A cdc2-like kinase distinct from cdk5 is associated with neurofilaments

1996 ◽  
Vol 109 (6) ◽  
pp. 1565-1573
Author(s):  
R. Starr ◽  
F.L. Hall ◽  
M.J. Monteiro
Keyword(s):  
Molecular Mass ◽  
Protein Kinases ◽  
Kinase Inhibitor ◽  
Rat Tissues ◽  
Immunoprecipitation Assay ◽  
Sds Page ◽  
Brain Extracts ◽  
Phosphoamino Acid Analysis ◽  
H Protein

An immunoprecipitation assay was used to identify protein kinases which are physically associated with neurofilaments (NF) in mouse brain extracts. Using this approach, we show that a cdc2-related kinase is associated with NF. The cdc2-related kinase was found to be distinct from cdk5 and the authentic cdc2 by a number of criteria. Firstly, it has a molecular mass on SDS-PAGE gels of 34 kDa, similar to that of cdc2, but differing from cdk5 (31 kDa). Secondly, it is not recognized by an antibody specific for cdk5. Thirdly, it is recognized by an antibody raised against the C-terminal region of authentic cdc2, but not by an antibody specific for the PSTAIRE motif. Using immunoblotting, we further show that the cdc2-related kinase copurifies with NF isolated from rat tissues. In vitro kinase assays further demonstrated that immunoprecipitated cdc2-related kinase phosphorylates recombinant NF-H protein. Phosphorylation of NF-H by the cdc2-like activity was not affected by 3 microM olomoucine but was inhibited by 10 microM of this kinase inhibitor. Phosphoamino acid analysis of in vitro phosphorylated NF-H indicates that the immunoprecipitated cdc2-related kinase phosphorylates serine residues.

scholarly journals Cyclin-dependent kinase inhibitor 1B (CDKN1B) gene variants in AIP mutation-negative familial isolated pituitary adenoma kindreds

2012 ◽  
Vol 19 (3) ◽  
pp. 233-241 ◽  
Author(s):  
Maria A Tichomirowa ◽  
Misu Lee ◽  
Anne Barlier ◽  
Adrian F Daly ◽  
Ilaria Marinoni ◽  
...  
Keyword(s):  
Pituitary Adenoma ◽  
Pituitary Adenomas ◽  
Kinase Inhibitor ◽  
Carney Complex ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Pituitary Tumorigenesis ◽  
Sds Page ◽  
Clinicopathological Profile ◽  
Low Incidence

Familial isolated pituitary adenoma (FIPA) occurs in families and is unrelated to multiple endocrine neoplasia type 1 and Carney complex. Mutations inAIPaccount only for 15–25% of FIPA families.CDKN1Bmutations cause MEN4 in which affected patients can suffer from pituitary adenomas. With this study, we wanted to assess whether mutations inCDKN1Boccur among a large cohort ofAIPmutation-negative FIPA kindreds. Eighty-eightAIPmutation-negative FIPA families were studied and 124 affected subjects underwent sequencing ofCDKN1B. Functional analysis of putativeCDKN1Bmutations was performed usingin silicoandin vitroapproaches. GermlineCDKN1Banalysis revealed two nucleotide changes: c.286A>C (p.K96Q) and c.356T>C (p.I119T).In vitro, the K96Q change decreased p27 affinity for Grb2 but did not segregate with pituitary adenoma in the FIPA kindred. The I119T substitution occurred in a female patient with acromegaly. p27I119Tshows an abnormal migration pattern by SDS–PAGE. Three variants (p.S56T, p.T142T, and c.605+36C>T) are likely nonpathogenic becauseIn vitroeffects were not seen. In conclusion, two patients had germline sequence changes inCDKN1B, which led to functional alterations in the encoded p27 proteinsin vitro. Such rareCDKN1Bvariants may contribute to the development of pituitary adenomas, but their low incidence and lack of clear segregation with affected patients makeCDKN1Bsequencing unlikely to be of use in routine genetic investigation of FIPA kindreds. However, further characterization of the role ofCDKN1Bin pituitary tumorigenesis in these and other cases could help clarify the clinicopathological profile of MEN4.

scholarly journals Major tegument protein VP8 of bovine herpesvirus 1 is phosphorylated by viral US3 and cellular CK2 protein kinases

2009 ◽  
Vol 90 (12) ◽  
pp. 2829-2839 ◽  
Author(s):  
Shaunivan L. Labiuk ◽  
Lorne A. Babiuk ◽  
Sylvia van Drunen Littel-van den Hurk
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Gene Product ◽  
Kinase Inhibitor ◽  
Bovine Herpesvirus ◽  
Kinase Assay ◽  
Bovine Herpesvirus 1 ◽  
Infected Cells ◽  
Herpesvirus 1

The UL47 gene product, VP8, is one of the major tegument proteins of bovine herpesvirus 1 (BoHV-1) and is subject to phosphorylation. Analysis of protein bands co-immunoprecipitated with VP8 from BoHV-1-infected cells by mass spectroscopy suggested that VP8 interacts with two protein kinases: cellular CK2 and viral US3. CK2 is a highly conserved cellular protein, expressed ubiquitously and known to phosphorylate numerous proteins. The US3 gene product is one of the viral kinases produced by BoHV-1 during infection. Interactions of CK2 and US3 with VP8 were confirmed outside the context of infection when FLAG–VP8 was expressed alone or co-expressed with US3–haemagglutinin tag in Cos-7 cells. Furthermore, VP8 and US3 were found to co-localize in the nucleus during viral infection. To explore the significance of these interactions, an in vitro kinase assay was performed, which demonstrated that VP8 is heavily phosphorylated by CK2. In the presence of the highly specific CK2 kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), phosphorylation of VP8 was significantly reduced. Phosphorylation of VP8 was also inhibited by the presence of kenpaullone, a less specific CK2 inhibitor, but not by protein kinase CK1 or protein kinase C inhibitors. When VP8 and US3 were both included in the kinase assay in the presence of DMAT, phosphorylation of VP8 was again observed. Autophosphorylation of US3 was also detected and was not inhibited by DMAT. Based on these results, it is proposed that VP8 interacts with cellular CK2 and viral US3 in BoHV-1-infected cells, and is in turn subject to kinase activities associated with both of these proteins.

scholarly journals The effects of 6 hours of hypoxia on protein synthesis in rat tissues in vivo and in vitro

1985 ◽  
Vol 228 (1) ◽  
pp. 179-185 ◽  
Author(s):  
V R Preedy ◽  
D M Smith ◽  
P H Sugden
Keyword(s):  
Protein Synthesis ◽  
Gastric Emptying ◽  
Skeletal Muscles ◽  
Rat Tissues ◽  
Tissue Nitrogen ◽  
The Brain ◽  
H Protein

Rates of protein synthesis were measured in vivo in several tissues (heart, skeletal muscles, liver, tibia, skin, brain, kidney, lung) of fed rats exposed to O2/N2 (1:9) for 6 h starting at 08:00-11:00 h. Protein synthesis rates were depressed by 15-35% compared with normoxic controls in all of the tissues studied. The decreases were greatest in the brain and the skin. Although hypoxia inhibited gastric emptying, its effects on protein synthesis could probably not be attributed to its induction of a starved state, because protein-synthesis rates in brain and skin were not decreased by a 15-18 h period of starvation initiated at 23:00 h. Furthermore, we showed that protein synthesis was inhibited by hypoxia in the rat heart perfused in vitro, suggesting a direct effect. The role of hypoxia in perturbing tissue nitrogen balance in various physiological and pathological states is discussed.

Heterotrophic nitrogen removal in Bacillus sp. K5: involvement of a novel hydroxylamine oxidase

2017 ◽  
Vol 76 (12) ◽  
pp. 3461-3467 ◽  
Author(s):  
Yunlong Yang ◽  
Ershu Lin ◽  
Shaobin Huang
Keyword(s):  
Molecular Mass ◽  
Electron Acceptor ◽  
Critical Role ◽  
Gel Filtration ◽  
Nitrite Accumulation ◽  
Bacillus Sp ◽  
Ammonium N ◽  
Sds Page ◽  
Bacillus Genus

Abstract An aerobic denitrifying bacterium isolated from a bio-trickling filter treating NOx, Bacillus sp. K5, is able to convert ammonium to nitrite, in which hydroxylamine oxidase (HAO) plays a critical role. In the present study, the performance for simultaneous nitrification and denitrification was investigated with batch experiments and an HAO was purified by an anion-exchange and gel-filtration chromatography from strain K5. The purified HAO's molecular mass was determined by SDS-PAGE and its activity by measuring the change in the concentration of ferricyanide, the electron acceptor. Results showed that as much as 87.8 mg L−1 ammonium-N was removed without nitrite accumulation within 24 hours in the sodium citrate medium at C/N of 15. The HAO isolated from the strain K5 was approximately 71 KDa. With hydroxylamine (NH2OH) as a substrate and potassium ferricyanide as an electron acceptor, the enzyme was capable of oxidizing NH2OH to nitrite in vitro when the pH varied from 7 to 9 and temperature ranged from 25 °C to 40 °C. This is the first time that an HAO has been purified from the Bacillus genus, and the findings revealed that it is distinctive in its molecular mass and enzyme properties.

Distribution of the adrenocortical inner zone antigen

1994 ◽  
Vol 141 (3) ◽  
pp. 459-466 ◽  
Author(s):  
M M Ho ◽  
S Barker ◽  
G P Vinson
Keyword(s):  
Molecular Mass ◽  
Adrenal Cortex ◽  
Rat Hepatocytes ◽  
Immunoblot Analysis ◽  
Detection Methods ◽  
Rat Tissues ◽  
Renal Tubules ◽  
Immunoreactive Protein

Abstract Previous studies using a mouse monoclonal antibody (IZAb) have identified inner zone-specific antigens (IZAg 1 and 2) in the rat adrenal cortex. The expression of IZAgl is enhanced by dibutyryl-cyclic AMP or ACTH in vitro and by in vivo ACTH treatment. These studies also suggest that IZAg may be involved in steroidogenesis in rat adrenals. Using sensitive detection methods, the distribution of IZAg in adrenals from other species and in other rat tissues was studied. Pig and rabbit adrenals expressed an IZAb-immunoreactive protein with molecular mass identical with that of IZAg1, but no immunoreactivity was detected in bovine, guinea-pig or mouse adrenals. In the rabbit adrenal, as in the rat, IZAb bound only to zonae fasciculata/reticularis cells. However, the whole adrenal cortex in the pig immunoreacted with IZAb. Although immunofluorescence was observed in human adrenal sections, predominantly in the zona reticularis, no significant immunoreactive protein band was detected by immunoblot analysis. Apart from the zonae fasciculata/reticularis in the adrenal gland, an IZAb-immunoreactive protein with a molecular mass of 26 000 Da, corresponding to IZAg1, was also found in rat hepatocytes, renal tubules, ovary (corpus luteum, interstitial cells, theca interna of mature follicles) and Leydig cells. The observations suggest that IZAg may be involved in the processes of biosynthesis, metabolism and/or excretion of steroid hormones. Journal of Endocrinology (1994) 141, 459–466

scholarly journals A Strain of Bacillus thuringiensis Containing a Novel cry7Aa2 Gene That Is Highly Toxic to Leptinotarsa decemlineata (Say) (Coleoptera; Chrysomelidae)

2019 ◽  
Author(s):  
Mikel Dominguez- ◽  
Maite Villanueva ◽  
Ana Beatriz Fernandez ◽  
Primitivo Caballero
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Molecular Mass ◽  
Leptinotarsa Decemlineata ◽  
Insect Pests ◽  
Amino Acid Residues ◽  
Cry Protein ◽  
Sds Page ◽  
Neonate Larvae

The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity respect to Cry7Aa1 protein and a predicted molecular mass of 129.4 kDa. The primary structure of this Cry7Aa2 protein, which revealed the presence of eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that, after in vitro trypsin incubation, it was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae two proteinase-resistant fragments of 60 and 65 kDa were produced. Spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 (18.8 μg/ml), which was statistically equal to the estimated LC50 (20.8 μg/mL) for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 4 times approximately (LC50 = 4.9 μg/mL). The advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.

scholarly journals Synapsins as major neuronal Ca2+/S100A1-interacting proteins

1999 ◽  
Vol 344 (2) ◽  
pp. 577-583 ◽  
Author(s):  
Jörg HEIERHORST ◽  
Ken I. MITCHELHILL ◽  
Richard J. MANN ◽  
Tony TIGANIS ◽  
Andrew J. CZERNIK ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Double Labelling ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Brain Extracts ◽  
Dependent Protein

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca2+-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca2+/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca2+-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5′-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca2+-dependent and Zn2+-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.

scholarly journals Synthesis of 35S-labelled macromolecules by polymorphonuclear neutrophils. Evidence for the production of [35S]sulphite which can modify both endogenous and exogenous proteins

1992 ◽  
Vol 288 (2) ◽  
pp. 577-583 ◽  
Author(s):  
E E Gardiner ◽  
H C Robinson ◽  
A Sriratana ◽  
S S Mok ◽  
D A Lowther ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Anion Exchange ◽  
Incubation Medium ◽  
Cell Extract ◽  
Polymorphonuclear Neutrophils ◽  
Anion Exchange Column ◽  
Core Proteins ◽  
Sds Page ◽  
Molecular Masses

The incorporation of [35S]sulphate into macromolecules by rabbit peritoneal polymorphonuclear neutrophils (PMN) in vitro revealed that two major groups of 35S-labelled macromolecules were synthesized by these cells. The first group did not bind to anion-exchange columns at pH 6.0 and contained 60-80% of the total incorporated radiolabel. The second group did bind to anion-exchange columns at pH 6.0 and eluted as a single peak of radioactivity at an ionic strength characteristic of sulphated proteoglycans; it accounted for the remaining incorporated radiolabel. Analysis of this material on Sepharose CL-6B demonstrated that 35S-labelled macromolecules isolated from the cell extract migrated with Kav. of 0.36, while corresponding material isolated from the medium migrated with Kav. of 0.51. When subjected to electrophoresis on SDS/polyacrylamide gels the intact proteoglycan had a molecular mass of approx. 90 kDa and yielded two core proteins of molecular mass 31 kDa and 28 kDa after digestion with chondroitinase ABC. The peak of labelled macromolecules which did not bind to the anion-exchange column was found, by SDS/PAGE, to comprise 35S-labelled proteins of various molecular masses. The 35S label was displaced from this fraction by treatment with 0.1 M-sodium sulphite, suggesting that the radiolabel was in the form of an S-sulpho sulphite derivative. Using the sulphite-trapping agents N-2,4-dinitroanilinomaleimide and cyst(e)ine, [35S]sulphite was detected in the incubation medium of PMN, indicating that these cells were able to synthesize [35S]sulphite from [35S]sulphate. The release of [35S]sulphite from neutrophil cultures was calculated to be 78 pmol/h per 10(6) cells. When exogenous proteins were included in the incubation medium of cell cultures, the [35S]sulphite reacted with these proteins to form a stable 35S-labelled conjugate.

scholarly journals Glutamyl-tRNA Reductase of Chlorobium vibrioforme Is a Dissociable Homodimer That Contains One Tightly Bound Heme per Subunit

2005 ◽  
Vol 187 (13) ◽  
pp. 4444-4450 ◽  
Author(s):  
Alaka Srivastava ◽  
Samuel I. Beale
Keyword(s):  
Molecular Mass ◽  
Column Chromatography ◽  
Aminolevulinic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Affinity Column ◽  
Chlorobium Vibrioforme ◽  
Sds Page

ABSTRACT δ-Aminolevulinic acid, the biosynthetic precursor of tetrapyrroles, is synthesized from glutamate via the tRNA-dependent five-carbon pathway in the green sulfur bacterium Chlorobium vibrioforme. The enzyme glutamyl-tRNA reductase (GTR), encoded by the hemA gene, catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. To characterize the GTR protein, the hemA gene from C. vibrioforme was cloned into expression plasmids that added an N-terminal His6 tag to the expressed protein. The His-tagged GTR protein was purified using Ni affinity column chromatography. GTR was observable as a 49-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The native molecular mass, as determined by gel filtration chromatography, appeared to be approximately 40 kDa, indicating that native GTR is a monomer. However, when the protein was mixed with 5% (vol/vol) glycerol, the product had an apparent molecular mass of 95 kDa, indicating that the protein is a dimer under these conditions. Purified His6-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNAGlu and purified recombinant Chlamydomonas reinhardtii glutamate-1-semialdehyde aminotransferase. The expressed GTR contained 1 mol of tightly bound heme per mol of pep tide subunit. The heme remained bound to the protein throughout purification and was not removed by anion- or cation-exchange column chromatography. However, the bound heme was released during SDS-PAGE if the protein was denatured in the presence of β-mercaptoethanol. Added heme did not inhibit the activity of purified expressed GTR in vitro. However, when the GTR was expressed in the presence of 3-amino-2,3- dihydrobenzoic acid (gabaculine), an inhibitor of heme synthesis, the purified GTR had 60 to 70% less bound heme than control GTR, and it was inhibited by hemin in vitro.

The multiple alternative oxidase proteins of soybean

1999 ◽  
Vol 26 (4) ◽  
pp. 337 ◽  
Author(s):  
M. Tanudji ◽  
I. N. Djajanegara ◽  
D. O. Daley ◽  
T. C. McCabe ◽  
P. M. Finnegan ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Alternative Oxidase ◽  
Cultured Cells ◽  
Liver Mitochondria ◽  
Apparent Molecular Mass ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Processing Peptidase ◽  
Sds Page ◽  
Multiple Alternative

The identity of the multiple alternative oxidase bands detected in various soybean tissues was investigated to determine if any modification that can alter the mobility on SDS-PAGE of the alternative oxidase occurs after mitochondrial import other than removal of the presequence. Comparison of the mature, in vitro imported products of AOX1, AOX2 and AOX3 in soybean cotyledons and rat liver mitochondria indicated that they had an identical apparent molecular mass to their in vitro expressed mature forms. This suggests that no modification specific to plant alternative oxidase altering the mobility on SDS-PAGE, took place. Changing the –2 and/or –3 Arg residue resulted in the inhibition of the generation of this mature form, suggesting that processing was most likely by the general mitochondrial processing peptidase. Comparison of the in vitro expressed mature forms to that detected by immunoblots of soybean tissues, required the induction of AOX1. Treatment of soybean cultured cells with antimycin A resulted in the induction of an additional band cross-reacting to monoclonal antibodies against the alternative oxidase. Comparison of the in vitro expressed mature forms to the alternative oxidase detected by western blotting indicated that they were identical in apparent molecular mass. These results indicated that no modification other than presequence removal, which alters mobility on SDS-PAGE, was required to generate the mature functional alternative oxidase proteins.

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