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K D Wilkinson

Publications and source records attributed to K D Wilkinson.

At least 55 records · Page 3Linked to original sources
PubMed1987

Protein ubiquitination: a regulatory post-translational modification.

The covalent attachment of ubiquitin to a variety of cellular proteins (ubiquitination) is a common post-translational modification in eukaryotic cells. Little is known about the function of these modifications in either the normal or the pathological state. The characteristics of ubiquitination in the nucleus, the cytoplasm, and on the plasma membrane are reviewed and discussed here. Also reported are studies on the enzymes which metabolize ubiquitin, using the ubiquitin-dependent proteolysis system as a model. Four enzymes which specifically recognize ubiquitin and hydrolyze carboxyl-terminal derivatives of ubiquitin have been partially purified from bovine thymus. These are thiol-containing proteases which will also release ubiquitin from ubiquitin-protein conjugates. The presence of these deconjugating enzymes and the proteases in the cytoplasm suggests that there is a partition of conjugates between proteolysis and deconjugation. To study the factors which determine the relative rates of proteolysis versus deconjugation, we have developed a general method of synthesizing large amounts of pure ubiquitin-protein conjugates. The structure/function relationships of ubiquitin have been probed by chemically modifying ubiquitin and examining its activity in the protein degradation system. These studies have identified regions of the ubiquitin molecule which are recognized by the enzymes of the proteolysis system, established that the molecule can be altered and used as a probe of such systems and will guide the design of site-directed mutant ubiquitins in order to more fully define the recognition sites on the ubiquitin molecule. It is likely that studies of these types will lead to an understanding of the molecular interactions required for proper ubiquitin function and allow design of drugs which could be useful in understanding the role of ubiquitination and its importance in normal and pathological states.

Histones↗
PubMed1986

Alcohol-induced conformational changes of ubiquitin.

Ubiquitin has been found to be soluble in ethylene glycol and alcohols as the perchlorate or hydrochloride salt. When the effect of alcohol on the structure of ubiquitin is examined, two reversible conformational transitions are observed. Upon lowering the dielectric constant of aqueous alcohol solutions of ubiquitin from 80 to 45, the native structure of ubiquitin is converted to a form consistent with 50% helical structure. This conformational change results in a change in exposure to solvent of the single methionine and the single tyrosine residues of ubiquitin. In agreement with crystallographic results, these residues are buried in the native conformation but become fully exposed to solvent upon undergoing this transition. Further lowering of the dielectric constant to 20 results in the accumulation of a conformation with almost complete helical structure. Thus, hydrophobic interactions cause facile conformational changes in the ubiquitin structure. These results are discussed in terms of a preferential solvation model. It is shown that the results obtained with different alcohols can be normalized by the use of a dielectric constant scale. This normalization corrects for the different molar volumes of different alcohols, allows comparison of results obtained with different alcohols, and should be useful in studying this phenomenon with different proteins.

Alcohols↗
PubMed1986

Role of ubiquitin conformations in the specificity of protein degradation: iodinated derivatives with altered conformations and activities.

Three iodinated derivatives of ubiquitin have been synthesized and these derivatives have been characterized in the ubiquitin-dependent protein degradation system. With chloramine-T as the oxidant, a derivative containing monoiodotyrosine is formed in the presence of 1 M KI and a derivative containing diiodotyrosine is produced in the presence of 1 mM KI. These derivatives exhibit phenolate ionizations at pH 9.2 and 7.9 with absorbance maxima at 305 and 314 nm, respectively. In addition to modification of the tyrosine residue, these conditions lead to the oxidation of the single methionine residue and iodination of the single histidine residue [M.J. Cox, R. Shapira, and K.D. Wilkinson (1986) Anal. Biochem. 154, 345-352]. Iodination of ubiquitin under these conditions renders the protein sensitive to hydrolysis by trypsin and results in an enhanced susceptibility to alcohol-induced helix formation. When the derivatives are tested in the ATP: pyrophosphate exchange reaction catalyzed by the ubiquitin adenylating enzyme, they are found to exhibit activity comparable to the native protein. When these derivatives are tested for the ability to act as a cofactor in the ubiquitin-dependent protein degradation system, they are both found to support a rate of protein degradation that is twice that of native ubiquitin. At high concentrations of derivatives, the rate of protein degradation is inhibited, while the steady state level of conjugates increases. Thus, the free derivatives inhibit the protease portion of the reaction, but are fully active in the activation and conjugation portions of the reaction. With iodine as the modification reagent, monoiodination of tyrosine is the predominant reaction. This derivative exhibits activity similar to native ubiquitin. Thus, it appears that modification of the histidine residue is responsible for the increased activity of the more highly iodinated derivatives. The enzymes of the system must recognize different portions of the ubiquitin structure, or different conformations of ubiquitin that are affected by the iodination of the histidine residue. These results suggest a conformational change of the ubiquitin molecule may be important in determining the rate and specificity of proteolysis.

Circular Dichroism↗
PubMed1986

Synthesis and characterization of ubiquitin ethyl ester, a new substrate for ubiquitin carboxyl-terminal hydrolase.

A new substrate for ubiquitin carboxyl-terminal hydrolase, the carboxyl-terminal ethyl ester of ubiquitin, has been synthesized by a trypsin-catalyzed transpeptidation. In the presence of 1.6 M glycylglycine ethyl ester, trypsin removes the carboxyl-terminal glycylglycine of ubiquitin and replaces it with the dipeptide ester. The equilibrium mixture under these conditions contains 30% ubiquitin ethyl ester and 70% hydrolysis product, the 74-residue fragment of ubiquitin. Ubiquitin ethyl ester can be purified by gel filtration and ion-exchange chromatography. The structure of this product has been verified by identification of the products of base hydrolysis, tryptic cleavage in aqueous solution, and peptide mapping. When ubiquitin ethyl ester is incubated with purified ubiquitin carboxyl-terminal hydrolase, specific cleavage of the ester linkage is observed. A rapid, sensitive assay is described utilizing high-performance liquid chromatography. By use of this assay, it has been shown that ubiquitin carboxyl-terminal hydrolase is inactivated in the absence of thiols. Optimal protective effects are seen with 10 mM dithiothreitol. The rate of catalysis is maximal at pH 8.5, with evidence for catalytically important groups with pK values of 5.2, 7.6, and 9.5. These findings are consistent with the participation of a thiol group in the active site. Native ubiquitin is a competitive inhibitor of ubiquitin ethyl ester hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocytes↗
PubMed1986

Structure and activities of a variant ubiquitin sequence from bakers' yeast.

Ubiquitin is an extremely conserved protein, with an identical sequence throughout the animal kingdom. However, the gene sequence of the yeast protein [Ozkaynak, E., Finley, D., & Varshavsky, A. (1984) Nature (London) 312, 663-666] predicts three amino acid differences. This implies that some functions or binding interactions of ubiquitin are different in yeast and animal cells. In an effort to define these differences, ubiquitin has been purified to homogeneity from bakers' yeast and characterized. Amino acid analysis of the protein and the isolated tryptic peptides confirms the primary structure of this protein as predicted from the gene sequence. This result indicates that the gene sequenced is the transcriptionally active gene from yeast. The conformation of yeast ubiquitin is similar to human ubiquitin as judged by circular dichroism, sensitivity to trypsin, and Stokes radius. Yeast and animal ubiquitins show identical activities in supporting ubiquitin-dependent protein degradation and in the ATP-pyrophosphate exchange reaction catalyzed by the purified ubiquitin-adenylating enzyme. Thus, the three conservative amino acid differences between yeast and animal ubiquitins have very little effect on the structure of ubiquitin or its activity in the ubiquitin-dependent proteolytic system. These results suggest that at least some of the evolutionary pressure preventing sequence variation among animal ubiquitins stems from one or more of its nonproteolytic functions.

Amino Acid Sequence↗
PubMed1986

Tryptic peptide mapping of ubiquitin and derivatives using reverse-phase high performance liquid chromatography.

The conditions for tryptic digestion and subsequent peptide mapping of the ATP-dependent proteolysis cofactor ubiquitin and its derivatives are described. In aqueous solution, the native ubiquitin which is composed of 76 amino acids undergoes only a single cleavage at arginine-74. Full digestion of ubiquitin was obtained in 6.5 M urea, although cleavages at lysine-33 and arginine-74 were slow. Peptide mapping was achieved by reverse-phase high-performance liquid chromatography with a C18 column using a trifluoroacetic acid/triethylamine buffer system and acetonitrile as eluants. The peptides, separated using a linear gradient, were identified by amino acid analysis. Derivatives analyzed by this method include oxidized, monoiodotyrosyl, and diiodotyrosyl ubiquitin. This technique will be useful in examining peptides of chemically modified ubiquitin with respect to extent and specificity of modification. In addition, this technique will be useful in comparing ubiquitin peptides of different organisms.

Amino Acid Sequence↗
PubMed1985

Ubiquitin-dependent proteolysis of native and alkylated bovine serum albumin: effects of protein structure and ATP concentration on selectivity.

The susceptibility of bovine serum albumin to degradation by the ubiquitin-dependent system of proteolysis depends on the severity of the iodination conditions [Wilkinson, K.D., & Audhya, T.K. (1981) J. Biol. Chem. 256, 9235-9241]. To evaluate if other modifications of the protein changed its susceptibility to degradation, chemically modified derivatives of bovine serum albumin have been synthesized, characterized, and tested as substrates for the ubiquitin-dependent system. Serum albumin was reduced or reduced and alkylated with iodoacetic acid or iodoacetamide. Only the alkylated derivatives exhibit saturation kinetics. Both alkylated proteins competitively inhibit the degradation of the other. These substrates are useful for assay of the intact proteolysis system in crude extracts and in assays for other substrates using competitive alternate substrate inhibition. The physical properties of these proteins suggest that charge, denaturation, or aggregation is not correlated with the degradation rate of these proteins by this system. However, the selectivity of the ubiquitin-dependent proteolysis depends strongly on the ATP concentration. At saturating substrate concentrations, both alkylated substrates are degraded equally. At low ATP concentrations, there is a 2.4-fold difference in the degradation rates of the alkylated proteins. The results presented here indicate that the ubiquitin-dependent protein degradation system is selective and responsive to ATP concentrations and that not all abnormal proteins are equally preferred substrates. Thus, the system may be more selective than previously thought.

Adenosine Triphosphate↗
PubMed1985

Three-dimensional structure of ubiquitin at 2.8 A resolution.

The three-dimensional structure of ubiquitin has been determined at 2.8 A resolution. X-ray diffraction data for the native protein and derivatives were collected with an automated diffractometer. Phases were obtained by use of a single isomorphous mercuric acetate derivative. The molecule contains a pronounced hydrophobic core. Prominent secondary structural features include three and one-half turns of alpha-helix, a mixed beta-sheet that contains four strands, and seven reverse turns. The histidine, tyrosine, and two phenylalanine residues are located on the surface of the molecule.

Amino Acid Sequence↗
PubMed1985

The large scale purification of ubiquitin from human erythrocytes.

A simple, reproducible method for the large-scale purification of active ubiquitin from human erythrocytes is described. Erythrocytes contain 100 micrograms free ubiquitin per cc of packed cells, of which 44% can be recovered in homogeneous form by a combination of heat treatment, ammonium sulfate fractionation, and ion exchange chromatography.

Chemical Precipitation↗
PubMed1984

Gentamicin diffusion across hydrogel bandage lenses and its kinetic distribution on the eye.

Soft contact lenses have been used as therapeutic bandages to aid epithelial healing following pentrating keratoplasty. Often the hydrogel lenses are used in conjunction with topical medications such as gentamicin. A reported complication is the persistence of infectious ulcers even though the eye is being treated with topical antibiotics. The purpose of this study was to measure the gentamicin diffusion coefficients for some hydrogel bandage lenses and to design a kinetic model to estimate the drug distribution on an eye covered with a hydrogel contact lens. The model includes the hydrogel diffusion coefficients and literature values for tear production, tear exchange per blink around the edge of a lens, fit, etc. From the computer generated data, it can be shown that the permeability of gentamicin sulfate through the Saulfon-80 hydrogel lens on a normal eye was only 0.002% of the amount of the drug under the contact lens after 10 minute intervals of topical drug application. The important drug distribution pathway was around the edge of the lens.

Biological Availability↗
PubMed1981

Stimulation of ATP-dependent proteolysis requires ubiquitin with the COOH-terminal sequence Arg-Gly-Gly.

It was previously shown that ubiquitin is very similar to the polypeptide cofactor of the ATP-dependent protein degradation system from rabbit reticulocytes (Wilkinson, K. D., Urban, M. K., and Haas, A. L. (1980) J. Biol. Chem. 255, 7529-7532). We have extended this work to show that the peptic peptide maps are identical for bovine ubiquitin and the polypeptide cofactor isolated from human erythrocytes. It was noted however that ubiquitin preparations were less active in stimulating proteolysis than preparations of the polypeptide cofactor. This decreased activity has been shown to be due to the presence of an inactive form of ubiquitin in some preparations. The two forms of ubiquitin are separable by high performance liquid chromatography. The active form of ubiquitin has the COOH-terminal sequence -Arg-Gly-Gly at residues number 74 to 76. The inactive form terminates in -Arg74 as previously reported in the sequence studies of ubiquitin. Limited tryptic digestion of active ubiquitin yields the inactive, later eluting form and the dipeptide glycylglycine. This preteolytic cleavage apparently occurs during purification from most tissues. We thus propose reserving the term ubiquitin for the intact 76-amino acid sequence and designating the 74-amino acid sequence as ubiquitin-t to indicate its derivation by a tryptic-like protease cleavage. This 76-residue sequence is consistent with the covalent structure of protein A-24, a conjugate where carboxyl group of the COOH-terminal glycylglycine of ubiquitin is linked by an amide bond to the epsilon-amino group of Lys-119 of histone H2A. Thus, the structural requirements of the protein and ubiquitin molecules are identical for formation of protein A-24 and for forming the covalent conjugates thought to be intermediates in ATP-dependent protein degradation.

Adenosine Triphosphate↗
PubMed1981

NADH inhibition and NAD activation of Escherichia coli lipoamide dehydrogenase catalyzing the NADH-lipoamide reaction.

A unique form of inhibition by NADH and partial reversal by NAD+ has been demonstrated with Escherichia coli lipoamide dehydrogenase. Substrate inhibition by NADH is consistent with its reduction of the active two-electron reduced enzyme intermediate to the inactive four-electron reduced form. NAD+ partially overcomes this inhibition by mass action reversal of this reduction. NAD+ activation is only partial since the presence of both NAD+ and NADH forces the accumulation of two binary enzyme-pyridine nucleotide complexes. These are intermediates in the two-electron to four-electron reduction of the enzyme and thus are not on the catalytic pathway. NAD+ is also shown to inhibit by binding to the oxidized enzyme to give a dead-end complex. From the steady state rate equations, it is apparent that the degree of inhibition will depend on the oxidation-reduction potential for two- to four-electron reduction of the enzyme. Thus, the wide variation in the severity of NADH inhibition between the E. coli and pig heart enzymes is explained by quantitative differences in the basic lipoamide dehydrogenase mechanism. A possible physiological role for this type of inhibition as a mechanism of control in E. coli is discussed.

Dihydrolipoamide Dehydrogenase↗
PubMed1980

Ubiquitin is the ATP-dependent proteolysis factor I of rabbit reticulocytes.

A small heat-stable polypeptide, ATP-dependent proteolysis factor 1 (APF-1), is an essential component of the ATP-dependent proteolytic system of rabbit reticulocytes (Ciechanover, A., Hod, Y., and Hershko. A. (1978) Biochem. Biophys. Res Commun. 81, 1100-1105). The following evidence supports the view that APF-1 is ubiquitin, a highly conserved heat-stable polypeptide found universally in nature: 1) APF-1 and ubiquitin (generously given by G. Goldstein) yield co-migrating bands on five polyacrylamide gel electrophoresis systems and in isoelectric focusing; 2) amino acid analysis shows excellent agreement between the two proteins; 3) APF-1 and ubiquitin give similar specific activity, in activating the ATP-dependent proteolysis system; 4) 125I-APF-1 and 125I-ubiquitin form electrophoretically identical covalent conjugates with endogenous reticulocyte proteins. Recently, such conjugates have been proposed as the active intermediates in ATP-dependent proteolysis (Ciechanover, A., Heller, H., Hershko, A., Haas, A.L., and Rose, I.A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1783-1786). Thus, ubiquitin is an essential component of the ATP-dependent system in reticulocytes and a similar role in degradation and proteolytic processing in other cells is likely.

Amino Acids↗
PubMed1980

Glucose exchange and catalysis by two crystalline hexokinase x glucose complexes. Evidence for an obligatory ATP-dependent conformational change in catalysis.

Hexokinase PI x glucose crystals grown with radiolabeled glucose under conditions similar to those used for x-ray diffraction studies (Bennett, W.S., Jr., and Steitz, T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852) have been shown to contain 1 mol of tightly bound glucose. These crystals exchange all of this glucose in a single exponential process (kobs = 0.7 min-1). In the crystalline form they are, however, inactive and do not catalyze formation of any bound glucose-6-P, suggesting that lattice forces prevent catalysis. A new catalytically active E x glucose complex has been crystallized in the presence of glucose and ADP, a competitive inhibitor of ATP. These crystals readily lose ADP upon washing with concentrated (NH4)2SO4. They exchange all of the tightly bound glucose more slowly than the form grown in the absence of ADP (kobs = 0.05 min-1). Addition of MgATP to the suspension in ammonium sulfate results in rapid conversion of half of the bound glucose to bound glucose 6-phosphate followed by further reaction as products are released. This agrees with the previously measured equilibrium constant of unity for enzyme-bound phosphoryl transfer catalyzed in solution (Wilkinson, K.D., and Rose, I.A. (1979) J. Biol. Chem. 254, 12567-12572). These results indicate that the two E x glucose crystals are distinguished by a nucleotide-dependent conformational difference, which is stabilized by lattice forces. The active crystals allow the facile dissociation of the ADP used to induce the change. This conformational change appears to be pevented in the E x glucose crystals and to be necessary to produce the active ternary complex.

Adenosine Triphosphate↗
PubMed1980

A suggestion for naming faces of ring compounds.

Discussion of the topology of interaction of ring compounds with macromolecules and receptors requires a system for naming the faces of the cyclic compound. An alpha/beta-face nomenclature is suggested that is based on the clockwise/counterclockwise direction of ascending numbering in the ring with the lowest numbered unshared ring atom. This system is applicable to a very broad range of compounds: sugars, cyclic bases, steroids, cyclitols, porphyrins, etc., and molecules with a single ring, fused rings, encompassing rings, and rings formed by head-to-tail polymerization.

Journal Article↗