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Title: Resolution of thylakoid polyphenol oxidase and a protein kinase

Abstract

The predominant protein kinase activity in octylglucoside (OG) extracts of spinach thylakoids has been attributed to a 64-kDa protein, tp64. Recent work calls into question the relation between tp64 and protein kinase activity, which were fractionated apart using fluid phase IEF and hydroxylapatite chromatography. Hind et al. sequenced tp64 from the cDNA and showed it to be a polyphenol oxidase (PPO) homolog. Its transit peptide indicates a location for the mature protein within the thylakoid lumen, where there is presumably no ATP and where it is remote from the presumed kinase substrates: the stromally exposed regions of integral PS-II membrane proteins. Here the authors suggest that the kinase is a 64-kDa protein distinct from tp64.

Authors:
; ;
Publication Date:
Research Org.:
Brookhaven National Lab., Upton, NY (United States)
Sponsoring Org.:
USDOE, Washington, DC (United States)
OSTI Identifier:
192405
Report Number(s):
BNL-62487; CONF-9508202-3
ON: DE96003576; TRN: 96:001567
DOE Contract Number:
AC02-76CH00016
Resource Type:
Conference
Resource Relation:
Conference: 10. international photosynthesis congress, Montpellier (France), 20-25 Aug 1995; Other Information: PBD: 1995
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; THYLAKOID MEMBRANE PROTEINS; BIOCHEMISTRY

Citation Formats

Race, H.L., Davenport, J.W., and Hind, G.. Resolution of thylakoid polyphenol oxidase and a protein kinase. United States: N. p., 1995. Web.
Race, H.L., Davenport, J.W., & Hind, G.. Resolution of thylakoid polyphenol oxidase and a protein kinase. United States.
Race, H.L., Davenport, J.W., and Hind, G.. 1995. "Resolution of thylakoid polyphenol oxidase and a protein kinase". United States. doi:. https://www.osti.gov/servlets/purl/192405.
@article{osti_192405,
title = {Resolution of thylakoid polyphenol oxidase and a protein kinase},
author = {Race, H.L. and Davenport, J.W. and Hind, G.},
abstractNote = {The predominant protein kinase activity in octylglucoside (OG) extracts of spinach thylakoids has been attributed to a 64-kDa protein, tp64. Recent work calls into question the relation between tp64 and protein kinase activity, which were fractionated apart using fluid phase IEF and hydroxylapatite chromatography. Hind et al. sequenced tp64 from the cDNA and showed it to be a polyphenol oxidase (PPO) homolog. Its transit peptide indicates a location for the mature protein within the thylakoid lumen, where there is presumably no ATP and where it is remote from the presumed kinase substrates: the stromally exposed regions of integral PS-II membrane proteins. Here the authors suggest that the kinase is a 64-kDa protein distinct from tp64.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = 1995,
month =
}

Conference:
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  • A 64 kDa phosphoprotein copurifies with histone kinase activity in detergent extract of thylakoid membranes and has been provisionally identified as the kinase (e.g. JBC 261 14062). The authors have prepared antibodies to this protein. As measured by western blotting, release of the kinase from thylakoids correlates with loss of kinase activity and preceeds solubilization of the bulk of the cytochrome b/f complex. Although treatment with NaBr, KI and urea did not release the protein, some was lost during NaOH washes and essentially all of it appeared in the aqueous fraction during the phase partitioning with Triton X-114. Although itmore » dissociates with relative ease from thylakoids, fractionation experiments indicate that the 64kDa is in fact a membrane protein and not an adventitiously bound stromal enzyme. Measured on the same 13% acrylamide gel, the kinase's molecular weight is 5 to 7 kDa smaller than that of the major stromal protein labeled with {sup 32}P-ATP. Unlike the stromal protein, the extent of labeling of the 64 kDa was unchanged by the subsequent chases with non-radioactive ATP. They conclude that while the 64 kDa protein may be a peripheral membrane protein, it is not a contaminating stromal enzyme with a phosphoenzyme intermediate like phosphoglucomutase. It is very likely that it is the kinase.« less
  • Control of state transitions in the thylakoid by reversible phosphorylation of the light-harvesting chlorophyll a/b protein complex of photosystem II (LHC-II) is modulated by a kinase. The kinase catalyzing this phosphorylation is associated with the thylakoid membrane, and is regulated by the redox state of the plastoquinone pool. The isolation and partial purification from spinach thylakoids of two protein kinases (CPK1, CPK2) of apparent molecular masses 25 kDa and 38 kDa has been reported. Neither enzyme utilizes isolated LHC-II as a substrate. The partial purification of a third protein kinase (LHCK) which can utilize both lysine-rich histones (IIIs and Vs)more » and isolated LHC-II as substrate has now been purified to homogeneity and characterized by SDS-polyacrylamide gel electrophoresis as a 64 kDa peptide. From a comparison of the two isolation procedures we have concluded that CPK1 is indeed a protein kinase, but has a lower specific activity than that of LHCK. 8 refs., 4 figs.« less
  • Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg/sup 2 +/ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg/sup 2 +/ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB)more » inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs.« less
  • Polyphenol oxidase activity (E.C. 1.14,18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. Sonication releases polyphenol oxidase from the membrane largely in the latent state. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time. Purified polyphenol oxidase utilizes o-diphenols asmore » substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K/sub m/. A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.« less
  • Coriolus versicolor, a white-rot basidiomycete, secretes ligno-celluloytic enzymes. Because these are valuable to paper-pulp agricultural industries, trials are in progress to substrate induce these enzymes enhance their secretions. Reported are attempts to develop an extracellular PPO (o-diphenols to 0-diquinones) purification protocol applicable to [open quote]batch-cultured[close quote] C. versicolor. Whereas dialysis (MW [open quote]cut-off[close quote], 14,000) of 13 day growth medium (GM) resulted in 2.17 fold PPO spc. act. increase, dialysis plus a 0-30% (NH[sub 4])[sub 2]SO[sub 4] [open quote]cut[close quote] yielded a 3.27 fold enhancement. Subsequent GM chromatography on DEAE CM-Sephadexes revealed that PPO exchanged with DEAE's counterion without enhancingmore » spc. act. Gel filtration of GM commercial PPOs on G-150 resulted in similar elutions indicating a substitute for ion exchange chromatography. Time-dependent fungal growth in liquid medium followed by viscometry utilizing CMC revealed a GM endocellulase 2 days after inoculation an activity rise to day 12. Filteration of Onozuka cellulase on G-150 yielded an elution profile similar to those of GM authentic PPO's compounding C. versicolor's PPO purification. SDS-PAGE of dialyzed GM revealed 4 proteins, one of which was removed by the (NH[sub 4])[sub 2]SO[sub 4]. The m[sub TS] of commercial Sigma's PPO Onozuka cellulase were 0.76 0.59, respectively, for comparison to C. versicolor's PPO. Affinity, hydroxylapatite hydrophobic interaction chromatographies may yield a single SDS-PAGE PPO band.« less