Subject protein

Cervera and Levine [81] studied the mechanism of oxidative modification of glutamine synthetase from Escherichia coli. It was found that active oxygen species initially caused inactivation of the enzyme and generated a more hydrophilic protein, which still was not a substrate for the protease. Continuous action of oxygen species resulted in the formation of oxidized protein subjected to the proteolytic attack of protease. 

Protein kinase A an enzyme that catalyzes the ATP-dependent addition of phosphate to other proteins subject to regulation by cAMP. 

Conventionally, the first attribute known about an enzyme used to be its function, usually in a crude extract. This property was screened for in microbial cultures or in tissue samples. The crude extract was then purified to homogeneity and the protein subjected to biochemical studies to learn of its pH and T profiles, its pi and subunit composition, catalytically important residues, and other properties. Proteolytic digestion of the protein with subsequent Edman degradation led to the primary sequence, but no information on the secondary structures such as a-heli-ces and [5-sheets or the folding in three dimensions of the polypeptide chain. The primary sequence could have been used to deduct the gene sequence but, with the degeneration of the code, several possibilities for certain amino acids occur, which makes prediction of the gene sequence a risk. 

Storm DR, Hansel C, Hacker B et al (1998) Impaired cerebellar long-term potentiation in type I adenylyl cyclase mutant mice. Neuron 20 1199-1210 Strittmatter SM, Valenzuela D, Kennedy TE et al (1990) GO is a major growth cone protein subject to regulation by GAP-43. Nature 344 836 11... 

A protein subject to NMR analysis may have 100-200 amino acid residues, which provide a 1H NMR spectrum of many hundreds of lines. Because the amino acid sequence can be assumed to have been determined previously by non-NMR methods, the first step in the NMR study is to assign each line in the spectrum to a specific moiety (NH, ot-CH, side chain CH3, etc.) of a specific amino acid residue. Without the 2D methods that we have discussed, it would be virtually impossible to make such assignments. For relatively small proteins ( 50—100 residues) it is often possible to use conventional homonuclear 2D methods, such as COSY and HOHAHA, to define some bonding paths and to supplement these results with NOE data for residues that are very close in space as a result of secondary structural elements such as a helices. However, for proteins of moderate size such techniques are insufficient, and special methods had to be developed and now constitute the standard method of making sequential assignments. 

Figure 11.10. Identification of proteins subject to S-nitrosyla-tion due to nNOS activity, a Experimental strategy. Protein samples were selectively biotinylated as before (cf Figure 11.9), enriched by adsorption to solid phase-bound streptavidin, blotted, and detected by specific antibodies, b Results for several proteins. SM Starting material (not passed through colunm both S-nitrosylated and unmodified protein molecules will show up here). El Colunm eluate - this will only detect the nitrosylated proteins, -i- Samples from wild-type mice (nNOS -I-/-I-), - samples from knockout mice (nNOS -/-). Data reproduced with permission from Nat Cell Biol. 3 193-7 (2001)...

Refers to the amount of protein subjected to SDS PAGE, the actual amount digested would be the product of this number and the % blotting efficiency. 

Figure 4.9. Staining of Proteins After Electrophoresis. Proteins subjected to electrophoresis on an SDS-polyacrylamide gel can be visualized by staining with Coomassie blue. [Courtesy of Kodak Scientific Imaging Systems.]...

Ninhydrin Assay for Adsorbed Proteins. Measurements were made by a colorimetric procedure based on the reaction of ninhydrin with amino acids (25). The films were hydrolyzed in 5 ml of 2.5N NaOH for 2 hrs in capped plastic tubes in a boiling water bath. Then 1.5 ml of glacial acetic acid was added and mixed next I ml of ninhydrin reagent was added and mixed. [The reagent was three times more concentrated in ninhydrin, SnCb, and citrate than prescribed by Moore and Stein (25)]. The tubes were capped and boiled 20 mins more. The solution was clarified by centrifugation, and the absorbance read immediately at 570 nm on a Beckman DB spectrophotometer. If necessary, the sample was diluted with 50-50 2-propanol-water. Calibration curves (absorbance vs. fig of protein) were constructed in the 0-30 and 0-100 fig range with known amounts of each type of protein subjected to this same analysis procedure. 

Oligomeric proteins subjected to HP tend to dissociate in subimits that are vulnerable to hydrolysis (Hayashi et al., 1987). However, once denatured, some oligomeric enzymes can reconstitute by means of a unimolecular step (fold) and a biomolecular step (aggregation). This is the case of the lactate dehydrogenase present in B. stearothermophilus treated with pressures of 280 MPa (Muller et al., 1984). 

Benjakul, S. and Bauer, E. 2000. Physicochemical and enzymatic changes of cod muscle proteins subjected to different freeze-thaw cycles. Journal of the Science of Food and Agriculture iO 1143-1150. 

In the Eastern-Western procedure, lipids are first applied to a solid support such as nitrocellulose. Next, proteins subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis are transferred by standard Western blotting techniques to the solid support in such a manner that the protein of interest is transferred to the lipid patch. During electrotransfer of protein to the solid support, protein, lipid, and sodium dodecyl sulfate mix and as transfer continues the sodium dodecyl sulfate is removed leaving behind the protein to refold in the presence of lipid. Attachment of the refolded protein to a solid support allows one to probe protein structure using conformation-sensitive antibodies or protein function... 

Blocking of amino groups by dimethylation is also being used to help understand changes in proteins subjected to dry heating. Trans-amidation of carboxyls with lysines, or of amides with lysines, can occur (38). Dimethylation should prevent this. 

Nakajima Y, Yoshida S, Inoue Y et al. Occupation of the Qg-binding pocket by a photosystem II inhibitor triggers dark cleavage of the Dl protein subjected to brief preillumination. J Biol Chem 1996 71 17383-17389. 

It is easy to destroy micellar systems irreversibly by adding water-miscible organic solvents such as, e.g., acetone and ethanol. This technique is exceptionally effective and convenient when it is important to extract the solubilized protein (enzyme) delicately so that it retains its structure and physiological (including catalytic) activity [28]. Here, it should be emphasized that, first, the ethanol and especially acetone should be cold. Second, during precipitation with acetone in neutral and basic media, formation of Schiff bases is possible besides which, side reactions involving modification of the protein subjected to isolation (including the loss of the protein solubility in water) are also probable. 

Protein Intake was 65 g/subject/day during all periods except when bran supplements were used. Amount of additional protein/ subject/day during these periods were as follows wheat bran, 2.01 corn bran, 2.93 and rice bran, 2.62. 

Biochemical analyses can be performed in parallel with or instead of morphological studies. At various time points during the course of in vitro disassembly, aliquots of the reaction mixture should be separated into supernatant and pellet fractions by sedimentation at 12,000 g and proteins subjected to SDS-PAGE and immunoblot analysis. An extract of stage 14 Drosophila oocytes contains only lamin Dm i, (Smith and Fisher, 1989 see also Lin and Fisher, 1990 Mans et at., 1995). Purified Drosophila nuclei contain lamins Dmi and Dm2 (Smith et al, 1987 see also Lin and Fisher, 1990 Maus et al, 1995). Lamin Dmmit migrates with a gel mobility intermediate to lamins Dm, and Dm2 (Fig. 4). 

Konnert s technique for refining the structure of proteins subject to known geometrical constraints has been developed by incorporating restraints on the variances of the interatomic distributions, in order to express the retention of local geometry that accompanies certain modes of motion." As as alternative to the sparse matrix approach, Hoad and Norman have utilized the fast Gauss-Seidel least-squares routine for the refinement of atomic co-ordinates." A comparison has been made of the structures obtained for bovine trypsin (EC 3.4.24.4) by the difference Fourier and real space refinement methods." ... 

Another thing that has impressed me is the way that work of a laboratory becomes channeled into new directions by the interest and enthusiasm of a good post-doctoral fellow, without having been guided that way by the Professor. Thus, Audry Stevens started the research which led to her co-discovery of RNA polymerase quite on her own. Harold Neu s coming led to a shift in the research of my laboratory away from nucleic acids and into osmotic shock and periplasmic enzymes, matters that were developed further by Don Hark-ness, Harold Dvorak, Wallace Brockman and Nancy Nossal (except that Nancy went back into nucleic acids, with much profit). Finally, in my last years at N.I.H., Yasuhiro Anraku completed the shift by initiating work on active transport and binding proteins, subjects which still occupy me today. 

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