scholarly journals Riboflavin-dependent expression of flavoenzymes of the nicotine regulon of Arthrobacter oxidans

1990 ◽  
Vol 270 (3) ◽  
pp. 673-678 ◽  
Author(s):  
R Brandsch ◽  
V Bichler
Keyword(s):  
Specific Activity ◽  
Enzyme Protein ◽  
Western Blots ◽  
Cell Extracts ◽  
Arthrobacter Oxidans ◽  
Steady State Levels ◽  
Minimal Concentration ◽  
Mutant Cells ◽  
In Cells

In cells of an Arthrobacter oxidans riboflavin-dependent mutant the specific activity of the DL-nicotine-inducible nicregulon enzymes nicotine dehydrogenase (NDH, EC 1.5.99.4), 6-hydroxy-L-nicotine oxidase (6-HLNO, EC 1.5.3.5) and 6-hydroxy-D-nicotine oxidase (6-HDNO, EC 1.5.3.6) was shown to be dependent on the supply of the vitamin in the growth medium. Experiments designed to identify at which level riboflavin directs the biosynthesis of these flavoenzymes revealed that the steady-state levels of enzyme protein analysed on Western blots correlated directly with riboflavin supply from the minimal concentration of 0.5 microns-riboflavin required for growth up to 8 microns-riboflavin. Mutant cells grown at the higher riboflavin concentration showed on dot-blots increased levels of RNA which hybridized to 32P-labelled probes derived from the nic-regulon genes. When cells grown at 2 microns-riboflavin were shifted to 8 microns-riboflavin, 6-HDNO expression increased as indicated by elevated enzyme and RNA levels. When the rates of synthesis of the 6-HDNO and 6-HLNO polypeptides after DL-nicotine induction was analysed in cells grown at 0.5 microns and 8 microns-riboflavin, only cells grown at the higher riboflavin concentration showed on Western blots an accumulation of the polypeptides. No 6-HDNO or 6-HLNO polypeptide was identified in cell extracts from cells grown on 0.5 microns-riboflavin. Pulse-chase experiments with [35S]methionine showed that 6-HDNO- and 6-HLNO synthesis was prevented in cells grown at the low riboflavin concentration. The absence of detectable enzyme levels seemed not to be caused by proteolytic breakdown. Incubation in vitro of apo-6HDNO with low- or high-riboflavin-grown-cell extracts showed no increased proteolytic activity in 0.5 microns-riboflavin-grown cells. From these results it is concluded that riboflavin supply co-regulates the expression of the nicregulon genes at the level of transcription and/or mRNA turnover.

scholarly journals Binding of FAD to 6-hydroxy-d-nicotine oxidase apoenzyme prevents degradation of the holoenzyme

1989 ◽  
Vol 258 (1) ◽  
pp. 187-192 ◽  
Author(s):  
R Brandsch ◽  
V Bichler ◽  
B Krauss
Keyword(s):  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Cysteine Proteinase Inhibitor ◽  
Western Blots ◽  
E Coli ◽  
Cell Extracts ◽  
Arthrobacter Oxidans ◽  
Cloned Gene

Expression of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene from Arthrobacter oxidans cloned into Escherichia coli showed a marked temperature-dependence. Transformed E. coli cells grown at 30 degrees C exhibited a several-fold higher 6-HDNO activity than did cells grown at 37 degrees C. This effect did not depend on the promoter used for expression of the cloned gene in E. coli, nor was it an effect of 6-HDNO mRNA instability at 37 degrees C. Studies performed in vivo and in vitro revealed that an increased susceptibility of apo-6-HDNO to proteolytic attack at 37 degrees C was responsible for the observed phenomenon. Extracts from cells grown at 37 degrees C showed on Western blots a decrease in immunologically detectable 6-HDNO polypeptide when compared with extracts from cells grown at 30 degrees C. The 6-HDNO polypeptide is covalently modified by attachment of the cofactor FAD to a histidine residue. It could be shown that covalent flavinylation of the apoenzyme in vitro, i.e. formation of holoenzyme, by incubation of cell extracts with FAD and phosphoenolpyruvate protected the 6-HDNO polypeptide from degradation at 37 degrees C. Of a variety of proteinase inhibitors tested only the cysteine-proteinase inhibitor L-3-trans-carboxyoxiran-2-carbonyl-L-leucylagmatine (E64) prevented degradation, by up to 70%, of the apoenzyme.

scholarly journals Optimization of an In Vitro Transcription/Translation System Based on Sulfolobus solfataricus Cell Lysate

Archaea ◽  
2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Giada Lo Gullo ◽  
Rosanna Mattossovich ◽  
Giuseppe Perugino ◽  
Anna La Teana ◽  
Paola Londei ◽  
...  
Keyword(s):  
High Efficiency ◽  
Specific Activity ◽  
Expression System ◽  
Sulfolobus Solfataricus ◽  
Essential Element ◽  
23S Rrna ◽  
Translation System ◽  
Cell Lysate ◽  
Cell Extracts

A system is described which permits the efficient synthesis of proteins in vitro at high temperature. It is based on the use of an unfractionated cell lysate (S30) from Sulfolobus solfataricus previously well characterized in our laboratory for translation of pretranscribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the S. solfataricus 16S/23S rRNA-encoding gene, from which specific mRNAs may be transcribed with high efficiency. The synthesis of two different proteins is reported, including the S. solfataricus DNA-alkylguanine-DNA-alkyl-transferase protein (SsOGT), which is shown to be successfully labeled with appropriate fluorescent substrates and visualized in cell extracts. The simplicity of the experimental procedure and specific activity of the proteins offer a number of possibilities for the study of structure-function relationships of proteins.

Omeprazole decreases H(+)-K(+)-ATPase protein and increases permeability of oxyntic secretory membranes in rabbits

1993 ◽  
Vol 265 (2) ◽  
pp. G231-G241 ◽  
Author(s):  
J. M. Crothers ◽  
D. C. Chow ◽  
J. G. Forte
Keyword(s):  
Atpase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Protein ◽  
Western Blots ◽  
Uptake Assay ◽  
Protein Incorporation ◽  
Gradient Medium ◽  
Density Gradient Sedimentation

Amounts and fractional distributions of gastric H(+)-K(+)-adenosinetriphosphatase (ATPase) activity and H(+)-K(+)-ATPase protein as well as properties of H(+)-K(+)-ATPase-containing membranes were studied in rabbits injected with omeprazole (OM; 1 mg/kg sc twice daily for 5 days). Total H(+)-K(+)-ATPase activity decreased to 22 +/- 2% of control (n = 4). Densitometry of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots showed H(+)-K(+)-ATPase protein was decreased to 60-70% of control. In vitro reduction of the enzyme-OM disulfide bond with 0.1 M 2-mercaptoethanol increased microsomal H(+)-K(+)-ATPase activity to 56 +/- 7% of control (n = 3), consistent with a substantial decrease in enzyme protein. Incorporation of 35S-labeled methionine for 30 min before death resulted in 2.2-fold more label per unit of microsomal alpha-subunit protein (5 days OM vs. control). Thus the decrease in enzyme protein resulted from increased breakdown rather than decreased synthesis. A striking shift in distribution of H(+)-K(+)-ATPase-containing microsomes (tubulovesicles) on sucrose gradients reflected slow equilibration of most control vesicles with the gradient medium and faster equilibration after 5 days OM, indicating increased permeability. After 5 days OM, microsomal vesicle acidification (by acridine orange uptake assay) was negligible, even with 2-mercaptoethanol treatment, and H+ leakage on sudden delta pH was faster than control. We conclude that extended OM treatment not only inhibits H(+)-K(+)-ATPase but accelerates its breakdown and renders H(+)-K(+)-ATPase-containing membranes more permeable. It is thus possible that increased backward H+ flux contributes to profound inhibition of acid secretion during extended omeprazole treatment. In parallel experiments, H(+)-K(+)-ATPase activity and density gradient sedimentation of tubulovesicles returned to near normal 3 days after OM withdrawal.

IN VITRO IMMUNE PARAMETERS OF MONOCLATEH, A, MONOCLONAL ANTIBODY PURIFIED HUMAN PLASMA FACTOR VIII:C THERAPEUTIC PREPARATION

1987 ◽  
Author(s):  
A B Schreiber ◽  
R Gillette ◽  
M E Hrinda
Keyword(s):  
Human Plasma ◽  
Specific Activity ◽  
Cell Mediated Immunity ◽  
Western Blots ◽  
Immune Parameters ◽  
Plasma Factor ◽  
Immune Impairment ◽  
Human Proteins ◽  
Lymphocyte Mitogenesis

MonoclateR is a highly purified human factor VIII:C preparation obtained by immunoaffinity chromatography of plasma cryoprecipitat followed by chromatography on aminohexyl sepharose. The resulting product is devoid of alloantigens, can be stabilized in the absence of extraneous human proteins and has a specific activity higher than 3000 U/mg.To ascertain product integrity, we set out to compare MonoclateR preparations with either unfractionated human plasma, cryoprecipitateor commercially available lyophilized antihemophilia factor (AHF) preparations by Western blots. As probes, we utilized a series of monoclonal antibodies to defined factor VIII:C epitopes. The MonoclateR VIII:C chain composition appeared indistinguishable from that in the native state of in commercial, clinically useful preparations.Patients with hemophilia are often afflicted with an impairment of cell-mediated immunity, when treated repeatedly with plasma cryoprecipitate preparations. Those preparations, containing at best 0.2% factor VIII:C in protein content, were found in our hands to dramatically inhibit both human mixed lymphocyte reactions and the phytohemagglutinin induced human lymphocyte mitogenesis. We obtained 50% inhibition at about 1 mg/ml protein concentration. These effects were not due to cytotoxicity nor related to procoagulant potency.MonoclateR, in contrast, was totally devoid of immunosuppressive activity at all concentrations tested. This in vitro finding may be related to clinical observation of corrections in immune impairment in adult hemophiliacs treated with MonoclateR. With this immunological profile, MonoclateR represents in our opinion, a promising novel treatment modality for hemophilia A.

scholarly journals The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.

1996 ◽  
Vol 16 (11) ◽  
pp. 6020-6028 ◽  
Author(s):  
S van Nocker ◽  
S Sadis ◽  
D M Rubin ◽  
M Glickman ◽  
H Fu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Protein ◽  
Degradation Pathway ◽  
26S Proteasome ◽  
Wild Type ◽  
Cell Extracts ◽  
Steady State Levels ◽  
Type Rate ◽  
Beta Galactosidase

The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.

Induction of stable microtubules in 3T3 fibroblasts by TGF-beta and serum

1994 ◽  
Vol 107 (3) ◽  
pp. 645-659 ◽  
Author(s):  
G.G. Gundersen ◽  
I. Kim ◽  
C.J. Chapin
Keyword(s):  
Growth Factors ◽  
Calf Serum ◽  
Tgf Beta ◽  
Wound Edge ◽  
Western Blots ◽  
Polypeptide Growth Factors ◽  
3T3 Fibroblasts ◽  
Mild Acid ◽  
In Cells

Previous studies have shown that fibroblasts induced to migrate into an in vitro wound rapidly generate an array of stable, post-translationally detyrosinated microtubules (Glu MTs) oriented toward the direction of migration. To understand how cells generate a stable array of MTs at a specific location, we have analyzed the contribution of media components to the formation of oriented Glu MTs in wounded monolayers of 3T3 fibroblasts. When confluent monolayers were placed in serum-free medium (SFM) for 2 days before wounding, the cells contained virtually no Glu MTs or nocodazole-resistant MTs and were incapable of generating Glu MTs in response to wounding. Such SFM-treated monolayers were capable of generating oriented Glu MTs within 1 hour of wounding, if calf serum (CS) was added back to the medium. The Glu MTs in the CS refed cells were oriented toward the wound in cells at the wound edge, and were juxtanuclear in cells within the monolayer, demonstrating that CS restored the Glu MT array characteristic of each cell type. To determine the nature of the ‘Glu MT-inducing’ factor in CS, we subjected CS to different treatments and found that the CS factor was nondialyzable, resistant to heat, mild acid and trypsin, but inactivated by treatment with dithiothreitol. The factor was not absorbed by charcoal and was present in lipoprotein-deficient serum. These properties are consistent with the properties of a number of polypeptide growth factors, so we screened purified growth factors for their ability to induce Glu MTs in wounded SFM-treated monolayers. Of all the growth factors tested, only TGF-beta 1 and TGF-beta 2 induced a significant level (> or = 70% of the CS response) of oriented Glu MTs. The SFM-treated cells were exquisitely sensitive to TGF-beta 1, with significant induction of Glu MTs observed at 0.01 ng/ml TGF-beta 1. Induction of Glu MTs observed by immunofluorescence after CS or TGF-beta treatments were paralleled by increases in Glu tubulin detected on western blots. The Glu MTs formed after either CS or TGF-beta 1 treatment showed enhanced resistance to nocodazole, confirming that both treatments increased the level of stable MTs in cells. The TGF-beta 1 induction of stable MTs was slower than that of CS (2-4 hours onset versus 1 hour onset), but by 24 hours the level of MT stabilization in TGF-beta 1 was even greater than that in CS.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Interleukin-6 mRNA and protein increase in vivo following induction of acute thrombocytopenia in mice

Blood ◽  
1991 ◽  
Vol 77 (2) ◽  
pp. 286-293 ◽  
Author(s):  
LH Cox ◽  
T Downs ◽  
K Dagg ◽  
J Henthorn ◽  
SA Burstein
Keyword(s):  
Specific Activity ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Experimental Animals ◽  
Steady State Levels ◽  
Phosphate Buffered Saline ◽  
Protein Serum ◽  
Significant Increment

Abstract Induction of experimental thrombocytopenia in rodents results in the enhancement of megakaryocytic growth and differentiation. Interleukin-3 (IL-3) and IL-6, cytokines with a broad spectrum of biologic activities, stimulate megakaryocytopoiesis in vitro. To determine if expression of these factors might increase in response to experimental thrombocytopenia, we measured steady-state levels of IL-3 and IL-6 mRNA following rabbit antiplatelet serum (APS) injection. Groups of mice were injected intravenously with 0.2 mL APS while control animals received rabbit antilymphocyte serum (ALS), normal rabbit serum (NRS), or phosphate-buffered saline (PBS). At various times up to 72 hours after injection mice were exsanguinated and splenectomized. Platelet counts in the experimental animals were less than 12% of controls. Splenic RNA was hybridized in solution to 32P-UTP-labeled cRNA probes for IL-3 and IL-6. RNase-resistant hybrids were resolved on denaturing gels and visualized autoradiographically. IL-3 hybrids were undetectable at all time points tested, irrespective of the film exposure time or specific activity of the probe. Conversely, IL-6 hybrids were easily visualized and showed peak expression at 1.5 to 2.0 hours. By 3 hours, IL-6 mRNA had returned almost to the level of the controls. Similar results were observed in the bone marrow, although maximal IL-6 mRNA in that tissue was observed 4 hours following APS administration. To determine if this mRNA increment was associated with a concomitant increase in bioactive protein, serum was tested for its ability to stimulate IL-6-dependent B9 cells. At 1.75 hours following injection, experimental animals showed a small but significant increment in IL-6 activity compared with controls (200 +/- 30 U/mL IL-6 compared with 129 +/- 17 U/mL in ALS-injected controls, 106 +/- 17 U/mL in NRS-injected controls and 84 +/- 17 U/mL in PBS-injected controls). The data show that IL-6 mRNA and bioactive protein increase in response to acute immunothrombocytopenia, while no increment in IL-3 is detectable. These results suggest that IL-6 may play a role in the physiologic response to acute immunothrombocytopenia.

scholarly journals The phosphorylation of Escherichia coli isocitrate dehydrogenase in intact cells

1984 ◽  
Vol 222 (3) ◽  
pp. 797-804 ◽  
Author(s):  
A C Borthwick ◽  
W H Holms ◽  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Isocitrate Dehydrogenase ◽  
Intact Cells ◽  
Cell Extracts ◽  
In Cells

The isocitrate dehydrogenase of Escherichia coli ML308 can be reversibly activated by addition of pyruvate to cells growing on acetate [Bennett & Holms (1975) J. Gen. Microbiol. 87, 37-51]. By using cells pulse-labelled with [32P]Pi we showed that the activation and inactivation of the enzyme in these conditions correlate with its dephosphorylation and rephosphorylation respectively. Incubation of cell extracts prepared during an activation/inactivation cycle with purified isocitrate dehydrogenase phosphatase confirmed that the pyruvate-induced activation of the dehydrogenase goes essentially to completion. The results show that the reversible changes in the activity of the dehydrogenase in cells grown on acetate are solely due to phosphorylation/dephosphorylation. Inactive 32P-labelled isocitrate dehydrogenase was isolated from cells incubated with [32P]Pi in the presence of acetate. Both this material and purified enzyme phosphorylated in vitro were digested with chymotrypsin, and the phosphopeptides were isolated and analysed. Only one phosphopeptide was observed in each case; the results show that the residue phosphorylated in vivo is identical with that phosphorylated by purified isocitrate dehydrogenase kinase in vitro.

N-Myristoylation is essential for protein phosphatases PPM1A and PPM1B to dephosphorylate their physiological substrates in cells

2013 ◽  
Vol 449 (3) ◽  
pp. 741-749 ◽  
Author(s):  
Toko Chida ◽  
Masakatsu Ando ◽  
Tasuku Matsuki ◽  
Yutaro Masu ◽  
Yuko Nagaura ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Specific Activity ◽  
Wild Type ◽  
Α Subunit ◽  
Nitrophenyl Phosphate ◽  
Dependent Protein ◽  
Amp Activated Protein Kinase ◽  
Physiological Substrates ◽  
In Cells

PPM [metal-dependent protein phosphatase, formerly called PP2C (protein phosphatase 2C)] family members play essential roles in regulating a variety of signalling pathways. While searching for protein phosphatase(s) that act on AMPK (AMP-activated protein kinase), we found that PPM1A and PPM1B are N-myristoylated and that this modification is essential for their ability to dephosphorylate the α subunit of AMPK (AMPKα) in cells. N-Myristoylation was also required for two other functions of PPM1A and PPM1B in cells. Although a non-myristoylated mutation (G2A) of PPM1A and PPM1B prevented membrane association, this relocalization did not likely cause the decreased activity towards AMPKα. In in vitro experiments, the G2A mutants exhibited reduced activities towards AMPKα, but much higher specific activity against an artificial substrate, PNPP (p-nitrophenyl phosphate), compared with the wild-type counterparts. Taken together, the results of the present study suggest that N-myristoylation of PPM1A and PPM1B plays a key role in recognition of their physiological substrates in cells.

scholarly journals A New Family of Bacteriolytic Proteins in Dictyostelium discoideum

2021 ◽  
Vol 10 ◽  
Author(s):  
Cyril Guilhen ◽  
Wanessa C. Lima ◽  
Estelle Ifrid ◽  
Xenia Crespo-Yañez ◽  
Otmane Lamrabet ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Phagocytic Cells ◽  
Bacterial Killing ◽  
Bacteriolytic Activity ◽  
New Family ◽  
Cell Extracts ◽  
New Strategy ◽  
Domain Of Unknown Function ◽  
In Cells

Phagocytic cells ingest and destroy bacteria efficiently and in doing so ensure the defense of the human body against infections. Phagocytic Dictyostelium discoideum amoebae represent a powerful model system to study the intracellular mechanisms ensuring destruction of ingested bacteria in phagosomes. Here, we discovered the presence of a bacteriolytic activity against Klebsiella pneumoniae in cellular extracts from D. discoideum. The bacteriolytic activity was detected only at a very acidic pH mimicking the conditions found in D. discoideum phagosomes. It was also strongly decreased in extracts of kil1 KO cells that were previously described to kill inefficiently internalized bacteria, suggesting that the activity observed in vitro is involved in killing of bacteria in phagosomes. We purified a fraction enriched in bacteriolytic activity where only 16 proteins were detected and focused on four proteins selectively enriched in this fraction. Three of them belong to a poorly characterized family of D. discoideum proteins exhibiting a DUF3430 domain of unknown function and were named BadA (Bacteriolytic D. discoideum A), BadB, and BadC. We overexpressed the BadA protein in cells, and the bacteriolytic activity increased concomitantly in cell extracts. Conversely, depletion of BadA from cell extracts decreased significantly their bacteriolytic activity. Finally, in cells overexpressing BadA, bacterial killing was faster than in parental cells. Together these results identify BadA as a D. discoideum protein required for cellular bactericidal activity. They also define a new strategy to identify and characterize bactericidal proteins in D. discoideum cells.

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