activity change with time throughout incubation. Corticosterone is itself metabolized, and hypotheses based on the rate of production of corticosterone may be therefore deceptive when the amount is measured at a single point in time. Thus disparities between the specific activities of products and intermediates may be expected which alone do not necessarily indicate differences between the biosynthetic pathways involved in production from endogenous precursors and those from added radioactive precursors. Other experiments suggest that steroids in incubation media, in concentrations comparable with those found in adrenal venous plasma, do not inhibit the continued synthesis of hormones.

scholarly journals Oxidative Metabolism in ‘Valencia’ Sweet Orange (Citrus sinensis Osbeck) Abscission Zone Tissue Treated with the Abscission Agent 5-Chloro-3-Methyl-4-Nitro-1H-Pyrazole

HortScience ◽  
2016 ◽  
Vol 51 (4) ◽  
pp. 377-382 ◽  
Author(s):  
Naveen Kumar ◽  
Robert C. Ebel
Keyword(s):  
Oxidative Metabolism ◽  
Indole Acetic Acid ◽  
Specific Activity ◽  
Sweet Orange ◽  
Malonic Dialdehyde ◽  
Citrus Fruit ◽  
Abscission Zone ◽  
Specific Activities ◽  
Orange Trees

5-Chloro-3-methyl-4-nitro-1H-pyrazole (CMNP) is an abscission agent, standardized for the mechanical harvesting of late season ‘Valencia’ sweet oranges in Florida. This work was conducted to investigate the role of CMNP to induce oxidative stress in the abscission zone (AZ) of ‘Valencia’ sweet orange. Fully mature ‘Valencia’ sweet orange trees in a commercial grove were sprayed with 2.0 mm of CMNP. The experiment was repeated three times during the Apr.–May 2013 harvest season. Fruit were harvested at 0, 1, 2, and 3 days after CMNP application. Hydrogen peroxide (H2O2) concentration and malonic dialdehyde (MDA) concentration, as well as superoxide dismutase (SOD), ascorbate peroxidase (APOD), glutathione reductase (GR), peroxidase (POD), and lipoxygenase (LOX) specific activities were measured 0, 1, 2, and 3 days after CMNP treatment (DAT). Rate of lipid peroxidation remains unchanged throughout the abscission period. However, LOX activity increased 1 DAT in AZ of treated fruit, which might produce jasmonic acid (JA), known to promote abscission in citrus. Levels of H2O2 were similar in the AZ of control and treated fruit except at 3 DAT. The specific activity of SOD declined at 2 DAT, which showed compromised SOD defense against superoxide radicals (O·−). APOD activity declined sharply at 3 DAT. Interestingly, GR activity was 1.9-fold higher in CMNP-treated fruit at 3 DAT. Higher GR and low APOD activity reflects limited functioning of the APOD/GR cycle (e.g., APOD and GR) in scavenging of H2O2 at 3 DAT. Guaiacol POD activity transiently increased at 1 DAT and then declined. POD plays an important role in cell wall lignification and indole acetic acid (IAA) oxidation. The decline in POD activity may cause a decrease in lignification while higher activity made the AZ sensitive to ethylene and thus promote abscission in citrus fruit. This work also showed that CMNP-induced abscission is a collaborative effort of oxidative metabolism in flavedo tissue (FT) and AZ.

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