Murray J. Ettinger

Protein methylation: a signal event in post-translational modification

Despite the data discussed here, little is known about the precise mechanisms that regulate protein methylation in eukaryotic cells. Ligand-stimulated regulation of a specific methyltransferase has not been directly demonstrated. In the case of the interferon receptor, histone methylating activity is constitutive and was not altered by treating intact cells for 5 min with interferon β. PRMT1 activity is qualitatively and quantitatively regulated by GST–TIS21 and GST–BTG1, two similar immediate-early gene fusion proteins, but these effects have only been demonstrated in vitro. The evidence for the involvement of protein methylation in differentiation is indirect, since these studies either rely on methylation inhibitors or measure the changes in methylation associated with unidentified, albeit specific, cellular proteins. The effects of methylation on the function of these proteins are also unknown. Thus, further efforts are required to establish the identities of both the enzymes and the substrates involved in regulated protein methylation in any given cellular model. For example, the nonreceptor agonist, elevated glucose, was recently shown to increase the incorporation of base-labile methyl groups (indicative of carboxyl methylation) into anti-CDC42 and anti-Rap1 immunoprecipitates from intact cells[24xKowluru, A. et al. J. Clin. Invest. 1996; 98: 540–555CrossrefSee all References][24]. Additional experiments indicate that carboxyl methylation of G proteins in this system is a requisite step in nutrient-induced insulin secretion. Similar approaches aimed at the identification of specific substrates altered by receptor-mediated events and the identification of the protein methyltransferase and methylesterase involved will further focus the molecular analysis of the role of regulated protein methylation in distinct cellular functions.The abundance of important signaling molecules that are methylated (including PP2A, G proteins, the Ras family of proteins, transcription factors and RNA processing proteins) and recent studies coupling growth factor receptors with protein methylation ensure that the role of protein methylation in cellular signaling events will receive a renewed and deserved interest in the near future.

The molecular properties of the copper enzyme galactose oxidase

Abstract Galactose oxidase (EC 1.1.3.9) has been purified 140-fold by DEAE- and CM-cellulose chromatography from cultures of Polyporus circinatus . The enzyme has a molecular weight of 68,000 ± 3,000 as determined by sedimentation equilibrium, sodium dodecyl sulfate-acrylamide gel electrophoresis, Sephadex G-150 chromatography, and osmometry. Galactose oxidase is a single-chain protein which does not self-associate. Charge isozymes of the enzyme are detected by ion-exchange chromatography and gel electrophoresis. The amino acid composition determined herein is significantly different from that previously reported (Kelly-Falcoz, F., Greenberg, H., And Horecker, B. L. (1965) J. Biol. Chem. 240 , 2966–2970). The enzyme contains 1% by weight of neutral carbohydrate. Galactose oxidase contains 1 g-atom of copper per 70,000 g of protein. The metal does not contribute to the electrophoretic or isozymic properties of the protein. However, the sedimentation coefficients of the holo- and apoenzymes, 4.76S and 4.83S, respectively, do suggest that small differences in protein conformation accompany the removal of the copper from the holoenzyme. Attempted sulfhydryl group titration of galactose oxidase shows that the holoenzyme is resistant to denaturation. However, in β-mercaptoethanol-guanidine HCl 5 half-cystine residues are titrated in the apoenzyme. On a dry-weight basis, the E 1cm 1% value for galactose oxidase at 280 nm is 15.4. Galactose oxidase has an isoelectric point above pH 10 which is a probable source of some of its anomalous behavior in physical measurements and enzyme-activity determinations.

Identification of an Apo-Superoxide Dismutase (Cu,Zn) Pool in Human Lymphoblasts

Copper incorporation (64Cu(II)) into Cu,Zn-superoxide dismutase (SOD) was studied in human lymphoblasts. Rapid incorporation of copper with a proportionate increase in SOD activity was detected. No copper incorporation or SOD activation was detected when 64Cu(II) was added to cell cytosols rather than to intact cells. Thus, incorporation of 64Cu was not due to isotopic exchange. Cycloheximide had no significant effect on copper incorporation and activation of SOD when the data were corrected for total cell copper. Thus, the data were consistent with copper incorporation into a preexisting apoSOD pool rather than newly synthesized SOD, and no new SOD synthesis was detected over a 15-h incubation period. The size of the apoSOD pool was estimated to be ≈35% of the total SOD in lymphoblasts. When cells were preincubated for 15 h with excess copper (15 μM Cu(II)), the size of the apo pool markedly decreased but was not eliminated, suggesting that the apoSOD was not due to copper deficiency. These experiments also indicated that newly arrived copper was preferentially incorporated into the apoSOD pool, while the function(s) of an apoSOD pool remains unknown. Copper binding to apoSOD may provide a rapid protective response against copper toxicity.

Copper Incorporation into Superoxide Dismutase in Menkes Lymphoblasts

The incorporation of copper into Cu,Zn-superoxide dismutase (SOD) was examined in Menkes lymphoblasts that express a genetic defect of copper metabolism. SOD activity was ≈40% higher in Menkes than normal lymphoblasts. Since Menkes lymphoblasts contain elevated copper levels, the higher SOD activity is most likely due to near copper saturation of an apoSOD pool that is in normal lymphoblasts. Cycloheximide markedly inhibited 64Cu(II) incorporation into SOD in Menkes lymphoblasts under conditions in which no significant, de novo synthesis of SOD protein was detected with normal lymphoblasts. The maximal amount of 64Cu incorporation into newly synthesized SOD in Menkes lymphoblasts was approximately equal to the maximal amount of 64Cu that could be incorporated into the apoSOD pool in normal lymphoblasts. The increased synthesis of SOD in Menkes lymphoblasts may play a protective role against copper toxicity in Menkes lymphoblasts. The protonophore, CCCP markedly inhibited 64Cu incorporation into SOD in both normal and Menkes lymphoblasts, which is consistent with 64Cu incorporation into SOD within a membrane-bounded compartment in both cell types. When 64Cu-incorporation into SOD was blocked with CCCP, copper accumulated in a Superose column fraction that contains S-adenosylhomocysteine hydrolase (SAHH), which has a high affinity for copper. SAHH may play a role in delivering copper to SOD.

Fluoride ion as a nmr relaxation probe of paramagnetic metalloenzymes: the binding of fluoride to galactose oxidase

Summary The use of fluoride ion, as a novel nuclear magnetic resonance relaxation probe of paramagnetic metalloenzymes, has been tested on galactose oxidase. The concentration dependence of the F − longitudinal relaxation rates, R 1 = 1/T 1 , and competition studies with CN demonstrate that F binds to the enzyme at or near the Cu 2+ site with a binding constant of the order of 1 M −1 . Competition studies with galactose indicate that a ternary or higher order complex between enzyme, galactose and F − is formed.

On the role of a cuprous ion intermediate in the galactose oxidase reaction

The Cu+2 electron spin resonance spectrum of galactose oxidase (galactose:O2 oxidoreductase, E.C. 1.1.3.9) indicates that the metal is in a pseudo-square planar environment. The electron g values are: gzz = 2.273, gxx = 2.058 and gyy = 2.048. The copper nuclear hyperfine constants are (in Gauss): Azz = 176.5, Axx = 28.8 and Ayy = 30.1. This spectrum is unaltered in either intensity or g or A values under conditions which cause the inhibition of galactose oxidase by superoxide dismutase. No combination of substrates (galactose and O2) and oxidant traps (superoxide dismutase and catalase) results in the reduction of the cupric ion resonance. Thus, a Cu+1-enzyme does not appear to be a stable intermediate along this enzymes reaction path.