Methods in Cell Biology

Robert H. Behal, … Douglas G. Cole, in Methods in Cell Biology, 2009

A Engineering Rationale

Biochemical analysis of IFT particle proteins has been challenging for multiple reasons. Biochemical analysis has been largely limited to Chlamydomonas flagella and, even so, the quantities available are not large. In addition, the particles readily dissociate into two major complexes, A and B, and complex B itself is not particularly stable. Thus, we have sought to use bacterial and yeast expression of recombinant proteins to study the structure and interactions of IFT particle proteins.

Coexpression of multiple proteins can be an especially powerful approach to generate protein complexes. To achieve this, we have adopted the Duet system of plasmids which allow coexpression of up to eight different proteins in one bacterial host; a number of these proteins can be engineered to contain a variety of affinity tags. The Duet vectors incorporate different origins of replication and antibiotic selection markers; therefore, stable cotransformation of a single host bacterium is possible. Furthermore, we have combined select Duet vectors with the pMal vector, which incorporates the maltose-binding protein (MBP), to take advantage of rapid and reversible amylose chromatography. Although we have used this approach to generate complexes containing as many as five different IFT particle proteins we will keep the discussion simple by presenting data on the production and purification of an IFT81/IFT74 subcomplex.View chapterPurchase book

Methods for Analysis of Golgi Complex Function

Peristera Roboti, … Martin Lowe, in Methods in Cell Biology, 2013

Summary

Biochemical analysis of trafficking within the secretory pathway remains an important approach to assess the functioning of this pathway. With the advent of technologies that can ablate the function of specific gene products, such as RNA interference or mutagenesis with zinc-finger nucleases, together with the development of increasing numbers of small molecule inhibitors, it is more important than ever that we can sensitively and quantitatively assess rates of protein trafficking within cells. The use of established biochemical approaches such as those described here affords this possibility for the secretory pathway. Therefore, we strongly believe that the continued use of such assays will be important for fully dissecting the mechanisms that underpin secretory trafficking in mammalian cells.View chapterPurchase book

A Sedentary Man with Acute Respiratory Failure and Myoglobinuria  

Tulio E. Bertorini MD, in Neuromuscular Case Studies, 2008

WHAT SHOULD BE DONE NEXT?

Biochemical analysis showed muscle carnitine levels of 28.5 moles/mg/ncp (normal, 11.6–26.4 moles/mg/ncp). Glycogen and glycogen enzyme content were normal.

CPT level was 7.70 pmol (normal, 43.8–100.7 pmol). This assay did not separate CPT I from CPT II.

It was concluded that this patient has CPT deficiency, or DiMauro’s disease.

Six months later, he had an episode of right lower lobe pneumonitis accompanied by fever, malaise, and generalized weakness. Sputum cultures for bacteria and fungi were negative; complement fixation titers for influenza, parainfluenza, ECHO, coxsackie and adenovirusesrespiratory syncytial virus titers, cytomegalic inclusion virus, and lymphochoriomeningitis were all normal and did not increase during convalescence. Titers for Mycoplasma pneumoniae rose from 1:16 to 1:64. During this episode, serum CK was 4000 U/L, but there was no evidence of myoglobinuria. He recuperated from this uneventfully.View chapterPurchase book

Intermediate Filament Proteins

Pavel Strnad, … Diana M. Toivola, in Methods in Enzymology, 2016

5.3 Biochemical Analysis of MDBs

Biochemical analysis constitutes another important aspect of MDB research. MDBs can be isolated by a combination of sucrose gradient centrifugation and subsequent isolation of insoluble proteins, as described in detail previously (Zatloukal et al., 2004). Given the rapidly expanding knowledge about keratin PTMs (Snider & Omary, 2014), careful consideration should be given regarding addition of appropriate inhibitors to the isolation buffers in order to maintain in vivo conditions. FACS-based MDB isolation represents another attractive tool to obtain pure aggregates; however, it has not been routinely used (Zatloukal, Bock, Rainer, Denk, & Weber, 1991). Lastly, the amount of p62, high-molecular weight K8 or ubiquitinated proteins (for list of useful antibodies, see Table 5) within the insoluble protein fraction, determined via immunoblotting, are increasingly being used as a tool to quantify the extent of MDB formation (Strnad et al., 2007).View chapterPurchase book

Primary Cilia

Bing Huang, … Nicholas LaRusso, in Methods in Cell Biology, 2009

C Characterization of Isolated Primary Cilia by Biochemical and Molecular Approaches

Biochemical analysis (i.e., western blot) is needed to confirm the purity of isolated cilia. Acetylated α-tubulin, a well-characterized ciliary marker, is used to establish that isolated pellets are indeed primary cilia. Moreover, a marker of the apical membrane (expressed only at the apical membrane but not on cilia) is also run on western blot in order to verify that the isolated ciliary fractions are not contaminated with fragments of cell membranes. Markers of the apical membrane of the particular cells used for isolation should be determined in advance. Figure 6 shows a verification of the purity of isolated cilia. We previously determined that the purinergic receptor, P2Y2, is exclusively localized to the apical membrane of cholangiocytes but not in cholangiocyte cilia. Thus, the P2Y2 receptor served as a marker of apical membrane of cholangiocytes. Western blots demonstrate the purity of primary cilia isolated by both approaches. After verification of the purity of isolated cilia, these fractions could be used for detection of any ciliary-associated proteins of interest by western blotting or by immunofluorescent confocal microscope. Figure 7 shows that isolated primary cilia of cholangiocytes are positively stained for ciliary-associated proteins, polycystin-1 and polycystin-2.

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Determination of Circulating Native and Denaturated HDL Concentrations and its Clinical Implications

Takanari Nakano, … Tsugikazu Komoda, in The HDL Handbook, 2010

Conclusion

Biochemical analysis of HDL or apoA-I have revealed that apoA-I, the major protein constitute of HDL particles, is readily oxidized in vivo. The oxidation is mediated by a variety of radicals or enzymes. Several pathways for the oxidation have been shown and target amino acids have been identified. The high susceptibility of HDL to oxidation and its association with loss of anti-atherogenic ability have attracted several groups as a mechanism of atherosclerosis. We and some other groups have been focusing on the clinical usefulness of its measurement. Assays for oxidized HDL have been developed and applied for the measurement of clinical specimens. Unexpectedly, some of the results are paradoxical. Oxidized HDL was decreased in patients who were supposed to have increased oxidative stress. Oxidized HDL may be removed from the circulation rapidly considering the more rapid clearance of oxidized HDL than native HDL. Moreover, decreased oxidized HDL in patients suggests that the pathway to clear or scavenge oxidized HDL is accelerated in some disease conditions. Thus, the quantitative measurement of oxidized HDL may be not useful as a clinical marker.

A number of amino acids in apoA-I can be oxidized. Some may be related to its dysfunction for reverse cholesterol transport, suggesting that such determinants are candidates to be examined as antibody targets for further development of immunoassays.View chapterPurchase book

Interactions of Parathyroid Hormone and Parathyroid Hormone-Related Protein with Their Receptors

MICHAEL CHOREV, … MICHAEL ROSENBLATT, in The Parathyroids (Second Edition), 2001

Cross-Linking to Position 13 in PTH

Biochemical analysis of the photocross-linking product of radiolabeled [Nle8,18,Lys13(Nɛp(3-I-Bz)-Bz),Nal23,Arg26,27,Tyr34]bPTH(1–34)NH2 [Lys13(pBz2)-PTH] with hPTH1-Rc expressed in HEK293/C-21 cells identifies a glycosylated radioactive band of ∼6 kDa, which is delimited by Lys-C and CNBr cleavage sites at the N and C termini. The theoretical cleavage restriction map of hPTH1-Rc reveals the minimal radiolabeled 125I-Lys13(pBz2)-PTH-hPTH1-Rc conjugated fragment, corresponding to hPTH1-Rc(173–189) located at the C-terminal region of the extracellular N terminus (26).

Site-directed mutagenesis within the 17 amino acid residues comprising hPTH1-Rc(173–189) combined with subsequent biochemical analysis further delineates the boundaries of the contact site for 125I-Lys13(pBz2)-PTH to hPTH1-Rc(182–189), an 8-amino acid sequence (263). Several single site-mutated receptors were generated, which include a new Lys-C-susceptible cleavage site. The mutant [R181K]hPTH1-Rc was stably expressed in HEK293 cells (∼200,000 Rcs/cell) and was fully functional. Compared to the wild-type receptor, Lys-C cleavage of the 125I-Lys13(pBz2)-PTH-[R181K] photoconjugate produces a smaller conjugated fragment (∼18 vs. ∼9 kDa, respectively), corresponding to a cleavage site upstream to the N-glycosylated Asn176. Interestingly, the only functional mutations that failed to cross-link to 125I-Lys13(pBz2)-PTH were the [R186K/A] mutants (263). However, [R186K]hPTH1-Rc stably expressed in HEK293 cells cross-links effectively to 125I-Bpa1-PTH and displays wild-type receptor-like cyclase activity and binding affinity similar to that in HEK293/C-21 cells. These findings suggest that R186 participates in an interaction with the ligand that either provides a contact site for position 13 in the ligand or provides an interaction that brings the ligand into the close spatial proximity required for cross-linking within the hPTH1-Rc(182–189) contact site (263). This interaction does not appear to be essential for a productive ligand–receptor interaction because [R186K] is fully functional and cross-links effectively with 125I-Bpa1-PTH.View chapterPurchase book

Mitochondrial Encephalomyopathies

Darryl C. De Vivo, … Salvatore DiMauro, in Neuromuscular Disorders of Infancy, Childhood, and Adolescence (Second Edition), 2015

Biochemistry

Biochemical analysis is usually conducted in frozen muscle tissue or in cultured fibroblasts. A detailed, step-by-step procedure for the measurement of the activities of complexes I–IV has been published286 and should facilitate comparison of data among laboratories, thus far a thorny issue.

Individual respiratory complex deficiencies are expected to occur in patients with mutations in mtDNA protein-coding genes and with mutations in genes encoding single respiratory chain subunits (direct hits) or assembly genes for specific complexes (indirect hits). While this is largely the case, because the respiratory chain is organized in supercomplexes, a severe defect in one complex is likely to be associated with less severe defects in one or more additional complexes. Thus, for example, mutations in the MTCYB gene cause defects of complex III but also of complex I.276

Impaired biochemical activities of all complexes containing subunits encoded by mtDNA suggests either mutations in mtDNA genes controlling protein synthesis globally (tRNA or rRNA genes, single deletions) or mutations in nDNA genes controlling mtDNA maintenance (multiple deletions, depletion, defects of mtDNA translation, defects of the MIM lipid milieu).

Some special patterns may be suggestive of specific problems. Combined defects of complex II + III are often associated with primary CoQ10 deficiency whereas combined defects of complexes I, II, and III may be due to defective assembly of the FeS cluster proteins, especially when they are accompanied by aconitase deficiency.159View chapterPurchase book

Surgical Retina

W. Richard Green, J. Sebag, in Retina (Fourth Edition), 2006

BIOCHEMISTRY OF VITREOUS

Biochemical analysis discloses that about 99% of the vitreous is water; about 1.0% is composed of inorganic salts and organic lipids of low molecular weight, and about 0.1% consists of soluble and insoluble proteins and hyaluronan. The hydrated volume of vitreous is dependent on the polymer of hyaluronan acid.17,23,26 Hyaluronan acid has a high negative electrostatic potential. When hydrated, the hyaluronan acid polymer maintains a certain spatial relationship with the dipolar water molecules, the hyaluronic acid containing the water much like a rigid sponge. Water is not chemically bound to the hyaluronic acid but is held to the coils of the polymer units like water in a screen wire. The hydrated volume of the vitreous can be reduced by altering the polymeric of structure with introduction of positively charged molecules, such as iron and protein. Alternatively, free radicals generated by incident light or metabolism27 could alter the conformation of hyaluronan and influence its association with proteoglycans and collagen, causing dissociation from the gel matrix. The collagen molecules are now able to cross-link and aggregate into packed bundles of parallel collagen fibrils (Fig. 114-9), while the dissociated hyaluronan molecules pool into lacunae of liquid vitreous (Fig. 114-10) as water is drawn with the hydrophilic hyaluronan molecules.28 The spatial integrity of the vitreous is then lost and results in collapse and condensation of the collagen framework (Fig. 114-10). Such degeneration occurs, to some degree, in the vitreous of virtually all adults.View chapterPurchase book

Biosynthetic Enzymes for (1-3)-β-Glucans, (1-3;1-6)-β-Glucans from Yeasts

Satoru Nogami, Yoshikazu Ohya, in Chemistry, Biochemistry, and Biology of 1-3 Beta Glucans and Related Polysaccharides, 2009

3.2. Regulatory Subunit

Biochemical analysis in early studies suggested the involvement of GTPase in (1,3)-β-glucan synthesis (Kang and Cabib, 1986; Mol et al., 1994). The GTP-binding regulatory subunit was partially purified and its molecular weight was estimated to be 20 kDa by photoaffinity labeling (Mol et al., 1994), but impurity of the sample prevented further detailed analysis of this regulatory component. However, genetic analysis in Schizosaccharomyces pombe suggested that geranylgeranyltransferase I (GGTase I) plays a role in the regulation of glucan synthase (Diaz et al., 1993; Ribas et al., 1991). One of the most important substrates of GGTase I is a Rho-type GTPase, acting as a molecular switch that monitors and receives upstream signals for cell morphogenesis (Inoue et al., 1996; Takai et al., 2001). These preceding studies suggested that the Rho type GTPase is the regulatory subunit of glucan synthase. Our group and that of Cabib’s revealed that Rho1p, one of the Rho-type GTPases in yeast, regulates glucan synthase activity (Drgonova et al., 1996; Qadota et al., 1996). Rho1 was detected in the purified glucan synthase fraction. Some alleles of the rho1 conditional mutant result in reduced glucan synthase activity and low glucan content (Qadota et al., 1996). The low glucan synthase activity can be restored by adding recombinant Rho1 to the reaction mixture (Mazur and Baginsky, 1996; Qadota et al., 1996). Low-glucan synthesis activity of a temperature-sensitive FKS1 mutant was restored by overexpressing the constitutive active allele of RHO1 (Sekiya-Kawasaki et al., 2002).View chapterPurchase book

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