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At least 685 records · Page 38Linked to original sources
PubMed1984

Chemical modification of dopamine beta-hydroxylase.

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.

Amino Acids↗
PubMed1986

The catalysis by heparin of the reaction between thrombin and antithrombin.

Fluorescence polarization has been used to study the kinetics of the combination of thrombin with antithrombin and its catalysis by the polysaccharide heparin. The heparin-catalysed combination of thrombin and antithrombin is saturable with respect to both thrombin and antithrombin. The rate-determining step of the reaction is approximately 1.7 s-1. The kinetics observed can be explained by proposing that the catalyst of the reaction is not heparin alone but a complex of heparin and antithrombin (bound at the high-affinity site). The temperature dependence of the heparin-catalysed reaction is indistinguishable from that of the uncatalysed reaction. This coincidence is consistent with the rate-limiting step being the same in both cases.

Antithrombins↗
PubMed2005

Characterization of advanced glycation end products for biochemical studies: side chain modifications and fluorescence characteristics.

Advanced glycation end products (AGEs) are known to be involved in the pathogenesis of several diseases and therefore effects of AGEs on cells are the objective of numerous investigations. Since AGEs used in biochemical studies are usually not chemically characterized, comparison of data is difficult if not impossible. To find a suitable characterization protocol, human serum albumin was reacted with different concentrations of glucose, methyl glyoxal, and glyoxylic acid. The obtained AGEs were characterized with respect to the extent of side chain modifications (lysine and arginine), the carboxymethyl lysine and carbonyl content, and the fibrillar state. Additionally, their fluorescence and absorbance characteristics were extensively studied. Although we found significant differences in the degree of modification and in AGE-specific fluorescence when using different modifiers, the results provide important information and allow comparing AGEs derived from different modifier concentrations. The results also suggest strong conformational changes within the modified proteins. In the present paper we propose a set of parameters that is sufficient to partially characterize AGEs used for biochemical studies.

Amino Acids↗
PubMed2004

Oxidation of phosphatidylserine: a mechanism for plasma membrane phospholipid scrambling during apoptosis?

Selective oxidation of phosphatidylserine (PS) during apoptosis precedes its externalization in plasma membrane and is essential for the engulfment of apoptotic cells. To experimentally test whether PS oxidation stimulates its externalization via its effects on aminophospholipid translocase (APT) or by enhanced PS scrambling, action of oxidized PS (PSox) was studied using leukemia HL-60 cells and lymphoma Raji cells. Both PS and PSox were equally well recognized by APT. PSox did not inhibit APT. Rate of transmembrane PS diffusion was fourfold higher in cells with integrated PSox than with PS. Thus, PSox acts as a "non-enzymatic scramblase" likely contributing to PS externalization.

Adenosine Triphosphate↗
PubMed2004

Immobilization of cell adhesive RGD peptide onto the surface of highly porous biodegradable polymer scaffolds fabricated by a gas foaming/salt leaching method.

A cell adhesive peptide moiety, Gly-Arg-Gly-Asp-Tyr (GRGDY), was immobilized onto the surface of highly porous biodegradable polymer scaffolds for enhancing cell adhesion and function. A carboxyl terminal end of poly(D,L-lactic-co-glycolic acid) (PLGA) was functionalized with a primary amine group by conjugating hexaethylene glycol-diamine. The PLGA-NH2 was blended with PLGA in varying ratios to prepare films by solvent casting or to fabricate porous scaffolds by a gas foaming/salt leaching method. Under hydrating conditions, the activated GRGDY could be directly immobilized to the surface exposed amine groups of the PLGA-NH2 blend films or scaffolds. For the PLGA blend films, the surface density of GRGDY, surface wettability change, and cell adhesion behaviors were characterized. The extent of cell adhesion was substantially enhanced by increasing the blend ratio of PLGA-NH2 to PLGA. The level of an alkaline phosphatase activity, measured as a degree of cell differentiation, was also enhanced as a result of the introduction of cell adhesive peptides.

Absorbable Implants↗
PubMed2004

Contribution of lymphatic drainage through stomata to albumin removal from pleural space.

The contribution of lymphatic drainage through the stomata of parietal mesothelium to the overall removal of labeled albumin from the pleural space was found 89% in sheep with very large hydrothoraces (10 ml/kg), a condition involving a approximately 20 times increase in lymphatic drainage [Broaddus et al., J. Appl. Physiol. 64 (1988) 384]. We determined this contribution in anesthetized rabbits with small (0.12 ml/kg) and large (2.4 ml/kg) hydrothoraces of Ringer-albumin with labeled albumin and labeled dextran-2000 kDa. This dextran was used as marker of liquid removal through the stomata because it should essentially leave the pleural space through the stomata only, owing to its size. The removal of labeled albumin by lymphatic drainage through the stomata was 39% of the overall removal in the small hydrothoraces, and 64% in the large ones. Hence, lymphatic drainage through the stomata does not contribute most of protein and liquid removal from the pleural space under physiological conditions, as it has been maintained. It markedly increases with the increase in pleural liquid volume.

Albumins↗
PubMed2000

Modeling the kinetics of acylation of insulin using a recursive method for solving the systems of coupled differential equations.

This paper describes a theoretical method for solving systems of coupled differential equations that describe the kinetics of complicated reaction networks in which a molecule having multiple reaction sites reacts irreversibly with multiple equivalents of a ligand (reagent). The members of the network differ in the number of equivalents of reagent that have reacted, and in the patterns of sites of reaction. A recursive algorithm generates series, asymptotic, and average solutions describing this kinetic scheme. This method was validated by successfully simulating the experimental data for the kinetics of acylation of insulin.

Acylation↗
PubMed1976

High-performance liquid chromatographic determination of kanamycin.

A fast, selective, and precise liquid chromatographic method for simultaneous, independent determination of kanamycins A and B is described. Sample components are separated on a pellicular cation exchanger and monitored by fluorescence using post-column on-line derivatization. Less than 0.35 mug of kanamycin B can be detected in as much as 7 mug kanamycin A injected. The detection limit for kanamycin A is less than 20 ng injected. Reproducibility of the entire chromatographic system is about 1% (2 theta) based upon repeated injections of standards. Precision of repeated process sample preparation is about 6% (2 theta). Chromatographic analysis time is less than 15 min per sample.

Chemical Phenomena↗
PubMed2005

Preparation of hybrid (19S-20S-PA28) proteasome complexes and analysis of peptides generated during protein degradation.

PA28 (also named REG or 11S) is a ring-shaped (180-kDa) interferon-gamma-induced complex that associates with the 20S proteasome and dramatically stimulates the breakdown of short peptides. Immunoprecipitation studies indicate that in vivo PA28 also exists in larger complexes that also contain the 19S particle, which is required for the ATP-ubiquitin-dependent degradation of proteins. However, because of its lability (e.g., it does not withstand exposure to high ionic strength buffers), this larger complex cannot be purified by standard biochemical protocols. Therefore, we developed a method to reconstitute in vitro such hybrid proteasomes (i.e., PA28-20S-19S) from highly purified components. This chapter describes conditions that allow the association of PA28 with "singly capped" 26S (i.e., 19S-20S) particles. In addition assays are described to measure absolute rates of degradation of several non-ubiquitinated proteins by 26S and 20S proteasomes and methods to analyze the pattern and size distribution of peptides generated during the degradation of these proteins.

Animals↗