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E L Gross

Publications and source records attributed to E L Gross.

At least 37 records · Page 2Linked to original sources
PubMed1982

Chemical modification of spinach plastocyanin: separation and characterization of four different forms.

Spinach plastocyanin has been chemically modified by using a water-soluble carbodiimide to form an amide bond between a protein carboxyl group and one amino group of ethylenediamine. This reaction produced four distinct species of chemically modified plastocyanin which could be separated either by polyacrylamide gel electrophoresis (in the absence of sodium dodecyl sulfate) or by ion-exchange chromatography on DEAE-Sephadex. The four forms contained 2.1, 3.2, 4.1, and 6.3 mol of ethylenediamine per mol of plastocyanin and were labeled fractions IV, III, II, and I, respectively. All four fractions showed a shift in midpoint redox potential of greater than +40 mV. In contrast, the Km for P700+ reduction decreased with increasing extents of modification. The addition of Mg2+ ions decreased the Km for the negatively charged fractions II, III, and IV but increased it for the positively charged fraction I. Fraction III showed an increased incorporation of ethylenediamine into residues 42-45 which are located approximately 18 A distant from the copper. This fraction also showed a maximal increase in redox potential. Potential calculations showed that the electrostatic effect of incorporating two positive charges at this distance was sufficient to produce a shift in redox potential of +40 mV. However, an electrostatically induced conformational change which would affect the environment of the copper cannot be ruled out.

Carbodiimides↗
PubMed1982

Effect of trypsin treatment of photosystem I particles on the electron donation to p700.

Proteolysis of photosystem I particles had no effect on P700 oxidation but did inhibit the rate of P700(+) reduction. The V(max) values were decreased for both dichlorophenol and plastocyanin, but the K(m) values were unaffected indicating that trypsin treatment altered electron transfer rather than the binding of the donor to the photosystem I complex. The salt dependence of P700(+) reduction was unaffected. The effects of P700(+) reduction were the same for the preparations of different workers (Shiozawa, Alberte, Thornber 1974 Arch Biochem Biophys 165: 388; and Bengis, Nelson 1975 J Biol Chem 250: 2783).In both cases, the 70-kilodalton, chlorophyll-containing polypeptide was digested confirming its role in transferring electrons from plastocyanin to P700. The fact that the preparation of Shiozawa et al. lacks subunit (III) but still used plastocyanin as the electron donor rules out a role for this subunit as "the plastocyanin binding protein." Subunit III was also digested in the Bengis and Nelson preparation.

Journal Article↗
PubMed1981

Effect of carboxyl group modification on redox properties and electron donation capability of spinach plastocyanin.

Spinach plastocyanin was chemically modified by using a water-soluble carbodiimide to form an amide bond between a protein carboxyl group and one amino group of ethylenediamine. On the average, four plastocyanin carboxyl groups were replaced with positively charged amino groups. Modified plastocyanin facilitated high rates of electron donation to the oxidized P700 reaction center of photosystem I particles in the absence of cations. Control plastocyanin was totally inactive in the absence of divalent cations due to charge repulsion between the negatively charged plastocyanin and photosystem I proteins. The Km for the binding of modified plastocyanin to photosystem I particles was 2.1 uM compared to 36.5 uM for control plastocyanin in the presence of MgCl2. Therefore, chemical modification was mre effective than charge shielding by cations in facilitating the binding of plastocyanin to photosystem I. Chemical modification also increased the midpoint redox potential of plastocyanin from +380 to +420 mV, which indicated an alteration of the copper environment. Ethylenediamine was covalently attached to tryptic peptides from plastocyanin that contained amino acid residues 42-45, 59-61, and 68 which are highly conserved glutamic acid and aspartic acid residues. Chemically altering the charge on these residues changed the activity of plastocyanin which indicates that these amino acids are involved in the ionic regulation of the plastocyanin-PSI interaction. Because chemical modification also altered the environment of the chromophore, certain of these amino acids must be located near the copper site.

Carbodiimides↗
PubMed1981

Use of chemical modification to study the relationship between activity and net protein charge of the photosystem I core complex.

The net charge on the photosystem I core complex of Shiozawa et al. (Shiozawa, J. A., Alberte, R. S., & Thornber, J. P. (1974) Arch. Biochem. Biophys. 165, 388-397) has been altered by using a water-soluble carbodiimide to form an amide bond between protein carboxyl groups and an amino group of ethylenediamine. This process replaces negatively charged carboxyl groups with positively charged free amino groups. Six hundred moles of ethylenediamine was incorporated per mole of P700 reaction center. The modification of the complex shifted the isoelectric pH of photosystem I from 5.0 to 9.5 without affecting the total amount of P700. The modified complex exhibited an increase in energy transfer from light-harvesting chlorophyll alpha molecules to the reaction center. This phenomenon has been previously observed upon addition of Mg2+ ions to the complex (Gross, E. L., & Grenier, J. (1978) Arch. Biochem. Biophys. 187, 387-398). The modification replaces the divalent cation requirement for electron donation to P700 by plastocyanin and lowered the Km for plastocyanin binding to 2.0 microM, compared to 32 microM for control photosystem I in the presence of Mg2+. In addition, the modification lowered the Km for the negatively charged electron donors dichlorophenolindophenol and ascorbate. These results suggest that changing the charge on the photosystem I complex from negative to positive stimulates both light utilization and electron transfer from electron donors to P700. We suggest that cation regulation of photosystem I activity occurs by a process in which cations alter the charge of the local environment around the complex.

Chlorophyll↗
PubMed1981

Effects of Proteolysis on the Photochemical Activity and Polypeptide Composition of the Photosystem II Core Complex Isolated from Spinach Chloroplasts.

The photosystem II core complex (TSF-IIa) is composed of polypeptides of molecular weight 54-, 47-, 42-, and 30 kilodaltons (kD) and cytochrome b-559. After treatment with trypsin or alpha-chymotrypsin for 20 hours, the TSF-IIa particles still retained their photochemical activity and the light-induced cytochrome b-559 signal, although all of the polypeptides of the complexes, except the 30 kD unit were extensively degraded. Proteolytic treatment decreased the apparent molecular weight of the complex from 250,000 to 100,000 daltons as determined by gel filtration, and also decreased the protein to chlorophyll ratio by 40%. Chlorophyll a appeared to be associated with the 47- and 42 kD polypeptides. Proteolysis of the complex produced a single chlorophyll a band with a slightly higher electrophoretic mobility. This band was not equivalent to the 30 kD polypeptide. Proteolysis also reduced the sensitivity of the TSF-IIa particles to 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), but did not completely abolish it.It was concluded that although the polypeptides of the photosystem II core complex were cleaved by the proteases, many of the peptide fragments do not dissociate from the complex, particularly the chlorophyll a-containing portions, leaving the complex photochemically active. This supports the concept that chlorophyll has a structural function in chlorophyll-protein complexes.

Journal Article↗
PubMed1978

Use of immobilized light-harvesting chlorophyll a/b protein to study the stoichiometry of its self-association.

D. J. Davis & E. L. Gross (1976) Biochim. Biophys. Acta 449, 554-564 previously observed that the light-harvesting chlorophyll a/b protein or chlorophyll protein complex II self-associated as determined by ultracentrifugation. We have determined the stoichiometry of complex formation by immobilizing the monomer on ethylenediamine-Sepharose 4B and determing the ability of immobilized protein to bind the free protein. The amount of soluble protein bound to the immobilized protein increased as the concentration of soluble protein increased. The binding was maximal between pH 7 and 8. The maximum binding was three molecules bound per one molecule of protein immobilized. These results indicate that a tetramer is the intrinsic structural unit of the light-harvesting chlorophyll a/b protein in the chloroplast membrane. Upon complex formation, the chlorophyll fluorescence was decreased without any spectral change. The maximum binding was approximately doubled upon addition of 0.5 mM CaCl2 whereas 5 mM NaCl had no effect. Addition of CaCl2 had no effect on the fluorescence of the monomer. The light-harvesting chlorophyll a/b protein can be isolated from a sodium lauryl sulfate extract of chloroplasts by affinity chromatography using the immobilized light-harvesting chlorophyll a/b protein.

Cations↗
PubMed1976

Protein-protein interactions of the light-harvesting chlorophyll a/b protein. II. Evidence for two stages of cation independent association.

In a previous paper, we observed a two-stage cation-independent association of the light-harvesting chlorophyll a/b protein from spinach chloroplasts based on concentration-dependent changes in the sedimentation coefficient. The two stages of association occurred between (2-4) and (4-7) mug/ml chlorophyll. In this paper, we provide further evidence for this association. This includes: (1) A decrease in the number of divalent cation binding sites in the second stage of association. (2) A corresponding decrease in the extent of the cation-dependent association. (3) A positive deviation from Beer's law for chlorophyll b for both stages of the cation-independent association and a positive deviation for chlorophyll a for the second stage of association only. (4) A change in the fluorescence emission of both chlorophyll a and b. The change for chlorophyll b was observed for both steps of association whereas that for chlorophyll a was observed for the second step of association only. Therefore, the first stage of association affects only chlorophyll b whereas the second stage alters the environment of both chlorophyll a and b. (5) In addition, divalent cations quenched chlorophyll fluorescence. However, the quenching which required 200-300 muM divalent cation for half-maximal effects was related neither to divalent cation binding nor to the divalent cation-induced association of the protein.

Aerobiosis↗
PubMed1976

The effect of mono- and divalent cations on the quantum yields for electron transport in chloroplasts.

We have previously shown (Gross, E.L. and Hess, S.C. (1973) Arch. Biochem. Biophys 159, 832-836) that low concentrations of monovalent cations (3-10 mM) caused changes in chlorophyll a fluorescence indicative of a transfer of excitation energy from Photosystem II to Photosystem I. These effects were reversed by divalent cations or higher concentrations of monovalent cations. In this study, we have examined the effects of cations on the relative quantum yields for Photosystem II (dichlorophenolindophenol reduction) and Photosystem I (diphenylcarbazone disproportionation) with the following results. (1) Low concentrations of monovalent cations decreased the quantum yield for dichlorophenolindophenol reduction and increased that for diphenylcarbazone disproportionation. These results confirm that cations promote excitation energy transfer to Photosystem I. (2) Higher concentrations of monovalent cations (30-100 mM) had no effect on electron transport. Therefore, the increases in chlorophyll a fluorescence observed at these concentrations may be due to a decrease in the rate constant for radiationless decay. (3) In the absence of Tricine, divalent cations also promote energy transfer from the light harvesting pigments to Photosystem I. However, a direct inhibition of Photosystem I and divalent cations can reverse the Tricine effects.m II photochemistry cannot be rules out. (4) Tricine biases the system in favor of Photosystem I and divalent cations can reverse the Tricine effects.

Calcium↗
PubMed1975

Protein-protein interactions of light-harvesting pigment protein from spinach chloroplasts. I.Ca-2+ binding and its relation to protein association.

The role of divalent cations in the regulation of the distribution of excitation energy between the two photosystems involved in green plants photosynthesis has led us to search for a better understanding of how such phenomena might occur at the molecular level. Since small changes in orientation of and distance between pigment molecules could greatly affect the distribution of excitation energy, we have decided to study the effects of ions on the light-harvesting pigment protein from spinach chloroplasts. The light-harvesting pigment protein is shown to have two types of binding sites for Ca-2+. Binding studies and analytical ultracentrifugation indicate that site I (K-d equals 2.5 mu-M, n equals 1.5-4.0 mu-mol Ca-2+ bound/mg chlorophyll) is lost as the protein associates. Site II (K-d equals 32 mu-M, n equals 9,5 mu-mol Ca-2+/mg chlorophyll) is not affected by the association of the protein. This site is responsible, however, for a further divalent cation-dependent association of the protein. The possible role of this protein in grana stacking and control of spillover is discussed.

Calcium↗
PubMed1975

The effect of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide on calcium binding and associated changes in chloroplast structure and chlorophyll a fluorescence in spinach chloroplasts.

1. Chemical modification of carboxyl groups on the chloroplast membrane with a water-soluble carbodiimide plus a nucleophile caused inhibition of Ca-2plus binding. 2. Both binding sites were affected and showed a decrease in the number of binding sites and an increase in the dissociation constant. 3. Cation-induced changes in chlorophyll a fluorescence and structural changes (deltaA540) were inhibited at the same carbodiimide concentrations as Ca-2plus binding, emphasizing the relationship between these processes. 4. Chloroplasts that were illuminated with high intensity light for short time periods showed a decrease in the carbodiimide-mediated inhibition of Ca-2plus binding.

Calcium↗
PubMed1975

Effects of sodium and magnesium cations on the "dark-" and light-induced chlorophyll a fluorescence yields in sucrose-washed spinach chloroplasts.

The effects of Na plus and Mg-2 plus on the "dark" level (O level) and light-induced (P level) fluorescence in sucrose-washed spinach clhoroplasts were studied. Low concentrations of NaCl (2-10 mM) cause a significant decrease in both the O and P levels in the chlorophyll fluorescence transient. The effect on the O level may reflect changes in the bulk chlorophyll a. At 77 degrees K NaCl increases the F735/F685 emission peak ratio in dark-adapted and preilluminated chloroplasts, but has no significant effect on this ratio in sucrose-washed Photosystem II particles. This evidence is consistent with a sodium-induced excitation-energy distribution in favor of Photosystem I. In the presence of MgCl2, with or without NaCl, there is a slight decrease in the O and P level fluorescence as compared with the salt-free control, but an increase as compared with the NaCl-treated sample. Magnesium appears to override the sodium-induced changes. At low temperatures in chloroplasts and Photosystem II particles, MgCl2 has different effects on the F735/F685 ratio apparently depending on the state of the membrane. Magnesium, however, always induces an increase in the F695/F685 ratio. These results suggest that magnesium may influence Photosystem II reaction centers as well as energy distribution between the two photosystems.

Chlorophyll↗