Low Concentrations Of Trypsin Research Articles

A Monomeric, Tightly Folded Stromal Intermediate on the △pH-dependent Thylakoidal Protein Transport Pathway

Two distinct mechanisms have been previously identified for the transport of proteins across the chloroplast thylakoid membrane, one of which is unusual in that neither soluble factors nor ATP are required; the system requires only the transthylakoidal delta pH. We have examined this mechanism by studying the properties of one of its substrates: the extrinsic 23-kDa protein (23K) of photosystem II. Previous work has shown that this protein can be transported into isolated thylakoids as the full-length precursor protein; we show that the stromal import intermediate form of this protein is similarly translocation-competent. Gel filtration tests indicate that the stromal intermediate is probably monomeric. Protease sensitivity tests on both the initial in vitro translation product and the stromal import intermediate show that the presequence is highly susceptible to digestion whereas the mature protein is resistant to high concentrations of trypsin. The mature protein becomes very sensitive to digestion if unfolded in urea, or after heating, and we therefore propose that the natural substrate for this translocation system consists of a relatively unfolded presequence together with a tightly folded passenger protein. The ability of thylakoids to import pre-23K is destroyed by prior treatment of the thylakoids with low concentrations of trypsin, demonstrating the involvement of surface-exposed proteins in the import process. However, we can find no evidence for the binding of pre-23K or i23K to the thylakoid surface, and we therefore propose that the initial interaction of these substrates with the thylakoidal translocase is weak, reversible, and probably delta pH-independent. In the second phase of the translocation mechanism, the delta pH drives either the translocation and unfolding of proteins, or the translocation of a fully folded protein.

Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase.

The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain. Previous studies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 micrograms/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain. At higher protease concentrations (1 micrograms/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus.

The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport. We have previously reported that rab5 regulates endosome-endosome recognition and/or fusion in vitro. Here, we show that this process depends on the rab5 N-terminal domain. Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide. Two-dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions. Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin-treatment of the cytosol. Trypsin-treated cytosol prepared from mock-transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments. Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N-terminus. A synthetic peptide of rab5 N-terminal domain inhibited endosome fusion in our cell-free assay. A version of the same peptide truncated at the N-terminus or a peptide of rab3 N-terminal domain were without effects. Altogether, these observations suggest that the N-terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery.

Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4-like heparan sulfate proteoglycan and a distinct chondroitin sulfate form.

Proteoglycan accumulation by thioglycollate-elicited mouse peritoneal macrophages and a panel of murine monocyte-macrophage cell lines has been examined to determine whether these cells express plasma membrane-anchored heparan sulfate proteoglycans. Initially, cells were screened for heparan sulfate and chondroitin sulfate glycosaminoglycans after metabolic labeling with radiosulfate. Chondroitin sulfate is secreted to a variable extent by every cell type examined. In contrast, heparan sulfate is all but absent from immature pre-monocytes and is associated predominantly with the cell layer of mature macrophage-like cells. In the P388D1 cell line, the cell-associated chondroitin sulfate is largely present as a plasma membrane-anchored proteoglycan containing a 55 kD core protein moiety, which appears to be unique. In contrast, the cell-associated heparan sulfate is composed of a proteoglycan fraction and protein-free glycosaminoglycan chains, which accumulate intracellularly. A fraction of the heparan sulfate proteoglycan contains a lipophilic domain and can be released from cells following mild treatment with trypsin, suggesting that it is anchored in the plasma membrane. Isolation of this proteoglycan indicates that it is likely syndecan-4: it is expressed as a heparan sulfate proteoglycan at the cell surface, it is cleaved from the plasma membrane by low concentrations of trypsin, and it consists of a single 37 kD core protein moiety that co-migrates with syndecan-4 isolated from NMuMG mouse mammary epithelial cells. Northern analysis reveals that a panel of macrophage-like cell lines accumulate similar amounts of syndecan-4 mRNA, demonstrating that this proteoglycan is expressed by a variety of mature macrophage-like cells. Syndecan-1 mRNA is present only in a subset of these cells, suggesting that the expression of this heparan sulfate proteoglycan may be more highly regulated by these cells.

DNA binding alters the protease susceptibility of the p50 subunit of NF-kappa B.

The subdomain structure of the p50 subunit of NF-kappa B (amino acids 35-381) was investigated by partial proteolysis of the native protein. Trypsin cleaves p50 at a limited number of sites with an initial cleavage at low trypsin concentration occurring after R362 and a second cleavage taking place at higher trypsin concentration after K77. The cleavage after R362 does not alter the DNA binding characteristics of p50 but removes the nuclear localisation signal indicating that this region occupies a highly exposed position on the surface of the protein. The second cleavage after K77 generates a protein that although dimeric is incapable of binding DNA, thus emphasising the importance of residues 35-77 in DNA recognition. However p50 dimers containing one molecule cleaved after K77 and one molecule with this region intact are capable of binding DNA. When very high concentrations of trypsin are employed p50 is completely degraded. However if p50 is bound tightly to DNA containing its specific recognition site prior to trypsin addition the cleavage after K77 is almost completely blocked and the protein becomes highly resistant to proteolysis. These data suggest that bound DNA may mask critical trypsin cleavage sites or that DNA binding is accompanied by a conformational change in protein structure that renders the protein resistant to proteolysis.

TRANSFORMED MURINE LEYDIG-CELLS IN SERUM-FREE CULTURE - USEFUL MODEL FOR ANALYSES OF STEROID HORMONE-INDUCED CELL-GROWTH AND CELL MORPHOLOGY (REVIEW)

A clonal cell line has been established from estrogen-induced testicular Leydig cell tumor of BALB/c mouse. This cell line termed as B-1F can be maintained in a serum-free culture condition without any authentic growth factor. The growth of B-1F cells is significantly enhanced by estrogen, androgen or retinoic acid. Biochemical and Northern blot analyses reveal that B-1F cells contain respective receptors for these stimulants, although estrogen receptor has an interesting mutation. The simultaneous addition of these stimulants in combination fails to elevate the maximum levels of B-1F cell growth, suggesting that these stimulants would share some common pathway to stimulate B-1F cell growth. One possible step affected by these stimulants is mapped to 5-lipoxygenase since inhibitors of this enzyme can stimulate B-1F cell growth and estrogen stimuli can lower its activity. B-1F cells grow in a serum-free medium as a cell clump irrespective of the presence of growth stimulants. Interestingly, the addition of low concentrations of elastase or trypsin in the medium changes the cell morphology from a round to well spreaded shape. Furthermore, treatment of conditioned medium from B-1F cell culture with trypsin or elastase results in an appearance of cell-spreading activity. In addition, a relatively high concentration of elastase abolishes estrogen-induced enhancemant of B-1F cell growth. In conclusion, a serum-free culture of B-1F cells can provide us with a model system quite useful for studying the effects of physiologically active molecules on cancer cells.

Stereo views and immunogold labeling of the pellicular microtubules at the inner surface of the plasma membrane of Leishmania as revealed by fracture-flip.

We used a modification of fracture-flip to reveal the nanoanatomy of the inner surface of the plasma membrane in promastigotes of Leishmania. After freeze-fracture, lightly fixed promastigotes were coated with a stabilizing layer of carbon evaporated from an electron gun, thawed, and washed. Fractured promastigotes attached to the carbon casts by the protoplasmic (i.e., inner) halves of their plasma membranes were treated with Triton X-100, followed by exposure to low concentrations of trypsin and thorough washing. This was followed by picking up and flipping of the replicas, followed by air-drying. The actual inner surfaces of the plasma membrane were then imaged by platinum shadowing. Extended, three-dimensional, high-resolution views of the inner surface of the plasma membrane showed parallel arrays of microtubules (average spacing 47 nm) closely apposed to the inner surface. Cytochemical labeling confirmed the morphological identification of both subpellicular and flagellar microtubules, as determined by treatment with mouse monoclonal anti-alpha- or anti-beta-tubulin, followed by labeling with goat anti-mouse IgG adsorbed to colloidal gold. Removal of the microtubules revealed parallel arrays of particles (average diameter 17 nm). We hypothesize that these particles represent the cytoplasmic portion of proteins that link the microtubules to the plasma membrane.

Ecto-phosphorylation on aortic endothelial cells. Exquisite sensitivity to staurosporine.

One- and two-dimensional gel electrophoresis of proteins from bovine aortic endothelial cells (BAEC) incubated with [gamma-32P]ATP revealed the preferential labelling of a cell-associated 21 kDa substrate. The labelling of this band was detectable within 30 s, increased up to 30 min and was stable for at least 3 h following the wash-out of the ATP. This protein was also labelled after incubation of the cells with [gamma-35S]ATP. Incorporation of radioactivity into the 21 kDa band did not occur if the endothelial cells were treated with low concentrations of trypsin (0.01%) before or after the labelling period. The pattern of BAEC protein phosphorylation by [gamma-32P]ATP was completely different from that of the fetal calf serum used for the cell culture. The presence of serum during the incubation of BAEC with [gamma-32P]ATP did not modify qualitatively the labelling pattern and, in particular, did not enhance the phosphorylation of the 21 kDa substrate; this suggests that neither the kinase nor the 21 kDa substrate are adsorbed serum proteins. Staurosporine, a protein kinase inhibitor with low specificity, decreased the labelling of the 21 kDa protein with an IC50 of 2 nM. In contrast, at 100 nM, staurosporine did not decrease the accumulation of inositol phosphates induced by ATP via the activation of P2y receptors. These data indicate the presence of aortic endothelial cells of an ecto-kinase which uses extracellular ATP to produce the selective and long-lived phosphorylation of a 21 kDa endothelial substrate. Ecto-phosphorylation of this protein might play a role in the modulation of endothelial cell functions by ATP, in addition to the P2y receptors [Boeynaems & Pearson (1990) Trends Pharmacol. Sci. 11, 34-37]. The exquisite sensitivity of ecto-phosphorylation to inhibition by staurosporine and its specific inhibition by some isoquinolinesulphonamide compounds provide potential pharmacological tools to investigate this hypothesis.

Occlusion of Rb + after extensive tryptic digestion of shark rectal gland Na,K-ATPase

Na,K-ATPase from rectal glands of Squalus acanthias has been subjected to proteolysis with trypsin. The E 1- and E 2-forms of the enzyme can be distinguished from the inactivation patterns at low trypsin concentrations, as previously seen with kidney enzyme. Extensive degradation by trypsin in the presence of 5 mM Rb + yields membrane fragments with a 19 kDa peptide as the major proteolytic fragment of the α-subunit. The sequence of the N-terminnal 40 residues of this peptide is almost identical to that of a similar proteolytic fragment isolated by Capasso et al. (Capasso, J.M., Hoving, S., Tal, D.M., Goldshleger, R. and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1150–1158) using kidney Na,K-ATPase. Rb + occlusion can be fully retained under these circumstances, supporting the findings with kidney enzyme that only minor parts of the α-subunit are required to form a functional occlusion-site.

Changes in Contractile Properties with Selective Digestion of Connectin (Titin) in Skinned Fibers of Frog Skeletal Muscle

Changes in contractile properties of mechanically skinned fibers were examined when connectin in the fibers was selectively digested by a low concentration (0.25 microgram/ml) of trypsin. Resting tension and isometric active tension were reduced as the digestion of the connectin progressed; the rate of reduction of active tension was larger than that of resting tension. Maximum shortening speed and calcium ion sensitivity of active tension were not changed by the digestion. Electron micrographs showed that A-bands in the fibers treated with trypsin are dislocated from I-bands. These results suggest that the digestion of connectin does not directly influence the reaction of actin-myosin-regulatory proteins, and thus the resultant reduction in the active tension is mainly due to disordering of the regular structure in a sarcomere.

Phosphorylation of a cell surface 112 kDa protein by an ecto-protein kinase in rat L6 myoblasts

Studies with subconfluent day 2 cultures of rat myoblasts revealed that a cell surface 112 kDa protein could be phosphorylated by extracellular ATP. Analysis of the phosphorylated 112 kDa protein suggested the involvement of a serine protein kinase. The following evidence indicated the cell surface location of this protein kinase: (i) extracellular ATP was unable to penetrate the cell membrane under our experimental conditions; (ii) the phosphorylated protein profile of intact cells differed significantly from that of broken cells; (iii) the phosphorylation of the 112 kDa protein could be abolished by pretreatment of cells with very low concentrations of trypsin; (iv) the phosphorylated 112 kDa protein could be dephosphorylated by exogenously added alkaline phosphatase; (v) the phosphorylation of the 112 kDa protein was inhibited by exogenously added proteins; and (vi) exogenously added proteins could be phosphorylated by intact cells under similar experimental conditions. The phosphorylated 112 kDa protein was detected only when the reaction was carried out in the presence of Ca2+, Mg2+, and F- ions. Kinetic analysis that revealed that the Km value of the ecto-protein kinase for ATP was 0.04 microM, and the Vmax. value for phosphorylation of the 112 kDa protein was 1.67 x 10(-4) pmol/min per 10(5) cells. Data presented in the accompanying paper [Chen & Lo (1991) Biochem. J. 279, 475-482] show that there was a constant and adequate supply of ATP on the cell surface of rat myoblasts for efficient functioning of this protein kinase, and that mutants defective in either the ecto-protein kinase or the 112 kDa protein were also impaired in myogenic differentiation. This and other biochemical studies suggest that the ecto-protein kinase and the 112 kDa protein might play important roles in myogenic differentiation.

Inactivation of HIV-1 by trypsin and its use in demonstrating specific virus infection of cells

HIV-1 was found sensitive to inactivation by low concentrations of trypsin. The use of trypsin was valuable for assessing non-specific binding of HIV virions to CD4+ cells. This effect was also helpful for eliminating input virus in experiments studying HIV infection of cells in vitro.

Recombinant rat guanidinoacetate methyltransferase: Structure and function of the NH 2-terminal region as deduced by limited proteolysis

Recombinant rat liver guanidinoacetate methyltransferase, a monomeric protein with M r 26,000, is inactivated upon incubation with low concentrations of trypsin. Examination of the reaction products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and highperformance liquid chromatography followed by amino acid analysis and sequencing of isolated peptides reveals that the inactivation is due to the cleavage of the NH 2terminal segment after Arg 20. The cleaved peptide is not tightly associated with the rest of the protein. The rate of inactivation is not affected by the presence of either S-adenosylmethionine (AdoMet) or guanidinoacetate, but a substantial retardation of inactivation is observed when both substrates are present. The cleavage at Arg 20 is also slowed by cross-linking Cys 15 and Cys 90 by a disulfide bond. An equilibrium binding study shows that guanidinoacetate methyltransferase in the free form binds AdoMet but not guanidinoacetate. The trypsin-modified enzyme, despite having no catalytic activity, can weakly bind AdoMet and guanidinoacetate in the presence of AdoMet. Chymotrypsin rapidly hydrolyzes the peptide bond after Trp 19, and elastase cleaves the bond after Ala 24, leading in both cases to loss of activity. The results obtained in this study suggest that the portion of the methyltransferase around residues 19–24 is highly exposed to the solvent and flexible. The results also indicate that the NH 2-terminal region is not directly involved in substrate binding but plays a role in catalysis.

Nuclear Ah receptor from mouse hepatoma cells: Effect of partial proteolysis on relative molecular mass and DNA-binding properties

The nuclear Ah receptor from mouse hepatoma (Hepa-1c1c9) cells is a 176-kDa multimeric protein which is stable under conditions of up to 1 m KCl. Under denaturing conditions, the Hepa-1 nuclear receptor can be dissociated into a ligand-binding subunit of M r ~ 91,000. The identity of subunits that compose the nuclear Ah receptor is currently unknown. We used partial proteolysis under nondenaturing conditions as an approach to study the domain organization of the nuclear form of Ah receptor from Hepa-1c1c9 cells treated with [ 3H]2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in culture. Low concentrations of trypsin (0.5 μg/mg nuclear protein) generated heterogeneous fragments with the main fragment having a Stokes radius ( R s) ≈ 6 nm. More discrete ligand-binding fragments of M r ~ 84,000 ( R s ≈ 4 nm/ ≈ 5 S) and M r ≈ 16,000 ( R s ≈ 2 nm/ ≈ 2 S) could be generated using higher concentrations of trypsin (5 μg/ mg nuclear protein). The relative concentration of the 84 and 16-kDa fragment was dependent on duration of protease treatment; formation of the 16-kDa fragment was accompanied by some loss in [ 3H]TCDD binding. Treatment of nuclear Ah receptor with α-chymotrypsin (1 μg/mg nuclear protein) generated a single, apparently homogeneous ligand-binding fragment of M r ~ 101,000 ( R s ≈ 5 nm/≈ 5 S). When analyzed by DNA-cellulose chromatography, the chymotryptic fragment eluted at a significantly higher KCl concentration (462 m m) compared to native untreated nuclear Ah receptor (385 m m). Despite this increased affinity for DNA-cellulose columns, the ligand-binding fragment generated by chymotrypsin treatment was unable to interact with a dioxin responsive element in a gel retardation assay. DNA-cellulose binding ability, therefore, does not appear to be a reliable indicator of specific DNA interactions for these protease-modified fragments.

Equilibrium Binding Characteristics of Monoclonal Antibodies Recognizing Melanoma Cell Surface Antigens

The equilibrium binding characteristics of a panel of six monoclonal antibodies (MAb) recognizing melanoma cell surface antigens (125 kdal cell surface melanoma associated glycoprotein antigen, 125kD-MAA; high molecular weight melanoma associated antigen, HMW-MAA; and a non-protein melanoma associated antigen, NP-MAA) were investigated using the cell lines SK-MEL-2, SK-MEL-5, and M21. The MAbs displayed equilibrium association constant (K) values ranging from 10(7) M-1 to 10(10) M-1 and maximum MAb binding values (Qmax) from 2 x 10(4) to 2 x 10(6) MAb molecules bound per cell. High trypsin concentrations were shown to have deleterious effects on Qmax values obtained for antibodies recognizing the 125kD-MAA, and even low trypsin concentrations affected Qmax values obtained for MAbs recognizing the HMW-MAA (although a complete linear recovery of HMW-MAA antigen was observed in 20-25 hours). Significant changes in Qmax were also noted for different cell passages. Except for MAb 43.2, little variation in K was observed when different cell lines were used. Linear Scatchard plots were obtained for all MAbs except 43.2 in which case concave down behavior was observed suggesting the existence of positive cooperativity between the binding sites of this MAb.

Import of chemically synthesized signal peptides into rat liver mitochondria.

The signal peptides of pre-aldehyde dehydrogenase (22-mer) and pre-ornithine transcarbamylase (27-mer) were chemically synthesized and their imports into rat liver mitochondria were studied. Both signal peptides were imported rapidly (within 2 min) in the absence of a membrane potential, exogenous ATP, or rabbit reticulocyte lysate. Signal peptides also were imported into mitochondria treated with a low concentration of trypsin which removed the outer membrane proteins. It was concluded that the chemically synthesized signal peptide could be imported differently than the precursor proteins. The imported signal peptide were found to be associated with both outer and inner membranes. Pulse-chase experiments showed that the import was unidirectional and that the signal peptides associated with inner membranes increased during the chase time. The signal peptides inhibited import of precursor proteins to different extents. Association of signal peptides with inner membrane near or at translocator sites might result in inhibition of precursor import.

Sequence, localization and function of the invasin protein of Yersinia enterocoiitica

The inv locus of Yersinia enterocolitica is sufficient to convert a non-invasive Escherichia coli K12 strain into a microorganism that is able to penetrate cultured mammalian cells. The nucleotide sequence of inv reveals an open reading frame corresponding to an 835-amino-acid protein that is homologous to the invasin protein from Yersinia pseudotuberculosis. A polyclonal antiserum elicited by a synthetic peptide corresponding to the C-terminal 88 amino acids of this open reading frame detected a unique 100 kD protein in cell lysates of Y. enterocolitica strain 8081 c and in an E. coli strain harbouring the cloned inv gene. This protein localized to the outer membranes of both microorganisms and was cleaved by low concentrations of extracellular trypsin. HEp-2 cells were shown to attach to surfaces coated with bacterial outer membranes containing invasin and this attachment was destroyed by treatment of the membranes with trypsin. Thus it appears that the invasin protein from Y. enterocolitica is able to mediate both attachment to and entry of cultured epithelial cells.

Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62

The precursor protein p62 of the prototype alphavirus Semliki Forest virus (SFV) undergoes during transport to the cell surface a proteolytic cleavage to form the mature envelope glycoprotein E2. To investigate the biological significance of this cleavage event, single amino acid substitutions were introduced at the cleavages site through mutagenesis of cDNA corresponding to the structural region of the SFV genome. The phenotypes of the cleavage site mutants were studied in BHK cells by using recombinant vaccinia virus vectors. Nonconservative substitutions completely abolished p62 cleavage. Uncleaved p62 was transported with normal kinetics to the cell surface, where it became accessible to low concentrations of exogenous trypsin. The proteolytic cleavage of envelope glycoprotein precursors has been shown to activate the membrane fusion potential of viral spikes in several virus families. Here we demonstrate that the fusion function of the SFV spike is activated by the cleavage of p62. Cleavage-deficient p62 expressed at the cell surface did not function in low-pH-triggered (pH 5.5) cell-cell membrane fusion; however, cleavage of the mutated p62 with exogenous trypsin restored the fusion function. We discuss a model for SFV assembly and fusion where p62 cleavage plays a crucial role in the stability of the multimeric association of the viral envelope glycoproteins.

Combined GTG-banding and nonradioactive in situ hybridization improves characterization of complex karyotypes

Nonradioactive in situ hybridization (ISH) using biotinylated centromere probes for chromosomes 1, 6, 7, 10, 16, 17, 18, and the X, respectively, was combined with GTG-banding to study cytogenetic changes in two different ovarian cancer cell lines. ISH was performed after GTG-banding on the same metaphase. The use of a low trypsin concentration (0.01%) in the banding procedure was essential for subsequent ISH. This combined approach allows the detection of subtle chromosomal rearrangements and appears to aid the identification of marker chromosomes.

Characteristics of the Mg2+-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor

The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg(2+)-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na(2)SO(3)-dependent Mg(2+)-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg(2+)-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg(2+)-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg(2+)-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t((1/2)) of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one.