Shmuel Shaltiel

DETERMINATION OF CARBONYL CONTENT IN OXIDATIVELY MODIFIED PROTEINS

Publisher Summary This chapter discusses methods to determine carbonyl content in oxidatively modified proteins. The methods described are (1) reduction of the carbonyl group to an alcohol with tritiated borohydride; (2) reaction of the carbonyl group with 2,4-dinitrophenylhydrazine to form the 2,4-dinitrophenylhydrazone; (3) reaction of the carbonyl with fluorescein thiosemicarbazide to form the thiosemicarbazone; and (4) reaction of the carbonyl group with fluorescein amine to form a Schiff base followed by reduction to the secondary amine with cyanoborohydride. Van Poelje and Snell have also quantitated protein-bound pyruvoyl groups through formation of a Schiff base with p-aminobenzoic acid followed by reduction with cyanoborohydride. Although a systematic investigation has not appeared, this method should also be useful in detecting other protein-bound carbonyl groups. Carbonyl content of proteins is expressed as moles carbonyl/mole subunit for purified proteins of known molecular weight. For extracts, the results may be given as nanomoles carbonyl/milligram protein. For a protein having a molecular weight of 50,000, a carbonyl content of 1 mol carbonyl/mol protein corresponds to 20 nmol carbonyl/mg proteins.

Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride

Oxidatively modified proteins have been implicated in a variety of physiologic and pathologic processes. Oxidative modification typically causes inactivation of enzymes and also the introduction of carbonyl groups into amino acid side chains of the protein. We describe a method to quantify oxidatively modified proteins through reduction of these carbonyl groups with tritiated borohydride. The technique was applied to purified, oxidatively modified glutamine synthetase and to bronchoalveolar lavage fluid from dogs and from humans. Since the protein content of lung lavage fluid is low, a very sensitive method was required to measure the oxidized residues. Reduction of the carbonyl group generated during oxidation of proteins with tritiated borohydride provided excellent sensitivity. Incorporation of tritium was directly proportional to the amount of protein with a range from 10 to 1000 micrograms. Should moieties other than amino acids be labeled, they are easily removed by rapid benchtop hydrolysis of the protein followed by chromatography on Dowex 50.

Hydrocarbon-coated Sepharoses. Use in the purification of glycogen phosphorylase

Abstract A homologous series of hydrocarbon-coated Sepharoses varying in the length of their alkyl side chains (Seph-NH(CH2)nH) was synthesized. These modified Sepharoses provide a versatile tool for the purification of proteins, since, by choosing the suitable member of the series, a desired protein can be extracted from a protein mixture. This is illustrated in the case of glycogen phosphorylase, which is not retained at all by methyl Sepharose (n=1), is retarded by propyl Sepharose (n=3), is adsorbed on butyl Sepharose (n=4) and can be eluted from the column by deforming buffers, and is so tightly adsorbed on hexyl Sepharose (n=6) that it could be eluted from the column only in the denatured form, by washing with 0.2 N CH3COOH. On a preparative scale, a hundred-fold purification of phosphorylase could be achieved in one step, by passage of a crude muscle extract on a small butyl Sepharose column.

Thiolysis of some dinitrophenyl derivatives of amino acids

Abstract FDNB ∗ has been widely used in structural and functional studies of peptides and proteins ( Sanger, 1945 ; Hirs et al . , 1961 ; Sokolovsky et al . , 1964 ; Di Prisco, 1967 ). Being a reactive aryl halide, this reagent may react with several of the functional groups of proteins such as α- and ϵ- amino groups, imidazoles, sulfhydryls and aliphatic or phenolic hydroxyls. This paper describes a method for quantitative removal of dinitrophenyl groups from histidine, tyrosine and cysteine side chains. The reaction is referred to as “thiolysis” since cleavage is brought about by thiols (e. g. 2-mercaptoethanol). In view of the mild conditions under which the reaction proceeds (aqueous medium, pH = 8.0 and 22°), it may find a variety of applications in peptide and protein chemistry.

The mode of binding of pyridoxal 5′-phosphate in glycogen phosphorylase

Abstract The absorption, fluorescence and chemical properties of the pyridoxal 5′-phosphate (PLP) site in glycogen phosphorylase at pH 7.0 can be simulated by Schiff base derivatives of PLP in non-aqueous solvents. It is proposed that at neutral pH,PLP in phosphorylase is embedded in a hydrophobic microenvironment and bound to an e-amino group of a lysine residue in the protein through a hydrogen-bonded Schiff base structure.

Ultrastructure of beaded agarose

Abstract Beaded agarose was dehydrated and embedded in Epon by a procedure that preserves the size, shape and, most likely, the native ultrastructure of the beads. Thin sections of the embedded beads reveal under the electron microscope a network sponge-like structure, uniform throughout the bead. The matrix skeleton is fairly rigid, though it occupies only a small percentage of the bead volume. This skeleton is composed of thin filaments (~20 A in diameter) bundled in a side-by-side assembly. The pores or channels between the filament bundles vary in shape and diameter (up to 0.3 μm). This structure accounts for some of the known physicochemical properties of beaded agarose.

Phosphorylation of Vitronectin by Casein Kinase II IDENTIFICATION OF THE SITES AND THEIR PROMOTION OF CELL ADHESION AND SPREADING

The cell adhesion protein vitronectin (Vn) was previously shown to be the major target in human blood for an extracellular protein kinase A, which is released from platelets upon their physiological stimulation with thrombin and also prevails as an ectoenzyme in several other types of blood cells. Because plasma Vn was shown to have only one protein kinase A phosphorylation site (Ser378) but to contain ∼3 mol of covalently bound phosphate, and because human serum and blood cells were shown to contain also a casein kinase II (CKII) on their surface, we studied the phosphorylation of Vn by CKII attempting to find out whether such phosphorylation modulates Vn function, an acid test for its having a physiological relevance. Here we show (i) that the CKII phosphorylation of Vn has a K m of 0.5–2 μm (lower than the Vn concentration in blood, 3–6 μm), (ii) that it is targeted to Thr50 and Thr57, which are vicinal to the RGD site of Vn, and (iii) that the phosphorylation of Thr57 facilitates the phosphorylation of Thr50. The maximal stoichiometry of the CKII phosphorylation of plasma Vn was found to be low, which, in principle, could be due to its partial prephosphorylation in vivo. However, for the detection of a functional modulation, we needed a comparison between a fully phosphorylated Vn (at Thr57 and Thr50) and a nonphosphorylated Vn. Therefore, we expressed Vn in a baculovirus system and show (i) that the CKII phosphorylation of wt-Vn enhances the adhesion of bovine aorta endothelial cells; (ii) that the double mutant T50E/T57E (in which the neutral Thr residues are replaced by the negatively charged Glu residues considered analogs of Thr-P) has a significantly enhanced capacity to promote cell adhesion and to accelerate cell spreading when compared with either wild-type Vn or to the neutral T50A/T57A mutant; and (iii) that, at least in the case of bovine aorta endothelial cells, the T50E/T57E mutant exhibits an enhanced adhesion, which seems to be due to an increased affinity toward the αvβ3 Vn receptors.

Unveiling the substrate specificity of meprin beta on the basis of the site in protein kinase A cleaved by the kinase splitting membranal proteinase.

The kinase splitting membranal proteinase (KSMP) is a metalloendopeptidase that inactivates the catalytic (C) subunit of protein kinase A (PKA) by clipping off its carboxyl terminal tail. Here we show that this cleavage occurs at Glu332-Glu333, within the cluster of acidic amino acids (Asp328-Glu334) of the kinase. The Km values of KSMP and of meprin β (which reproduces KSMP activity) for the C-subunit are below 1 μM. The Km for peptides containing a stretch of four Glu residues are in the micromolar range, illustrating the significant contribution of this cluster to the substrate recognition of meprin β. This conclusion is supported by a systematic study using a series of the C-subunit mutants with deletions and mutations in the cluster of acidics. Hydrophobic amino acids vicinal to the cleavage site increase the Kcat of the proteinase. These studies unveil a new specificity for meprin β, suggesting new substrates that are 1-2 orders of magnitude better in their Km and Kcat than those commonly used for meprin assay. A search for substrates having such a cluster of acidics and hydrophobics, which are accessible to meprin under physiological conditions, point at gastrin as a potential target. Indeed, meprin β is shown to cleave gastrin at its cluster of five glutamic acid residues and also at the M-D bond within its WMDF-NH2 sequence, which is indispensable for all the known biological activities of gastrins. The latter meprin cleavage will lead to the inactivation of gastrin and thus to the control of its activity.

Plasmin cleavage of vitronectin Identification of the site and consequent attenuation in binding plasminogen activator inhibitor‐1

Plasmin is shown to specifically cleave vitronectin at the Arg361‐Ser362 bond, 18 amino acid residues upstream from the site of the endogenous cleavage which gives rise to the two‐chain form of vitronectin in plasma. The cleavage site is established using the exclusive phosphorylation of Ser378 with protein kinase A. As a result of the plasmin cleavage, the affinity between vitronectin and the type‐1 inhibitor of plasminogen activator (PAI‐1) is significantly reduced. This cleavage is stimulated by glycosaminoglycans, which are known to anchor vitronectin to the extracellular matrix. A mechanism is proposed through which plasmin can arrest its own production by feedback signalling, unleashing PAI‐1 from the immobilized vitronectin found in the vascular subendothelium, which becomes exposed at the locus of a hemostatic event.

YOS9, the Putative Yeast Homolog of a Gene Amplified in Osteosarcomas, Is Involved in the Endoplasmic Reticulum (ER)-Golgi Transport of GPI-anchored Proteins*

The OS-9 gene maps to a region (q13–15) of chromosome 12 that is highly amplified in human osteosarcomas and encodes a protein of unknown function. Here we have characterized a homolog designated as YOS9 (YDR057w) from Saccharomyces cerevisiae. The yeast protein (Yos9) is a membrane-associated glycoprotein that localizes to the endoplasmic reticulum (ER). YOS9 interacts genetically with genes involved in ER-Golgi transport, particularly SEC34, whose temperature-sensitive mutant is rescued by YOS9overexpression. Interestingly, Yos9 appears to play a direct role in the transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus. Yos9 binds directly to Gas1 and Mkc7 and accelerates Gas1 transport and processing in cells overexpressingYOS9. Correspondingly, Gas1 processing is slowed in cells bearing a deletion in YOS9. No effect upon the transport and processing of non-GPI-anchored proteins (e.g. invertase and carboxypeptidase Y) was detected in cells either lacking or overexpressing Yos9. As Yos9 is not a component of the Emp24 complex, it may act as a novel escort factor for GPI-anchored proteins in ER-Golgi transport in yeast and possibly in mammals.