Nature of the anchors of membrane-bound aminopeptidase,amylase, and trypsin and secretory mechanisms in Spodoptera frugiperda (Lepidoptera) midgut cells

Spodoptera frugiperda larvae have a microvillar aminopeptidase and both soluble and membrane-bound forms of amylase and trypsin. Membrane-bound aminopeptidase is solubilized by glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) and detergents, suggesting it has a GPI anchor. Membrane-bound trypsin is not affected by GPI-PLC, although it is solubilized by papain and by different detergents. Membrane-bound amylase is similar to trypsin, although once solubilized in detergent it behaves as a hydrophilic protein. Musca domestica trypsin antiserum cross-reacts with only one polypeptide from S. frugiperda midgut. With this antiserum, trypsin was immunolocalized in the anterior midgut cells at the microvillar surface and on the membranes of secretory vesicles found in the apical cytoplasm and inside the microvilli. The data suggest that in this region trypsin is bound to the secretory vesicle membrane by a hydrophobic anchor. Vesicles migrate through the microvilli and are discharged into the lumen by a pinching-off process. Trypsin is then partly processed to a soluble form and partly, still bound to vesicle membranes, incorporated into the peritrophic membrane. In posterior midgut cells, trypsin immunolabelling is randomly distributed inside the secretory vesicles and at the microvilli surface, suggesting exocytosis. Amylase probably follows a route similar to that described for trypsin in anterior midgut, although membrane-bound forms (peptide anchor) solubilize apparently as a consequence of a pH increase inside the vesicles.     

Characterization,subsite mapping and N-terminal sequence of miliin,a serine-protease isolated from the latex of Euphorbia milii

Miliin is a serine protease purified from the latex of Euphorbia milii. This work reports the effect of pH and temperature on the catalytic activity of miliin, using fluorescence resonance energy transfer (FRET) substrates. Miliin displayed the highest activity at pH 9 and 35 °C. Subsite mapping shows that subsites S₂ to S₂′ prefer uncharged residues. The S₂ subsite prefers hydrophobic aliphatic amino acids (Val, Pro and Ile) and defines the cleavage site. This work is the first one that reports subsite mapping of Euphorbiacea proteases. The N-terminal sequence showed higher similarity (40%) with the serine protease LIM9 isolated from Lilium. The presence of Tyr, Pro and Lys at positions 2, 5 and 10 respectively, were observed for most of the serine proteases used for comparison. The N-terminal sequence has striking differences with those reported previously for milin and eumiliin, other serine proteases isolated from the latex of E. milii.     

Synthesis and subcellular distribution of heparan sulfate in the rat exocrine pancreas.

A kinetic study was conducted to investigate the properties of subsites S₁′ and S₂′ of α-chymotrypsin and subtilisin BPN′, which were deduced from model complexes with a pancreatic trypsin inhibitor and a hexapeptide substrate, respectively. For this purpose,

and

(AA, various amino acid residues) were synthesized. Since they were susceptible to cleavage at the positions shown by the arrows, we could examine the effect of P₁′ or P₂′ amino acid residue on hydrolysis [amino acid residues in peptide substrates and the corresponding subsites in enzymes are numbered according to the system of Schechter and Berger (1967)Biochem. Biophys. Res. Commun.27, 157–162]. The results agreed well with interactions of the leaving group with the corresponding subsites in both enzymes, which were deduced from the model complexes.     

Proteins of the kidney microvillar membrane. The amphipathic form of dipeptidyl peptidase IV.

Dipeptidyl peptidase IV was solubilized from the microvillar membrane of pig kidney by Triton X-100. The purified enzyme was homogeneous on polyacrylamide-gel electrophoresis and ultracentrifugation, although immunoelectrophoresis indicated that amino-peptidase M was a minor contaminant. A comparison of the detergent-solubilized and proteinase (autolysis)-solubilized forms of the enzyme was undertaken to elucidate the structure and function of the hydrophobic domain that serves to anchor the protein to the membrane. No differences in catalytic properties, nor in sensitivity to inhibition by di-isopropyl phosphorofluoridate were found. On the other hand, several structural differences could be demonstrated. Both forms were about 130,000 subunit mol.wt., but the detergent form appeared to be larger by no more than about 4,000. Electron microscopy showed both forms to be dimers, and gel filtration revealed a difference in the dimeric mol.wt. of about 38 000, mainly attributable to detergent molecules bound to the hydrophobic domain. Papain converted the detergent form into a hydrophilic form that could not be distinguished in properties from the autolysis form. A hydrophobic peptide of about 3500 mol.wt. was identified as a product of papain treatment. The detergent and proteinase forms differed in primary structure. Partial N-terminal amino acid sequences were shown to be different, and the pattern of release of amino acids from the C-terminus by carboxypeptidase Y was essentially similar. The results are consistent with a model in which the protein is anchored to the microvillar membrane by a small hydrophobic domain located within the N-terminal amino acid sequence of the polypeptide chain. The significance of these results in relation to biosynthesis of the enzyme and assembly in the membrane is discussed.     

Insertion of a Glycosylphosphatidylinositol-Anchored Enzyme into Liposomes

Incorporation of alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein, into liposomes containing detergent, followed by detergent removal with hydrophobic resin was performed. Incorporation media were collected during different steps of detergent removal and were analyzed by flotation in sucrose gradient. The presence of protein was checked by measuring enzymatic activity, while the presence of ³H-radio-labelled liposomes was followed by determination of the radioactivity. The incorporation yield of the protein into liposomes increased with incubation time in presence of hydrophobic resin. Protein was also incorporated at different protein/lipid ratios. At the highest protein lipid ratio, our data showed that 260 molecules of GPI-linked AP (AP-GPI) could be associated with one liposome, corresponding to 65% vesicle coverage. Finally, observations by electron cryomicroscopy indicated (i) that the protein seemed exclusively associated with the lipid bilayer via the GPI-anchor, as shown by the distance—about 2.5 nm—between the protein core and the liposome membrane; (ii) that the AP-GPI distribution was heterogeneous on the liposome surface, forming clusters of protein. Abbreviations: AP, alkaline phosphatase; AP-GPI, glycosylphosphatidylinositol-linked alkaline phosphatase; EM, electron microscopy; EPA, egg phosphatidic acid; GPI, glycosylphosphatidylinositol; OctGlc, n-octyl -D-glucoside; PtdCho, egg yolk phosphatidylcholine; PtdIns-PLC, glycosylphosphatidylinositol-specific phospholipase C. Enzymes: Alkaline phosphatase, orthophosphoric-monoester phosphohydrolase (EC 3.1.3.1); glycosylphosphatidylinositol-specific phospholipase C (EC 3.1.4.10).     

Sialic Acid on the Glycosylphosphatidylinositol Anchor Regulates PrP-mediated Cell Signaling and Prion Formation

The prion diseases occur following the conversion of the cellular prion protein (PrP^C) into disease-related isoforms (PrP^Sc). In this study, the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP^C in prion formation was examined using a cell painting technique. PrP^Sc formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells) and in scrapie-infected primary cortical neurons was increased following the introduction of PrP^C. In contrast, PrP^C containing a GPI anchor from which the sialic acid had been removed (desialylated PrP^C) was not converted to PrP^Sc. Furthermore, the presence of desialylated PrP^C inhibited the production of PrP^Sc within prion-infected cortical neurons and ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrP^C contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrP^C. Desialylated PrP^C was less sensitive to cholesterol depletion than PrP^C and was not released from cells by treatment with glimepiride. The presence of desialylated PrP^C in neurons caused the dissociation of cytoplasmic phospholipase A₂ from PrP-containing membrane rafts and reduced the activation of cytoplasmic phospholipase A₂. These findings show that the sialic acid moiety of the GPI attached to PrP^C modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP^Sc formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases.     

The carboxyl terminus of Dictyostelium discoideum protein 1I encodes a functional glycosyl-phosphatidylinositol signal sequence

The 1I gene is expressed in the prespore cells of culminating Dictyostelium discoideum. The open reading frame of 1I cDNA encodes a protein of 155 amino acids with hydrophobic segments at both its NH(2)- and COOH-termini that are indicative of a glycosyl-phosphatidylinositol (GPI)-anchored protein. A hexaHis-tagged form of 1I expressed in D. discoideum cells appeared on Western blot analysis as a doublet of 27 and 24 kDa, with a minor polypeptide of 22 kDa. None of the polypeptides were released from the cell surface with bacterial phosphatidylinositol-specific phospholipase C, although all three were released upon nitrous acid treatment, indicating the presence of a phospholipase-resistant GPI anchor. Further evidence for the C-terminal sequence of 1I acting as a GPI attachment signal was obtained by replacing the GPI anchor signal sequence of porcine membrane dipeptidase with that from 1I. Two constructs of dipeptidase with the 1I GPI signal sequence were constructed, one of which included an additional six amino acids in the hydrophilic spacer. Both of the resultant constructs were targeted to the surface of COS cells and were GPI-anchored as shown by digestion with phospholipase C, indicating that the Dictyostelium GPI signal sequence is functional in mammalian cells. Site-specific antibodies recognising epitopes either side of the expected GPI anchor attachment site were used to determine the site of GPI anchor attachment in the constructs. These parallel approaches show that the C-terminal signal sequence of 1I can direct the addition of a GPI anchor.     

Does the tail wag the dog? How the structure of a glycosylphosphatidylinositol anchor affects prion formation

There is increasing interest in the role of the glycosylphosphatidylinositol (GPI) anchor attached to the cellular prion protein (PrP^C). Since GPI anchors can alter protein targeting, trafficking and cell signaling, our recent study examined how the structure of the GPI anchor affected prion formation. PrP^C containing a GPI anchor from which the sialic acid had been removed (desialylated PrP^C) was not converted to PrP^Sc in prion-infected neuronal cell lines and in scrapie-infected primary cortical neurons. In uninfected neurons desialylated PrP^C was associated with greater concentrations of gangliosides and cholesterol than PrP^C. In addition, the targeting of desialylated PrP^C to lipid rafts showed greater resistance to cholesterol depletion than PrP^C. The presence of desialylated PrP^C caused the dissociation of cytoplasmic phospholipase A₂ (cPLA₂) from PrP-containing lipid rafts, reduced the activation of cPLA₂ and inhibited PrP^Sc production. We conclude that the sialic acid moiety of the GPI attached to PrP^C modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP^Sc formation.     

Ectoenzymes of the kidney microvillar membrane. Isolation and characterization of the amphipathic form of renal dipeptidase and hydrolysis of its glycosyl-phosphatidylinositol anchor by an activity in plasma.

Renal dipeptidase (EC 3.4.13.11) has been solubilized from pig kidney microvillar membranes with n-octyl-beta-D-glucopyranoside and then purified by affinity chromatography on cilastatin-Sepharose. The enzyme exists as a disulphide-linked dimer of two identical subunits of Mr 45,000 each. The purified dipeptidase partitioned into the detergent-rich phase upon phase separation in Triton X-114 and reconstituted into liposomes consistent with the presence of the glycosyl-phosphatidylinositol membrane anchor. The N-terminal amino acid sequence of the amphipathic, detergent-solubilized, form of renal dipeptidase was identical with that of the hydrophilic, phospholipase-solubilized, form, locating the membrane anchor at the C-terminus of the protein. The glycosyl-phosphatidylinositol anchor of both purified and microvillar membrane renal dipeptidase was a substrate for an activity in pig plasma which displayed properties similar to those of a previously described phospholipase D. The cross-reacting determinant of the glycosyl-phosphatidylinositol anchor was generated by incubation of purified renal dipeptidase with bacterial phosphatidylinositol-specific phospholipase c, whereas the anchor-degrading activity in plasma failed to generate this determinant.     

Glycosyl-phosphatidylinositol-anchored proteins exist in the plasma membrane of Chlorella saccharophila (Krüger) Nadson: Plasma-membrane-bound nitrate reductase as an example

Experiments with plasma-membrane vesicles were performed in order to identify the attachment of hydrophobic nitrate reductase at the plasma membrane of Chlorella saccharophila. The enzyme was successfully removed from the plasma membrane with phosphoinositol-specific phospholipase C, and showed cross-reactivity with a monoclonal antibody (clone aGPI-3) raised against the glycosyl-phosphatidylinositol (GPI) anchor of Trypanosoma variant surface protein. The enzyme was labelled in vivo by feeding [³H]ethanolamine to the cells and underwent an hydrophobicity shift after treatment with phosphoinositol-specific phospholipase C. The attachment of this form of nitrate reductase to the plasma membrane via a GPI anchor was demonstrated.Abbreviations GPI glycosyl-phosphatidylinositol - NR nitratereductase - PI-PLC phosphoinositol-specific phospholipase C - PMNR Plasma-membrane-bound nitrate reductase The research was supported by a grant from Deutsche Forschungsgemeinschaft to R.T.     

Sunflower Seed Aminopeptidase-Catalyzed Hydrolysis of Phe-Xaa Dipeptides — Exploring the S1′ Specificity of the Enzyme

The S₁′ substrate specificity of the sunflower seed major aminopeptidase was studied with a series of dipeptide substrates with phenylalanine at P₁ and a hydrophobic amino acid at P₁′ position. The kinetic parameters of hydrolysis are significantly affected by the structure, side chain hydrophobicity and configuration of the P₁′ moiety. Its binding during enzyme-substrate complex formation takes place at a hydrophobic site of limited size following an extraction mechanism as seen from the applied structure-activity correlation. Attempts to establish such dependencies for the catalytic step of the reaction reveal the presence of additional S₁′-P₁′ enzyme-substrate interactions of greater complexity.     

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Abstract

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.     

Ectoenzymes of the kidney microvillar membrane. Differential solubilization by detergents can predict a glycosyl-phosphatidylinositol membrane anchor.

The pattern of solubilization of nine kidney microvillar ectoenzymes by a range of detergents distinguished two classes of membrane proteins: those released from the membrane by bacterial phosphatidylinositol-specific phospholipase C and those not so released. The latter group of transmembrane proteins were solubilized efficiently (greater than 80%) by all the detergents examined. In contrast, proteins released by phosphatidylinositol-specific phospholipase C were solubilized effectively only by octyl glucoside, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate and sodium deoxycholate. Octyl glucoside solubilized the amphipathic forms of the ectoenzymes examined, suggesting that this may be a useful detergent in the purification of glycosyl-phosphatidylinositol-anchored ectoenzymes.     

Prostasin regulates epithelial monolayer function: cell-specific Gpld1-mediated secretion and functional role for GPI anchor

Prostasin, a trypsinlike serine peptidase, is highly expressed in prostate, kidney, and lung epithelia, where it is bound to the cell surface, secreted, or both. Prostasin activates the epithelial sodium channel (ENaC) and suppresses invasion of prostate and breast cancer cells. The studies reported here establish mechanisms of membrane anchoring and secretion in kidney and lung epithelial cells and demonstrate a critical role for prostasin in regulating epithelial monolayer function. We report that endogenous mouse prostasin is glycosylphosphatidylinositol (GPI) anchored to the cell surface and is constitutively secreted from the apical surface of kidney cortical collecting duct cells. Using site-directed mutagenesis, detergent phase separation, and RNA interference approaches, we show that prostasin secretion depends on GPI anchor cleavage by endogenous GPI-specific phospholipase D1 (Gpld1). Secretion of prostasin by kidney and lung epithelial cells, in contrast to prostate epithelium, does not depend on COOH-terminal processing at conserved Arg³²². Using stably transfected M-1 cells expressing wild-type, catalytically inactive, or chimeric transmembrane (not GPI)-anchored prostasins we establish that prostasin regulates transepithelial resistance, current, and paracellular permeability by GPI anchor- and protease activity-dependent mechanisms. These studies demonstrate a novel role for prostasin in regulating epithelial monolayer resistance and permeability in kidney epithelial cells and, furthermore, show specifically that prostasin is a critical regulator of transepithelial ion transport in M-1 cells. These functions depend on the GPI anchor as well as the peptidase activity of prostasin. These studies suggest that cell-specific Gpld1- or peptidase-dependent pathways for prostasin secretion may control prostasin functions in a tissue-specific manner. serine protease; epithelial sodium channel; glycosylphosphatidylinositol anchor; transepithelial resistance; tight junction     

Proteins of the kidney microvillar membrane. Reconstitution of endopeptidase in liposomes shows that it is a short-stalked protein.

Pig kidney microvillar proteins were extracted with octyl beta-glucoside and reconstituted in liposomes prepared from microvillar lipids of known composition. Four peptidases, namely endopeptidase (EC 3.4.24.11), aminopeptidases N (EC 3.4.11.2) and A (EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), were shown to be reconstituted. At lipid/protein ratios greater than 4:1, about half the detergent-solubilized protein and nearly all of the activity of the four peptidases were reconstituted. Dissolution of the liposomes with Triton X-100 did not increase the activity of any of these peptidases, a result consistent with an asymmetric, 'right-side-out', orientation of these enzymes. When purified, endopeptidase was subjected to the same procedure; the two amphipathic forms of the enzyme (the detergent form and the trypsin-treated detergent form) were fully reconstituted. The amphiphilic form, purified after toluene/trypsin treatment, failed to reconstitute. Electron microscopy of microvilli showed that the appearance of the surface particles was profoundly altered by treatment with papain. Before treatment, the microvilli were coated with particles of stalk lengths ranging from 2.5 to 9 nm. After papain treatment nearly all the particles had stalks of 2-3 nm. Reconstituted microvillar proteins in liposomes showed the same heterogeneity of stalk length. In contrast, liposomes containing reconstituted endopeptidase revealed a very homogeneous population of particles of stalk length 2 nm. Since the smallest dimension of a papain molecule is 3.7 nm, the ability of papain, and other proteinases of similar molecular size, to release microvillar enzymes is crucially affected by the length of the junctional peptide that constitutes the stalk of this type of membrane protein.     

Activation of the glycosylphosphatidylinositol-anchored membrane proteinase upon release from Herpetomonas samuelpessoai by phospholipase C

We have analyzed the effects of exogenous phospholipase C (PLC) on the cell-surface polypeptides and proteinases of Herpetomonas samuelpessoai grown in chemically defined conditions by SDS-PAGE gels. Live parasites treated with PLC released into the extracellular medium a complex profile of glycosylphosphatidylinositol (GPI)-anchored polypeptides ranging from 15 to 100 kDa, some of them with proteolytic activity. Two major metalloproteinases with apparent molecular masses of 63 and 115 kDa were observed after PLC hydrolysis. Interestingly, only the PLC-released soluble form of the 115-kDa metalloenzyme, and not the membrane-anchored form, displayed proteolytic activity, demonstrating that cleavage of the GPI anchor can lead to enzymatic activation. Received: 19 December 2001 / Accepted: 25 January 2002     

Solubility and posttranslational regulation of GP130/F11--a neuronal GPI-linked cell adhesion molecule enriched in the neuronal membrane skeleton.

GP130 (renamed contactin) has previously been identified by its detergent insolubility and retention with the actin-containing "membrane skeleton" isolated from chicken neurons and brain. The contactin sequence predicted a transmembrane and cytoplasmic domain for the molecule. Recently, F11 was shown to have an identical sequence except for the C terminus, and it was predicted to be linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) group. Here we describe that GP130 can be released both from brain membranes and the detergent-insoluble membrane skeleton by a phosphoinositol-specific phospholipase C (PI-PLC) indicating that F11 and GP130/contactin are probably identical and that surprisingly the lipid anchor is partly or totally responsible for its non-ionic detergent insolubility. The "membrane skeleton" is a rich source of GPI-linked glycoproteins as judged by 1) most glycoproteins can be released by a PI-PLC and 2) most [3H]ethanolamine-labeled glycoproteins are present in, or enriched in the membrane skeleton. Thus, detergent insolubility appears to be a characteristic of GPI-anchored glycoproteins. No evidence has been obtained that GP130/F11 is released or secreted in vivo or in culture. In addition, GP130/F11 has an unusually long half-life in culture of greater than 3 days. The structure of the neuronal membrane skeleton and the potential function of GPI-anchored glycoproteins is discussed.     

Exploring the binding preferences/specificity in the active site of human cathepsin E

Aspartic proteinases are produced in the human body by a variety of cells. Some of these proteins, examples of which are pepsin, gastricsin, and renin, are secreted and exert their effects in the extracellular spaces. Cathepsin D and cathepsin E on the other hand are intracellular enzymes. The least characterized of the human aspartic proteinases is cathepsin E. Presented here are results of studies designed to characterize the binding specificities in the active site of human cathepsin E with comparison to othermechanistically similar enzymes. A peptide series based on Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu was generatedto elucidate the specificity in the individual binding pockets with systematic substitutions in the P_5? P₂ and P₂′-P₃′ based on charge, hydrophobicity, and hydrogen bonding. Also, to explore the S₂ binding preferences, asecond series of peptides based on Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu was generated with systematic replacements in the P₂ position. Kinetic parameters were determined forboth sets of peptides. The results were correlated to a rule-based structural model of human cathepsin E, constructed on the known three-dimensional structures of several highly homologous aspartic proteinases; porcine pepsin, bovine chymosin, yeast proteinase A, human cathepsin D, andmouse and human renin. Important specificity-determining interactions were found in the S₃ (Glu13) and S₂ (Thr-222, Gln-287, Leu-289, Ile-300)subsites. © 1995 Wiley-Liss, Inc.     

Two isoforms of eukaryotic phospholipase C in Paramecium affecting transport and release of GPI-anchored proteins in vivo

Surface proteins anchored by a glycosylphosphatidylinositol (GPI) residue in the cell membrane are widely distributed among eukaryotic cells. The GPI anchor is cleavable by a phospholipase C (PLC) leading to the release of such surface proteins, and this process is postulated to be essential in several systems. For higher eukaryotes, the responsible enzymes have not been characterized in any detail as yet. Here we characterize six PLCs in the ciliated protozoan, Paramecium, which, in terms of catalytic domains and architecture, all show characteristics of PLCs involved in signal transduction in higher eukaryotes. We show that some of these endogenous PLCs can release GPI-anchored surface proteins in vitro: using RNA_i to reduce PLC expression results in the same effects as the application of PLC inhibitors. With two enzymes, PLC2 and PLC6, RNA_i phenotypes show strong defects in release of GPI-anchored surface proteins in vivo. Moreover, these RNA_i lines also show abnormal surface protein distribution, suggesting that GPI cleavage may influence trafficking of anchored proteins. As we find GFP fusion proteins in the cytosol and in the surface protein extracts, these PLCs obviously show unconventional translocation mechanisms. This is the first molecular data on endogenous Paramecium PLCs with the described properties affecting GPI anchors in vitro and in vivo.     

Inhibition of Photosystem II by formate. Possible evidence for a direct role of bicarbonate in photosynthetic oxygen evolution

In broken chloroplasts the presence of 100 mM sodium formate at pH 8.2 will specifically lengthen the Photosystem II relaxation times of the reactions S′₂ → S₃ and S′₃ → S₀. Rates of reactions S′₀ → S₁ and S′₁ → S₂ remain unaffected. Evidence is presented which indicates the discrimination among S-states by formate cannot be attributed to a block imposed on the reducing side of Photosystem II. The results are interpreted in context of the known interaction of formate and CO₂ which is bound to the Photosystem II reaction center complex. It is proposed that those S-state transitions which show extended relaxation times in the presence of formate must result in the momentary release and rebinding of CO₂. Furthermore since formate is acting on the oxygen-evolving side of Photosystem II, it would seem that CO₂ is released in reactions that occur there. A chemical model of oxygen evolution is presented. It is based on the hypothesis that hydrated CO₂ is the immediate source of photosynthetically evolved oxygen and explains why, under certain conditions formate slows only the S-state transitions S′₂ → S₃ and S′₃ → S₀.