Using the detergents n-dodecyl ␤-D-maltoside and heptyl thioglycopyranoside, a subcore complex of photosystem II (PSII) has been isolated that contains the chlorophyll-binding protein, CP47, and the reaction center components, D1, D2, and cytochrome b 559 . We have found, by using sucrose density centrifugation, that the resulting preparation consisted of a mixture of dimeric and monomeric forms of the CP47 reaction center (RC) complex, having molecular masses of 410 ؎ 30 and 200 ؎ 28 kDa, respectively, as estimated by size exclusion chromatography. The level of the dimer in the preparation is significantly higher than the monomeric form. Both the monomer and dimer contain the proteins CP47, D1, and D2 and the ␣-and ␤-subunits of cytochrome b 559 . Analyses by mass spectrometry and N-terminal sequencing showed that both forms of the CP47-RC complex contain the products of the psbI, psbT c (chloroplast gene), and psbW with molecular masses of 4195.5, 3849.6, and 5927.4 Da, respectively. In contrast to the monomeric form, the CP47-RC dimer contained two extra proteins with low molecular weights, identified as the products of the psbL and psbK genes having molecular masses of 4365.5 and 4292.1, respectively. It was also found that the dimer contained slightly more molecules of chlorophyll a (21 ؎ 2.5) than the monomer (18 ؎ 1.5), a characteristic also observed in the room temperature absorption spectrum by comparing the ratio of absorption at 416 and 435 nm. Of particular note was the finding that the dimer, but not the monomer, contained plastoquinone-9 (estimated to be 1.5 ؎ 0.3 molecules per RC). The results indicate that the CP47-RC monomer is derived from the dimeric form of the complex, and therefore the latter is likely to represent an in vivo conformation. The PsbT c as well as the PsbI and PsbW proteins are identified as being intimately associated with the D1 and D2 proteins, and in the case of the dimer, importance is placed on the PsbL and PsbK proteins in sustaining plastoquinone binding and maintenance of the dimeric organization. Assuming only one copy of the ␣-and ␤-subunits of cytochrome b 559 , the monomeric and dimeric forms of the complex would be expected to contain 21 and 23 ؋ 2 transmembrane helices, respectively.The light-induced splitting of water and the consequential release of dioxygen is a fundamental reaction in photosynthesis taking place in all higher plants, algae, and cyanobacteria. This reaction is catalyzed by a complex known as photosystem II (PSII) 1 which is embedded in the lipid bilayer of the thylakoid membrane. The PSII complex is composed of at least 25 different proteins and binds a large number of pigments (1). At the heart of this complex is the reaction center (RC), consisting of the D1 and D2 proteins, where primary charge separation occurs (2). Closely associated with the D1 and D2 heterodimer are the two chlorophyll a-binding proteins, CP47 and CP43, and a range of small hydrophobic polypeptides including the ␣-and ␤-subunits of cytochrome b 559 . CP47 and CP43 act ...

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Mass spectrometry techniques have been applied in a protein mapping strategy to elucidate the majority of the primary structures of the D1 and D2 proteins present in the photosystem II reaction center. Evidence verifying the post-translational processing of the initiating methionine residue and acetylation of the free amino group, similar to those reported for other higher plant species, are presented for the two subunits from pea plants (Pisum sativum L.). Further covalent modifications observed on the D1 protein include the COOHterminal processing with a loss of nine amino acids and phosphorylation of Thr 2 . In addition, the studies reported in this paper provide the first definitive characterization of oxidations on specific amino acids of the D1 and D2 proteins. We believe that these oxidations, and to a much lesser extent the phosphorylations, are major contributors to the heterogeneity observed during the electrospray analysis of the intact subunits reported in the accompanying paper (Sharma, J., Panico, M., Barber, J., and Morris, H. R. (1997) J. Biol. Chem. 272, 33153-33157). Significantly, all of the regions that have been identified as those particularly susceptible to oxidation are anticipated (from current models) to be in close proximity to the redox active components of the photosystem II complex.The accurate molecular mass information afforded by techniques such as electrospray ionization (ESI) 1 mass spectrometry (MS) is particularly useful in cases where gene sequences are available; a correlation between experimental and expected measurements can be sufficient in confirming the identity and homogeneity of intact proteins. However, in situations where molecular weight discrepancies are detected or more detailed structural characterization is required, an MS peptide mass mapping strategy is usually applied (1). This procedure uses suitable MS techniques to analyze the peptides generated by enzymatic and/or chemical digestion of the protein under investigation. The molecular weights for the products must match within experimental error those predicted from the protein sequence; those that do not are then presumed to correspond to peptides containing sequence errors or post-translational modifications, or they may provide evidence for contaminating proteins.Detailed structural information of proteins is of fundamental importance in developing an understanding of their biological activities. It therefore follows that analysis of the primary structural features that govern the higher orders of structures of proteins are of considerable interest. The application of a wide range of biochemical and molecular biological studies has identified several post-translational modifications on the D1 and D2 proteins of photosystem II (PSII) reaction centers, for example NH 2 -and COOH-terminal proteolytic processing (2, 3), acetylation (2), lipid attachment and acylation (4, 5), and phosphorylation (2, 6). Most of these covalent processes have been associated with the biosynthesis and assembly of active photosy...

A sensitive and simple reverse phase HPLC purification scheme was developed for the rapid separation of the small protein subunits from photosystem II reaction center preparations. The precise molecular masses of the ␣-and ␤-subunits of cytochrome b 559 and the psbI gene product from pea plants, found to be 4394.6 ؎ 0.6, 9283.6 ؎ 0.7, and 4209.5 ؎ 0.5 Da, respectively, were then successfully determined for the first time by electrospray-and fast atom bombardment-mass spectrometry. Discrepancies between the molecular weights assigned and those calculated from the respective DNA sequences were observed for ␣-and ␤-subunits of cytochrome b 559 . Currently, the nucleotide sequence of the psbI gene product from pea plants is not available. Application of novel mapping and sequencing strategies has assured the elucidation of full primary structures of all of the purified subunits. The modifications identified here include the post-translational processing of the initiating methionine on both subunits of cytochrome b 559 , NH 2 -terminal acetylation and an mRNA editing site at residue 26 (Ser 3 Phe) on the ␤-subunit, and retention of the NH 2 -terminal formyl-Met on the psbI gene product. In addition, specific oxidation of a single amino acid residue was identified on the psbI gene product and the ␤-subunit purified from light-treated reaction center preparations. Overall, these studies provide the first detailed primary structural characterization of the small subunits of the reaction center complex and their associated light-induced modifications. Photosystem II (PSII)1 catalyzes the photoinduced splitting of water into molecular oxygen and reducing equivalents. This complex is embedded in the thylakoid membrane of plants, algae, and cyanobacteria and is composed of a large number of subunits (1, 2). At the heart of the complex is the reaction center where primary and secondary electron transfer processes occur. The cofactors that support these reactions are bound to the D1 and D2 proteins. These proteins are the products of the psbA and psbD genes, respectively, and are comparable with the L and M subunits of the reaction center of purple photosynthetic bacteria (3).In its isolated form, the reaction center of PSII contains, in addition to the D1 and D2 proteins, the ␣-and ␤-subunits of cytochrome b 559 , which are the products of the psbE and psbF genes, and the PSII-I protein, which is encoded by the psbI gene. In a recent paper (4), a sixth component has been claimed, which has an apparent molecular mass of about 6.5 kDa and is the product of the nuclear gene recently named psbW.Sites in the D2 and D1 proteins, named Q A and Q B respectively, bind plastoquinones that facilitate secondary electron flow. However, in the case of the isolated reaction center complex the plastoquinone binding affinity is significantly reduced, and no secondary electron flow occurs unless appropriate acceptors are added (5). In their absence, the photochemical reaction is restricted to the formation of the radical pair state P680 ϩ Phe...

A reverse phase high pressure liquid chromatography purification system for the rapid separation of photosystem II reaction center proteins free of salts and detergents is described. This procedure results in the isolation of the three small subunits: ␣-and ␤-subunits of cytochrome b 559 and PsbI protein, with near base-line resolution between each peak, although the D1 and D2 proteins were partially deconvoluted. Photosystem II (PSII)1 is a pigment-protein complex that catalyzes the unique reactions resulting in the splitting of water molecules into dioxygen and reducing equivalents (2). This complex is embedded in the thylakoid membrane of plants, algae, and cyanobacteria and is made up of more than 20 different subunits (3). It is now accepted that the photochemical reactions of PSII take place in a reaction center comprised of two proteins, D1 and D2 (4), which show many characteristics similar to those of the L and M subunits of purple photosynthetic bacteria. These subunits bind all of the redox factors required for primary electron transfer.In higher plants the D1 and D2 subunits, encoded by the chloroplast psbA and psbD genes, respectively, are synthesized on the 70 S ribosomes that are attached to the stromal thylakoids. Evidence from several biochemical and molecular biological studies has shown that upon translation both proteins undergo a variety of structural modifications. It is believed that most of these covalent changes are involved in the development and assembly of active PSII complexes or in controlling the triggering and degradation of the photodamaged PSII components (for review, see Ref. 5).Mass spectrometry is a highly valuable technique in the field of structural biochemistry. Electrospray ionization mass spectrometry (ESI-MS), with an accuracy of about 0.01%, provides an extremely sensitive method for determining the precise molecular mass of biological molecules Ͼ100 kDa. In many cases, the measurement of the intact molecular mass of a protein and comparison with the predicted value have been used to verify gene sequences, locate mRNA processing and editing events (1), identify mutations (6), detect post-translational modifications, and provide confirmation of start and stop signals of a gene, which, in some instances such as that of the psbC gene encoding the CP43 protein, have been difficult to define by other methods (7). In fact, now a substantial number of publications, concerned mainly with hydrophilic proteins, describe the successful application of this technique.ESI-MS of very hydrophobic species such as membrane proteins has, however, proved significantly more challenging than related studies on hydrophilic proteins. This characteristic can be attributed mainly to the incompatibility between most mass spectrometric techniques and the presence of detergents and/or salts required to retain the analytes in solution. Thus, the successful analysis of hydrophobic proteins and peptides relies upon developing successful sample handling protocols that are compatible with the techniqu...

The developed in situ gelling system as a promising ophthalmic formulation to prolong the drug lowering effect on the intraocular pressure.

Gemcitabine (2,2-difluorodeoxycytidine) is a deoxycytidine analog, currently being used as a first-choice drug in pancreatic metastatic cancer. Gemcitabine is administered weekly as 30-minute infusion with starting dose ranging from 800 to 1250 mg/m2. The aim of the present work was to develop starch nanoparticles (NPs) for the delivery of gemcitabine hydrochloride that could reduce its dose related side effects and may prolong its retention time (24 hrs) for the treatment of pancreatic cancer. Nanoparticles were prepared by emulsification diffusion method with slight modifications. Size and morphology of nanoparticles were investigated. Particles were spherical in shape with slightly rough surfaces. Particle size and polydispersity index were 231.4 nm and 1.0, respectively while zeta potential of blank NPs and drug loaded NPs were found to be −11.8 mV and −9.55 mV, respectively. Percent entrapment efficiency of different formulations was around ∼54% to 65%. In vitro release profile studies showed that around 70%–83% of drug was released from different formulations. Anticancerous cell line studies were also performed in human pancreatic cell lines (MIA-PA-CA-2).

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