Protein direct transfer

Further improvements can be achieved by replacing the oxygen with a non-physiological (synthetic) electron acceptor, which is able to shuttle electrons from the flavin redox center of the enzyme to the surface of the working electrode. Glucose oxidase (and other oxidoreductase enzymes) do not directly transfer electrons to conventional electrodes because their redox center is surroimded by a thick protein layer. This insulating shell introduces a spatial separation of the electron donor-acceptor pair, and hence an intrinsic barrier to direct electron transfer, in accordance with the distance dependence of the electron transfer rate (11) ... 

Biocompatible nanosized polyamidoamine (PAMAM) dendrimer films provided a suitable microenvironment for heme proteins to transfer electron directly with underlying pyrolytic graphite electrodes. The Mb-PAMAM film can catalytically reduced oxygen, hydrogen peroxide, and nitrite, indicating that the potential applicability of the film can be used to fabricate a new type of biosensor or bioreactor based on the direct electron transfer of Mb [234],... 

Lipids are transported between membranes. As indicated above, lipids are often biosynthesized in one intracellular membrane and must be transported to other intracellular compartments for membrane biogenesis. Because lipids are insoluble in water, special mechanisms must exist for the inter- and intracellular transport of membrane lipids. Vesicular trafficking, cytoplasmic transfer-exchange proteins and direct transfer across membrane contacts can transport lipids from one membrane to another. The best understood of such mechanisms is vesicular transport, wherein the lipid molecules are sorted into membrane vesicles that bud out from the donor membrane and travel to and then fuse with the recipient membrane. The well characterized transport of plasma cholesterol into cells via receptor-mediated endocytosis is a useful model of this type of lipid transport. [9, 20]. A brain specific transporter for cholesterol has been identified (see Chapter 5). It is believed that transport of cholesterol from the endoplasmic reticulum to other membranes and of glycolipids from the Golgi bodies to the plasma membrane is mediated by similar mechanisms. The transport of phosphoglycerides is less clearly understood. Recent evidence suggests that net phospholipid movement between subcellular membranes may occur via specialized zones of apposition, as characterized for transfer of PtdSer between mitochondria and the endoplasmic reticulum [21]. 

Photosystem I is a membrane pigment-protein complex in green plants, algae as well as cyanobacteria, and undergoes redox reactions by using the electrons transferred from photosystem II (PS II) [1], These membrane proteins are considered to be especially interesting in the study of monomolecular assemblies, because their structure contains hydrophilic area that can interact with the subphase as well as hydrophobic domains that can interact either with each other or with detergent and lipids [2], Moreover, studies with such proteins directly at the air-water interface are expected to be a valuable approach for their two-dimensional crystallization. 

There exists a wide variety in the setup of ELISA assays (direct binding or competition setups) and the enzymatic reaction utilized [148]. A similar principle to enhance sensitivity by enzymatic coupling is realized after gel electrophoretic separation of proteins. Here proteins are transferred to nitrocellulose ( western blot ) and detected by antibody-coupled enzymes. 

Fig. 4.1. Fundamentals of the ubiquitin system. Adapted from Ref [5]. Figure 4.1 shows the fundamentals of the ubiquitin system. (1) Ubiquitin is synthesized in linear chains or as the N-terminal fusion with small ribosomal subunits that are cleaved by de-ubiquitylating enzymes to form the active protein. Ubiquitin is then activated in an ATP-dependent manner by El where a thiolester linkage is formed. It is then transthiolated to the active-site cysteine of an E2. E2s interact with E3s and with substrates and mediate either the indirect (in the case of HECT E3s) or direct transfer of ubiquitin to substrate. A number of factors can affect this process. We know that interactions with Hsp70 can facilitate ubiquitylation in specific instances and competition for lysines on substrates with the processes of acetylation and sumoylation may be inhibitory in certain instances. (2) For efficient proteasomal targeting to occur chains of ubiquitin linked internally through K48 must be formed. This appears to involve multiple...

S ATP -I- [DNA-directed eukaryotic RNA polymerase II subunit Ila] (<4> distinct from other protein phosphokinases, transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in C-terminal domain of MW 220000 subunit of RNA-polymerase II [7] <4> substrates are RNA-polymerase II subunits of wheat germ, soy bean, pea and human [7] phosphorylates predominantly Ser-residues [1-3,5,7] <1> kinase CTDKl almost exclusively phosphorylates Ser-residues [5] <1> kinase CTDK2 phosphorylates to a lesser extent Thr-resi-dues [1] <3-5> phosphorylates to a lesser extent Thr-residues [1,5,7] <1> phosphorylates Ser- and Thr-residues equally [6] <1,3,5> phosphorylates not Tyr-residues [1,6] <1> kinase CTDKl 33 mol phosphate per mol IIA-subunit [5] <1> kinase CTDK2 40-50 mol phosphate per mol IIA-subunit, i.e. 1 phosphate per heptapeptide repeat [5] <4> no substrate is GTP [7] <2,4> no substrates are CTP and UTP [3,7] <2> no substrates are dTTP and AMP-PNP [3] <4> no substrates are bovine serum albumin and calf thymus histone [7] <5> no substrate is phosvitin... 

The transfer of the oligosaccharide to protein is followed in vivo by the excision of the D-glucosyl residues the nine D-mannosyl residues present in the transferred oligosaccharide probably remain in the inner core of the D-mannan. However, the possibility that some D-mannosyl residues could be almost simultaneously removed and added cannot be precluded.117,167 Finally, terminal, < -d-(1 - 3)-linked D-mannosyl groups are added to the trimmed, protein-bound oligosaccharide.118 The outer chain of the d-mannan is formed after the inner-core oligosaccharide has been transferred to protein and processed, apparently by direct transfer of D-mannosyl groups from GDP-D-mannose.168 The similarity of the structures of the outer chain... 

Specific detection of nitrocellulose membrane-bound proteins using a conjugated enzyme. (1) Proteins are transferred from electrophoresis gel to nitrocellulose membrane. Blocker proteins bind to unoccupied sites on the membrane. (2) The membrane is incubated with a primary antibody directed against the protein of interest. (3) A secondary antibody is directed against the primary antibody. (4) The second antibody is conjugated with an enzyme to provide a detection mechanism. Substrate solution is added to the blot. The conjugated enzyme (HRP or AP) catalyzes the conversion of substrate (S) to product (P) to form a colored precipitate at the site of the protein-antibody complex. 

Kinases Direct transfer of terminal phosphoryl group of ATP to substrate Creatine kinase Adenylate kinase Hexokinasc Phosphoglycerate kinase Pyruvate kinase Protein kinase Myokinase Phosphofructokinase Type 1 (M-S-E) Type 1 Type 1 Type 1 Type 2 (S-M-E)... 

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