Fernando Henriques

Characterization of three new chlorophyll-protein complexes.

Abstract Three new chlorophyll-proteins with electrophoretic mobilities intermediate between those of the P700 chl a-protein and the light-harvesting chl a,b-protein complexes are reported and their absorption spectra and polypeptide composition are characterized. Two of these chlorophyll-proteins, bands IIb and IIa, contain approximately equal amounts of chl a and b, have polypeptide compositions similar to that of the light-harvesting chl a,b-protein and probably represent oligomers of the latter complex. The third new chlorophyll-protein contains only chl a and its major polypeptide(s) is in the 42 kd region. Indirect evidence indicates this chlorophyll-protein is associated with the reaction-center of photosystem II.

Compositional characteristics of a chloroform/methanol soluble protein fraction from spinach chloroplast membranes

Extraction of an aqueous suspension of spinach chloroplast lamellae with a chloroform/methanol mixture leads to solubilization of about 1/3 of the total membrane protein. Amino acid analysis of the chloroform/methanol-soluble protein shows that this fraction is largely enriched in the hydrophobic residues proline, leucine, alanine and phenylalanine and considerably depleted in polar amino acids, namely lysine and arginine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized material reveals the presence of a variety of low molecular weight polypeptides (molecular weight less than or equal to 25 000), with more than 50% of the total fraction being contributed by a 25 000 dalton band. This band, which accounts for about 25% of the total chloroplast lamellar protein, has recently been identified as the main component of the light-harvesting chlorophyll-protein complex. The physiological role of most of the chloroform/methanol-soluble protein fraction is not known at present. From its chemical properties and apparent biological inertness, we propose that it plays mainly a structural role in situ, interacting with the lipid moiety of the chloroplast membrane. The material insoluble in the aqueous chloroform/methanol mixture is largely enriched in manganese, iron, cytochrome and water-soluble proteins, such as chloroplast coupling factor and ribulose diphosphate carboxylase.

Identification of chloroplast membrane peptides with subunits of coupling factor and ribulose-1,5 diphosphate carboxylase

Coupling factor 1 and ribulose-diphosphate carboxylase are the main peripheral proteins associated with chloroplast internal membranes. The two proteins were sequentially solubilized and purified by gel filtration and their subunit structure was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The differences between the polypeptide profiles of the insoluble membrane fraction, before and after extraction of these oligomeric proteins, allowed identification of original membrane peptides with specific protein subunits. The 52,000 and 14,000 molecular weight peaks are identical to the large and small subunits, respectively, of ribulose-diphosphate carboxylase; the 56,000 and 53,000 peaks are identified with the α and β subunits, respectively, of the coupling factor protein. These identifications, together with earlier studies on the 25,000 Mr band, assign a physiological role to the most prominent peptides of chloroplast internal membranes. Now it becomes apparent that the major membrane polypeptides do not directly relate to photosynthetic electron transport components, but rather to enzymatic capacities associated with this process and to the light-gathering antenna of the photosynthetic unit. The observation that chloroplast coupling factor 1 dissociates during gel filtration, with preferential loss of the smaller subunits (Mr < 50,000) is discussed in relation to the possible function of these subunits in situ in the thylakoid membrane.

Effects of virus infection on the chlorophyll content, photosynthetic rate and carbon metabolism of Tolmiea menziesii

Abstract Leaves from variegated Tolmiea menziesii plants, previously found to be virus-infected, have severely lowered chlorophyll (Chl) levels. Yet, infected plants appeared to grow as rapidly in a greenhouse as green, uninfected control plants. On a Chl basis, the photosynthetic (PS) rate of variegated leaves significantly exceeded that of control leaves. The area-based PS rate of the variegated leaves was less than that of the controls at low light levels, but approached the latters rate at higher intensity. Both types of leaves had similar CO2 compensation points and extents of oxygen inhibition of PS. The variegated leaves had an increased Chl a b ratio and a decreased content of light-harvesting Chi a + b protein. Leaf discs prepared from variegated leaves, when photosynthesizing with 14CO2, produced more labelled glycine and less sucrose than did control discs. The level of PS carbon reduction cycle intermediates was approximately equal in both disc types. We conclude that variegated leaves have a significantly lower amount of antenna Chl but not less reaction center Chl. The increase in labelled glycine in the variegated discs apparently results from viral demand for that metabolite in coat protein and nucleic acid synthesis. Withdrawal of glycine by the virus may be responsible for the lower Chl content of the infected leaves.

Polypeptide cross-linking in chloroplast membranes

Abstract Washed chloroplast membranes from romaine lettuce leaves were treated with the cross-linking reagent dimethyladipimidate (DMA) for various periods of time and subsequently analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparative examination of the electrophoretic profiles from control and treated membranes revealed that the light-harvesting chlorophyll-protein complex (LHCPC) was readily cross-linked to yield “dimers” and “oligomers” of higher molecular weight. Two polypeptides, of 25 and 23 kilodaltons, previously identified as two subunits of the LHCPC, were the major cross-linked species; other peptides were also cross-linked, but to a much lesser extent. These results suggest that cross-linking of chloroplast membranes with DMA, under our conditions, occurs primarily among the components of the LHCPC. We also measured the photosystem II activity in control and DMA-treated chloroplasts and found no impairment of this photochemical activity in the cross-linked chloroplasts as compared with controls.