In vivo modifications of the maize mitochondrial small heat stress protein, HSP22.

A maize (Zea mays L.) small heat shock protein (HSP), HSP22, was previously shown to accumulate to high levels in mitochondria during heat stress. Here we have purified native HSP22 and resolved the protein into three peaks using reverse phase high performance liquid chromatography. Mass spectrometry (MS) of the first two peaks revealed the presence of two HSP22 forms in each peak which differed in mass by 80 daltons (Da), indicative of a monophosphorylation. Phosphorylation of HSP22 by [gamma-(32)P]ATP was also observed in mitochondria labeled in vitro, but not when purified native HSP22 was similarly used, demonstrating that HSP22 does not autophosphorylate, implicating a kinase involvement in vivo. Collisionally induced dissociation tandem MS (CID MS/MS) identified Ser(59) as the phosphorylated residue. We have also observed forms of HSP22 that result from alternative intron splicing. The two HSP22 proteins in the first peak were approximately 57 Da larger than the two HSP22 proteins in the second peak. MS analysis revealed that the +57-Da forms have an additional Gly residue directly N-terminal of the expected Asp(84), which had been converted to an Asn residue. These results are the first demonstrations of phosphorylation and alternative intron splicing of a plant small HSP.

Heat-stress induced synthesis of chloroplast protein synthesis elongation factor (EF-Tu) in a heat-tolerant maize line.

A heat-tolerant maize (Zea mays L.) line, ZPBL 1304, synthesizes a unique set of five heat-shock polypeptides of 45 kDa. Previous studies suggested that these polypeptides might play a role in the development of thermotolerance in maize (Ristic et al., 1996, J. Plant Physiol. 149:424-432; Ristic et al., 1998, J. Plant Physiol. 153:497-505). In the present study, we isolated these polypeptides, sequenced them, and investigated their subcellular distribution and origin. Of the five polypeptides of 45 kDa, three polypeptides, including the two most abundant ones, yielded amino acid sequences similar to the chloroplast and bacterial protein synthesis elongation factor (EF-Tu). This was further confirmed using an antibody raised against maize EF-Tu, which showed a very strong reaction with the 45-kDa heatshock protein(s). Studies on subcellular distribution and origin revealed that the 45-kDa polypeptides were localized to the chloroplasts, and were likely of nuclear origin. A full-length maize EF-Tu cDNA (Zmeftu1), previously isolated from the B73 line of maize, was used as a probe for northern blot analysis of RNA extracted from the ZPBL 1304 maize line (the nucleotide and deduced amino acid sequences of Zmeftu1 are 88% identical to the rice EF-Tu sequence). Northern blots showed a 1.85-fold increase in steady-state levels of EF-Tu mRNA during heat stress. An increase in EF-Tu transcript levels during heat stress was accompanied by increased levels of the EF-Tu protein. Isolated chloroplasts from heat-stressed plants also had higher levels of EF-Tu as compared to control chloroplasts. The maize EF-Tu polypeptides showed > 80% sequence similarity with the bacterial EF-Tu, which has recently been shown to function as a molecular chaperone and to play a role in the protection of other proteins from thermal denaturation (Caldas et al., 1998, J. Biol. Chem. 273:11478-11482). It is hypothesized that chloroplast EF-Tu of the ZPBL 1304 maize line plays an important role in the development of thermotolerance.

The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain.

The dihydrolipoamide S-acetyltransferase (E2) subunit of the maize mitochondrial pyruvate dehydrogenase complex (PDC) was postulated to contain a single lipoyl domain based upon molecular mass and N-terminal protein sequence (Thelen, J. J., Miernyk, J. A., and Randall, D. D. (1998) Plant Physiol. 116, 1443-1450). This sequence was used to identify a cDNA from a maize expressed sequence tag data base. The deduced amino acid sequence of the full-length cDNA was greater than 30% identical to other E2s and contained a single lipoyl domain. Mature maize E2 was expressed in Escherichia coli and purified to a specific activity of 191 units mg(-1). The purified recombinant protein had a native mass of approximately 2.7 MDa and assembled into a 29-nm pentagonal dodecahedron as visualized by electron microscopy. Immunoanalysis of mitochondrial proteins from various plants, using a monoclonal antibody against the maize E2, revealed 50-54-kDa cross-reacting polypeptides in all samples. A larger protein (76 kDa) was also recognized in an enriched pea mitochondrial PDC preparation, indicating two distinct E2s. The presence of a single lipoyl-domain E2 in Arabidopsis thaliana was confirmed by identifying a gene encoding a hypothetical protein with 62% amino acid identity to the maize homologue. These data suggest that all plant mitochondrial PDCs contain an E2 with a single lipoyl domain. Additionally, A. thaliana and other dicots possess a second E2, which contains two lipoyl domains and is only 33% identical at the amino acid level to the smaller isoform. The reason two distinct E2s exist in dicotyledon plants is uncertain, although the variability between these isoforms, particularly within the subunit-binding domain, suggests different roles in assembly and/or function of the plant mitochondrial PDC.

Heat-stress response of maize mitochondria.

We have identified maize (Zea mays L. inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. During heat stress (42 degrees C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly. In contrast, levels of two 22-kD proteins increased dramatically (HSP22). Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22. The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species. Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress. Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a +13 degrees C heat stress. Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery. Under continuous heat-stress conditions, the level of HSP22 remained high. A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database. Sequence analysis indicated that HSP22 is a member of the plant small heat-shock protein superfamily.

Purification, Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase.

An NADH dehydrogenase activity from red beet (Beta vulgaris L.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays L. B73) mitochondria to a 58-kD protein doublet, a 45-kD protein, and a few other less prevalent proteins. Polyclonal antibodies prepared against the 58-kD protein of red beet roots were found to immunoprecipitate the NAD(P)H dehydrogenase activity. The antibodies cross-reacted to similar proteins in mitochondria from a number of plant species but not to rat liver mitochondrial proteins. The polyclonal antibodies were used in conjunction with maize mitochondrial fractionation to show that the 58-kD protein was likely part of a protein complex loosely associated with the membrane fraction. A membrane-impermeable protein cross-linking agent was used to further show that the majority of the 58-kD protein was located on the outer surface of the inner mitochondrial membrane or in the intermembrane space. Analysis of the cross-linked 58-kD NAD(P)H dehydrogenase indicated that specific proteins of 64, 48, and 45 kD were cross-linked to the 58-kD protein doublet. The NAD(P)H dehydrogenase activity was not affected by ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime] -tetraacetic acid or CaCl2, was stimulated somewhat (21%) by flavin mononucleotide, was inhibited by p-chloromercuribenzoic acid (49%) and mersalyl (40%), and was inhibited by a bud scale extract of Platanus occidentalis L. containing platanetin (61%).

Purification, Characterization, and Submitochondrial Localization of the 32-Kilodalton NADH Dehydrogenase from Maize.

Plant mitochondria have the unique ability to directly oxidize exogenous NAD(P)H. We recently separated two NAD(P)H dehydrogenase activities from maize (Zea mays L.) mitochondria using anion-exchange (Mono Q) chromatography. The first peak of activity oxidized only NADH, whereas the second oxidized both NADH and NADPH. In this paper we describe the purification of the first peak of activity to a 32-kD protein. Polyclonal antibodies to the 32-kD protein were used to show that it was present in mitochondria from several plant species. Two-dimensional gel analysis of the 32-kD NADH dehydrogenase indicated that it consisted of two major and one minor isoelectric forms. Immunoblot analysis of submitochondrial fractions indicated that the 32-kD protein was enriched in the soluble protein fraction after mitochondrial disruption and fractionation; however, some association with the membrane fraction was observed. The membrane-impermeable protein cross-linking agent 3,3[prime] -dithiobis-(sulfosuccinimidylpropionate) was used to further investigate the submitochondrial location of the 32-kD NADH dehydrogenase. The 32-kD protein was localized to the outer surface of the inner mitochondrial membrane or to the intermembrane space. The pH optimum for the enzyme was 7.0. The activity was found to be severely inhibited by p-chloromercuribenzoic acid, mersalyl, and dicumarol, and stimulated somewhat by flavin mononucleotide.

Monoclonal Antibodies to the [alpha]- and [beta]-Subunits of the Plant Mitochondrial F1-ATPase.

We have generated nine monoclonal antibodies against subunits of the maize (Zea mays L.) mitochondrial F1-ATPase. These monoclonal antibodies were generated by immunizing mice against maize mitochondrial fractions and randomly collecting useful hybridomas. To prove that these monoclonal antibodies were directed against ATPase subunits, we tested their cross-reactivity with purified F1-ATPase from pea cotyledon mitochondria. One of the antibodies ([alpha]-ATPaseD) cross-reacted with the pea F1-ATPase [alpha]-subunit and two ([beta]-ATPaseD and [beta]-ATPaseE) cross-reacted with the pea F1-ATPase [beta]-subunit. This established that, of the nine antibodies, four react with the maize [alpha]-ATPase subunit and the other five react with the maize [beta]-ATPase subunit. Most of the monoclonal antibodies cross-react with the F1-ATPase from a wide range of plant species. Each of the four monoclonal antibodies raised against the [alpha]-subunit recognizes a different epitope. Of the five [beta]-subunit antibodies, at least three different epitopes are recognized. Direct incubation of the monoclonal antibodies with the F1-ATPase failed to inhibit the ATPase activity. The monoclonal antibodies [alpha]-ATPaseD and [beta]-ATPaseD were bound to epoxide-glass QuantAffinity beads and incubated with a purified preparation of pea F1-ATPase. The ATPase activity was not inhibited when the antibodies bound the ATPase. The antibodies were used to help map the pea F1-ATPase subunits on a two-dimensional map of whole pea cotyledon mitochondrial protein. In addition, the antibodies have revealed antigenic similarities between various isoforms observed for the [alpha]- and [beta]-subunits of the purified F1-ATPase. The specificity of these monoclonal antibodies, along with their cross-species recognition and their ability to bind the F1-ATPase without inhibiting enzymic function, makes these antibodies useful and invaluable tools for the further purification and characterization of plant mitochondrial F1-ATPases.

Tissue-specific expression of the alternative oxidase in soybean and siratro.

Alternative oxidase activity (cyanide-insensitive respiration) was measured in mitochondria from the shoots, roots, and nodules of soybean (Glycine max L.) and siratro (Macroptilium atropurpureum) plants. Activity was highest in the shoots and lowest in the nodules. Alternative oxidase activity was associated with one (roots) or two (shoots) proteins between 30 and 35 kilodaltons that were detected by western blotting with a monoclonal antibody against Sauromatum guttatum alternative oxidase. No such protein was detected in nodule mitochondria. Measurements of oxygen uptake by isolated soybean root and nodule cells in the presence of cyanide and salicylhydroxamic acid indicated that alternative oxidase activity was confined to the uninfected cortex cells of the nodule. Immunoprecipitation of translation products of mRNA isolated from soybean shoots revealed a major band at 43 kilodaltons that is assumed to be the precursor of an alternative oxidase protein. This band was not seen when mRNA from nodules was treated in the same fashion. The results indicate that tissue-specific expression of the alternative oxidase occurs in soybean and siratro.

Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria.

Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak I, is capable of oxidizing NADPH and the beta form of NADH. The other two activities, peaks II and III, oxidize only beta-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component to peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca(2+) or Mg(2+). Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak II gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca(2+) and Mg(2+). As with peak I, platanetin had no effect on peak II activity. Peak III was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca(2+) and Mg(2+), but was inhibited by platanetin. The peak III enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak III activity is likely the exogenous NADH dehydrogenase.

Mitochondrial malate dehydrogenase from corn : purification of multiple forms.

A method to fractionate corn (Zea mays L. B73) mitochondria into soluble proteins, high molecular weight soluble proteins, and membrane proteins was developed. These fractions were analyzed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assays of mitochondrial enzyme activities. The Krebs cycle enzymes were enriched in the soluble fraction. Malate dehydrogenase has been purified from the soluble fraction by a two-step fast protein liquid chromatography method. Six different malate dehydrogenase peaks were obtained from the Mono Q column. These peaks were individually purified using a Phenyl Superose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks showed that three of the isoenzymes consisted of different homodimers (I, III, VI) and three were different heterodimers (II, IV, V). Apparent molecular masses of the three different monomer subunits were 37, 38, and 39 kilodaltons. Nondenaturing gel analysis of the malate dehydrogenase peaks showed that each Mono Q peak contained a band of malate dehydrogenase activity with different mobility. These observations are consistent with three nuclear genes encoding corn mitochondrial malate dehydrogenase. Polyclonal antibodies raised against purified malate dehydrogenase were used to identify the gene products using Western blots of two-dimensional gels.

Monoclonal antibodies to the alternative oxidase of higher plant mitochondria.

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alternative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent M(r) of 37, 36, and 35 kilodaltons (kD). Monoclonal antibodies have now been prepared to these proteins and designated as AOA (binding all three proteins of the alternative oxidase cluster), AOU (binding the upper or 37 kD protein), and AOL (binding the lower or 36 and 35 kD proteins). All three antibodies bind to their respective alternative oxidase proteins whether the proteins are in their native or denatured states (as on protein blots). AOA and AOU inhibit alternative oxidase activity around 49%, whereas AOL inhibits activity only 14%. When coupled individually to Sepharose 4B, all three monoclonal resins were capable of retaining the entire cluster of alternative oxidase proteins, suggesting that these proteins are physically associated in some manner. The monoclonals were capable of binding similar mitochondrial proteins in a number of thermogenic and nonthermogenic species, indicating that they will be useful in characterizing and purifying the alternative oxidase of different systems. The ability of the monoclonal-Sepharose 4B resins to retain the cluster of previously identified alternative oxidase proteins, along with the inhibition of alternative oxidase activity by these monoclonals, supports the role of these proteins in constituting the alternative oxidase.

Mitochondrial events during development of thermogenesis inSauromatum guttatum (Schott).

Changes in the mitochondrial electrontransport chain were followed in the thermogenic inflorescence ofSauromatum guttatum Schott from 5d before thermogenesis to 3d thereafter. The capacities of the alternative and cytochrome pathways of mitochondrial electron transport were found to be developmentally coordinated to contribute to the thermogenic events in the appendix and the sterile floral regions. Electron flow through the alternative pathway, is believed primarily responsible for heat production, and this pathway was expressed to the highest degree in both tissues during thermogenesis. In the appendix, the cytochrome chain was shut down considerably during thermogenesis, forcing electron flow through the alternative pathway and thus yielding maximum heat production. The shut-down of the cytochrome chain does not occur in the sterile floral region which may explain why this region is not as thermogenic as the appendix. Cytochrome-oxidase difference spectra indicated that the cytochrome oxidase of appendix mitochondria was not capable of accepting electrons on the day of thermogenesis, and that this capacity was partially restored by the following day even though the tissue was senescing at this time point. Relative levels of messenger RNAs for cytochrome-oxidase subunits I and II were found to decrease the day before thermogenesis, which could result in lower levels of these proteins in appendix mitochondria on the day of thermogenesis.The capacity for overall mitochondrial protein synthesis was also investigated and was found to drop continuously from 5d before thermogenesis to 3d thereafter, even though the capacities of the electron-transport chain were changing dramatically. The levels of mitochondrial ribosomal RNA levels decreased during development, which could explain the overall drop in mitochondrial translational efficiency. Experiments concerning the synthesis of the alternative-oxidase proteins indicated that they were most likely nuclearly encoded, and that their expression could be induced by salicylic acid.

Identification of the alternative terminal oxidase of higher plant mitochondria.

In addition to cytochrome oxidase, plant mitochondria have a second terminal oxidase called the alternative oxidase. The alternative oxidase is of great interest in that energy is not conserved when electrons flow through it. The potential energy of the system is thus lost as heat, and, in plants with high levels of the alternative oxidase, this results in thermogenesis. We have purified the alternative oxidase from mitochondria of the thermogenic spadix of Sauromatum guttatum and have identified its polypeptide constituents by using polyclonal antibodies. A 166-fold purification was achieved through a combination of cation-exchange (carboxymethyl-Sepharose) and hydrophobic-interaction (phenyl-Sepharose) chromatography. Polyclonal antibodies raised to the CM-Sepharose fractions readily immunoprecipitated alternative oxidase activity and immunoprecipitated four of the proteins that copurify with the activity. These proteins have apparent molecular masses of 37, 36, 35.5, and 35 kDa. Polyclonal antibodies raised individually to the 37-, 36-, and 35.5- plus 35-kDa proteins cross-reacted with all of these proteins, indicating the presence of common antigenic sites. The 37-kDa protein appears to be constitutive in Sauromatum, whereas expression of the 36- and 35-kDa proteins was correlated with presence of alternative pathway activity. The 35.5-kDa protein appears with loss of alternative pathway activity during senescence, indicating that this protein may be a degradation product of the 36-kDa protein. Binding of anti-36-kDa protein antibodies to total mitochondrial protein blots of five plant species indicated that similar proteins were always present when alternative pathway activity was observed.

Characterization and Solubilization of the Alternative Oxidase of Sauromatum guttatum Mitochondria.

The alternative oxidase activity of Sauromatum guttatum spadix mitochondria has been investigated as to its developmental expression and tissue localization. Mitochondria rich in alternative oxidase activity were found in a yellow cortex tissue present to varying degrees within the appendix, male floral, and sterile floral regions of the spadix. During a 5-day period just prior to anthesis, the alternative oxidase activity present in the appendix region was found to increase over 10-fold. On the following day when the appendix region becomes thermogenic, cytochrome oxidase activity was found to decrease by 92%, effectively forcing electron flow through the alternative oxidase. A procedure for efficient solubilization of the alternative oxidase from appendix region mitochondria was developed. The alternative oxidase thus solubilized was sensitive to heat inactivation and trypsin digestion. The activity showed inhibition characteristics expected of the alternative oxidase in that it was sensitive to salicylhydroxamic acid and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, but relatively insensitive to KCN and antimycin A. Essential sulfhydryl group(s) were indicated by reversible inhibition by p-chloromercuribenzoic acid. The solubilized alternative oxidase was most active in the detergent N,N-bis-(3-d-glucoamidopropyl)-deoxycholamide and had a pH optimum of 6.8.

Alternative Respiratory Path Capacity in Plant Mitochondria: Effect of Growth Temperature, the Electrochemical Gradient, and Assay pH.

Influence of growth temperature on the capacity of the mitochondrial alternative pathway of electron transport was investigated using etiolated corn (Zea mays L.) seedlings. These seedlings were grown to comparable size in either a warm (30 degrees C) or a cold (13 degrees C) temperature regime, and then their respiration rates were measured as O(2) uptake at 25 degrees C. The capacity of the alternative pathway (KCN-insensitive O(2) uptake) was found essentially to double in shoots of cold-grown seedlings. This increased capacity slowly developed over several days growth in the cold, but was lost within 1 day when the seedlings were exposed to a warm regime. When mitochondria were isolated from the shoots of these seedlings, a greater potential for flow through the alternative path was observed in mitochondria from the cold-grown seedlings with all substrates used (an average increase of 84%). Using exogenous NADH as the substrate, the effect of the electrochemical gradient on measurable capacities of the cytochrome and alternative pathways was investigated in mitochondria from both etiolated seedlings and thermogenic spadices. The uncoupler FCCP (p-trifluoromethoxycarbonylcyanide phenylhydrazone) was used to diminish the electrochemical gradient when desired. In corn (Zea mays L.) shoot and mung bean (Vigna radiata L.) hypocotyl mitochondria, which have relatively low capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the electrochemical gradient was found not to influence the potential for flow through the alternative path. However, in mitochondria from skunk cabbage (Symplocarpus foetidus L.) and voodoo lily (Sauromatum guttatum Schott) spadices, which have high capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the gradient occurred at the expense of flow through the alternative pathway. These results suggest that in mitochondria of thermogenic spadices, the combined capacities of the cytochrome and alternative paths exceed the capacity of the exogenous NADH dehydrogenase. The effect of assay pH on measurable capacities of the cytochrome and alternative paths was determined over a pH range of 5.6 to 8.8 using exogenous NADH as the mitochondrial substrate. When the electrochemical gradient was present, it limited the electron transport rate and little effect of assay pH was observed. However, when formation of the gradient was prevented through inclusion of FCCP, measurable capacities of the cytochrome and alternative paths were found to be greatly influenced by pH. This experiment also revealed that the potential for respiratory control is largely dependent upon the assay pH.

Energetics of proline transport in corn mitochondria.

The mechanism of proline entry into the matrix region of isolated corn mitochondria (Zea mays L. Mo17 x B73) was investigated by measuring osmotically induced changes of mitochondrial size (changes in A(520)) in combination with oxygen uptake measurements. Using NADH oxidation to generate the electrochemical gradient, we have determined that proline transport is stereospecific and that it can be inhibited by the proline analog l-thiazolidine-4-carboxylic acid.The energetics of proline transport was investigated by measuring the effects of FCCP (p-trifluoromethoxycarbonyl cyanide phenylhydrazone) and valinomycin on mitochondrial swelling and substrate oxidation. Proline transport and resulting oxidation were found to be partially dependent upon the energy of the electrochemical gradient. At low proline concentrations, entry was found to be primarily independent of the gradient (based on insensitivity to FCCP), whereas at higher proline concentrations a gradient-dependent mechanism became involved. Results with valinomycin indicated that proline transport and oxidation are dependent upon the pH potential across the membrane rather than the electrical (membrane) potential.

Effects of the Proline Analog l-Thiazolidine-4-carboxylic Acid on Proline Metabolism.

The effect of various proline analogs on proline oxidation in mitochondria isolated from etiolated barley (Hordeum vulgare) shoots was investigated. Of the analogs tested, only l-thiazolidine-4-carboxylic acid (T4C) was an effective inhibitor. T4C (1 millimolar) inhibited proline (10 millimolar) -dependent 0(2) uptake an average of 67%. T4C was also oxidized to some degree (12.9 nanoatoms oxygen per minute per milligram protein for 10 millimolar). The effect of T4C on the oxidation of other mitochondrial substrates was also tested. T4C inhibited big up tri, open(1)-pyrrolidine-5-carboxylic acid-dependent oxygen uptake slightly (13%), the oxidation of malate plus pyruvate even less (6%), and stimulated the oxidation of succinate (+11%), exogenous NADH (+19%), and citrate (+20%). Thus, inhibition by T4C in mitochondria is relatively specific to proline oxidation. T4C was found to inhibit proline dehydrogenase and not the transport of proline into the matrix.The effect of T4C on proline metabolism in detached green barley leaves was investigated. T4C inhibited proline oxidation in turgid leaves, increasing the proline content of these leaves slightly. In wilted leaves (that are synthesizing proline rapidly), T4C inhibited proline synthesis, which resulted in a decrease in the proline content of the leaves. big up tri, open(1)-pyrrolidine-5-carboxylic acid reductase (the last enzyme in proline synthesis) was not inhibited by T4C, and thus T4C's influence is prior to that step of the synthetic pathway. T4C had no influence on the incorporation of proline into protein.

Proline Oxidation in Corn Mitochondria : Involvement of NAD, Relationship to Ornithine Metabolism, and Sidedness on the Inner Membrane.

Proline-dependent oxygen uptake in corn mitochondria (Zea mays L. B73 x Mo17 or Mo17 x B73) occurs through a proline dehydrogenase (pH optimum around 7.2) bound to the matrix side of the inner mitochondrial membrane. Sidedness was established by determining the sensitivity of substrate-dependent ferricyanide reduction to antimycin and FCCP (P-trifluoromethoxycarbonylcyanide phenylhydrazone). Proline dehydrogenase activity did not involve nicotinamide adenine dinucleotide reduction, and thus electrons and protons from proline enter the respiratory chain directly. Delta(1)-Pyrroline-5-carboxylate (P5C) derived from proline was oxidized by a P5C dehydrogenase (pH optimum approximately 6.4). This enzyme was found to be similar to proline dehydrogenase in that it was bound to the matrix side of the inner membrane and fed electrons and protons directly into the respiratory chain.Ornithine-dependent oxygen uptake was measurable in corn mitochondria and resulted from an ornithine transaminase coupled with a P5C dehydrogenase. These enzymes existed as a complex bound to the matrix side of the inner membrane. P5C formed by ornithine transaminase was utilized directly by the associated P5C dehydrogenase and was not released into solution. Activity of this dehydrogenase involved the reduction of nicotinamide adenine dinucleotide.

Submitochondrial location and electron transport characteristics of enzymes involved in proline oxidation.

Isolated corn mitochondria (Zea mays cv. B73 x Mo17) were fractionated and the fragments were separated on a 20-45% (weight/weight) continuous sucrose gradient. Soluble enzymes remained at the top of the gradient overlapping with the outer membranes, while inner membrane vesicles and intact inner membranes were distributed farther down the gradient. Proline oxidase and Delta(1)-pyrroline-5-carboxylic acid dehydrogenase activities were associated only with the inner mitochondrial membrane. Glutamate dehydrogenase was confirmed as a matrix enzyme.Both proline and Delta(1)-pyrroline-5-carboxylic acid supported oxygen uptake in isolated mitochondria. Proline dependent oxygen uptake was relatively independent of pH with a maximum rate at pH 7.2. In contrast, Delta(1)-pyrroline-5-carboxylic acid-dependent oxygen uptake was sensitive to pH with an optimum at pH 6.1. The oxidation of proline and Delta(1)-pyrroline-5-carboxylic acid was inhibited by 10 micromolar rotenone. This indicates that electrons from these substrates enter the respiratory chain prior to at least one of the rotenone sensitive iron-sulfur proteins. Both substrates yielded ADP:O ratios of around 1.9 as compared to malate plus pyruvate (2.1), succinate (1.3), and exogenous NADH (1.2).