Programmed cell death in cereal aleurone

Progress in understanding programmed cell death (PCD) in the cereal aleurone is described. Cereal aleurone cells are specialized endosperm cells that function to synthesize and secrete hydrolytic enzymes that break down reserves in the starchy endosperm. Unlike the cells of the starchy endosperm, aleurone cells are viable in mature grain but undergo PCD when germination is triggered or when isolated aleurone layers or protoplasts are incubated in gibberellic acid (GA). Abscisic acid (ABA) slows down the process of aleurone cell death and isolated aleurone protoplasts can be kept alive in media containing ABA for up to 6 months. Cell death in barley aleurone occurs only after cells become highly vacuolated and is manifested in an abrupt loss of plasma membrane integrity. Aleurone cell death does not follow the apoptotic pathway found in many animal cells. The hallmarks of apoptosis, including internucleosomal DNA cleavage, plasma membrane and nuclear blebbing and formation of apoptotic bodies, are not observed in dying aleurone cells. PCD in barley aleurone cells is accompanied by the accumulation of a spectrum of nuclease and protease activities and the loss of organelles as a result of cellular autolysis.     

Active oxygen and cell death in cereal aleurone cells

The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H(2)O(2) is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.     

Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation

Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.     

Binding of hydrolases from wheat aleurone to concanavalin A- and wheat-germ agglutinin-sepharose

We have studied the ability of hydrolases (acid phosphatase and glycosidases) from the aleurone layers of resting wheat grains to interact with Con A- and WGA-Sepharose as a way to examine their glycoprotein nature. Aliquots (6–85% depending on the enzyme) of all the enzymes interacted with Con A-Sepharose. The major part of α-mannosidase activity (85%) was present in this form. Aliquots (2–20% depending on the enzyme) of the following four enzymes, β-galactosidase, α-mannosidase, β-N-acetylglucosaminidase and acid phosphatase, interacted with WGA-Sepharose. All the enzymes were found in forms which were unable to interact with either lectin. No forms of hydrolases interacting with both lectins were found in the crude extract. The specific activities of most of the enzymes recovered from the lectin-Sepharose gels were greater than those measured in the crude extract. In particular, the highest specific activities were found for β-N-acetylglucosaminidase and β-galactosidase recovered from WGA-Sepharose. Different lectin-binding forms of hydrolases were compared with respect to pH optimum and stability under various conditions (heat and guanidine hydrochloride treatments). The lectin-binding pattern of the hydrolases released in the incubation medium by the aleurone layers was similar to that reported above for the enzymes extracted from these tissues, suggesting that none of the hydrolase forms found in the aleurone layers is selectively released during incubation of these tissues.     

A gibberellin-induced nuclease is localized in the nucleus of wheat aleurone cells undergoing programmed cell death

The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis revealed PCD of aleurone cells in maize grains, although the process was delayed as compared with wheat. Aleurone cells undergoing PCD showed a rapid vacuolation with high lytic activity in the cytoplasm, whereas the nucleus, which adopted an irregular shape, appeared essentially intact and showed symptoms of degradation at the end of the process. A nuclease activity was identified localized in the nucleus of aleurone cells undergoing PCD, just prior to the appearance of DNA laddering. This nuclease was induced by gibberellic acid treatment and was not detected when gibberellin synthesis was inhibited or in gibberellic acid-insensitive mutants. This nuclease was activated by Ca(2+) and Mg(2+), strongly inhibited by Zn(2+), and showed optimum activity at neutral pH, resembling nucleases involved in apoptosis of animal cells.     

Nitric oxide acts as an antioxidant and delays programmed cell death in barley aleurone layers

Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of alpha-amylase. alpha-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.     

Cell death of barley aleurone protoplasts is mediated by reactive oxygen species

The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.     

Major antifungal activity from the bulbs of Indian squill Urginea indica is a chitinase

We have identified a chitinase with antifungal activity in the bulbs of the plant Urginea indica(Indian squill) and purified it about 26-fold. The purified preparation contained a Mr 29 kDa protein that was an active growth inhibitor of the fungal pathogens Fusarium oxysporum and Rhizoctonia solani in an in vitro assay. Amino acid sequence analysis of the Mr 29 kDa protein revealed it to be highly homologous to the family 19 glycoside hydrolases, which are known to possess chitinase activity. The U. indica chitinase lacked a cysteine-rich N-terminal domain (characteristic of class I chitinases) and contained a conserved motif indicative of the signature 1 of family 19 glycoside hydrolases. It shared a approximately 70% sequence identity with the 26 kDa endochitinase of Hordeum vulgare, a typical class II chitinase of family 19. The five cysteines in the partial sequence of the Mr 29 kDa chitinase were found to be identical in location to five of the seven cysteines present in the catalytic domain of the H. vulgare enzyme. The molecular weight, the lack of an N-terminal cysteine-rich sequence, and the striking identity to the H. vulgare endochitinase suggest that the Mr 29 kDa U. indica protein is a putative class II chitinase. The antifungal activity is presumably mediated through the chitinolytic activity of the Mr 29 kDa protein.     

Purification and properties of a beta-glycosidase purified from midgut cells of Spodoptera frugiperda (Lepidoptera) larvae

Two beta-glycosidases (BG) (Mr 47,000 and Mr 50,000) were purified from Spodoptera frugiperda (Lepidoptera: Noctuidae) midguts. These two polypeptides associate or dissociate depending on the medium ionic strength. The Mr 47,000 BG probably has two active sites. One of the putative active sites (cellobiase site) hydrolyses p-nitrophenyl beta-D-glucoside (NPbetaGlu) (79% of the total activity in saturated enzyme), cellobiose, amygdalin and probably also cellotriose, cellotetraose and cellopentaose. The cellobiase site has four subsites for glucose residue binding, as can be deduced from cellodextrin cleavage data. The enzymatic activity in this site is abolished after carbodiimide modification at pH 6.0. Since the inactivation is reduced in the presence of cellobiose, the results suggest the presence of a carboxylate as a catalytic group. The other active site of Mr 47,000 BG (galactosidase site) hydrolyses p-nitrophenyl beta-D-galactoside (NPbetaGal) better than NPbetaGlu, cleaves glucosylceramide and lactose and is unable to act on cellobiose, cellodextrins and amygdalin. This active site is not modified by carbodiimide at pH 6.0. The Mr 47,000 BG N-terminal sequence has high identity to plant beta-glycosidases and to mammalian lactase-phlorizin hydrolase, and contains the QIEGA motif, characteristic of the family of glycosyl hydrolases. The putative physiological role of this enzyme is the digestion of glycolipids (galactosidase site) and di- and oligosaccharides (cellobiase site) derived from hemicelluloses, thus resembling mammalian lactase-phlorizin hydrolase.     

Angiotensin II-producing proteases from granulomatous tissue reaction in mice infected with Schistosoma mansoni

1. Angiotensin I hydrolases, Mr 140,000 and Mr 70,000 were separated by gel filtration from Tris-HCl buffer extract of hepatic granulomas developed in mice with schistosomiasis. Two enzymes had different substrate specificity. 2. Mr 140,000 hydrolase activity was inhibited by captopril as reported for angiotensin converting enzyme (ACE), while that of Mr 70,000 hydrolase activity was inhibited by potato carboxypeptidase inhibitor. 3. An intermediary, des-Leu10-angiotensin I and then angiotensin II were formed from angiotensin I by Mr 70,000 hydrolase. 4. The findings suggest that Mr 70,000 enzyme is tissue carboxypeptidase A, and it generates angiotensin II in granulomatous inflammation as does ACE.     

Defense-related signaling by interaction of arabinogalactan proteins and beta-glucosyl Yariv reagent inhibits gibberellin signaling in barley aleurone cells

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at the plasma membrane and in extracellular spaces. A synthetic chemical, beta-glucosyl Yariv reagent (beta-GlcY), binds specifically to AGPs. We previously reported that gibberellin signaling is specifically inhibited by beta-GlcY treatment in barley aleurone protoplasts. In the present study, we found that beta-GlcY also inhibited gibberellin-induced programmed cell death (PCD) in aleurone cells. We examined the universality and specificity of the inhibitory effect of beta-GlcY on gibberellin signaling using microarray analysis and found that beta-GlcY was largely effective in repressing gibberellin-induced gene expression. In addition, >100 genes were up-regulated by beta-GlcY in a gibberellin-independent manner, and many of these were categorized as defense-related genes. Defense signaling triggered by several defense system inducers such as jasmonic acid and a chitin elicitor could inhibit gibberellin-inducible events such as alpha-amylase secretion, PCD and expression of some gibberellin-inducible genes in aleurone cells. Furthermore, beta-GlcY repressed the gibberellin-inducible Ca2+-ATPase gene which is important for gibberellin-dependent gene expression, and induced known repressors of gibberellin signaling, two WRKY genes and a NAK kinase gene. These effects of beta-GlcY were also phenocopied by the chitin elicitor and/or jasmonic acid. These results indicate that gibberellin signaling is under the regulation of defense-related signaling in aleurone cells. It is also probable that AGPs are involved in the perception of stimuli causing defense responses.     

Programmed cell death during endosperm development

The endosperm of cereals functions as a storage tissue in which the majority of starch and seed storage proteins are synthesized. During its development, cereal endosperm initiates a cell death program that eventually affects the entire tissue with the exception of the outermost cells, which differentiate into the aleurone layer and remain living in the mature seed. To date, the cell death program has been described for maize and wheat endosperm, which exhibits common and unique elements for each species. The progression of endosperm programmed cell death (PCD) in both species is accompanied by an increase in nuclease activity and the internucleosomal degradation of nuclear DNA, hallmarks of apoptosis in animals. Moreover, ethylene and abscisic acid are key to mediating PCD in cereal endosperm. The progression of the cell death program in developing maize endosperm follows a highly organized pattern whereas in wheat endosperm, PCD initiates stochastically. Although the essential characteristics of cereal endosperm PCD are now known, the molecular mechanisms responsible for its execution remain to be identified.     

Proteomics analysis of rice lesion mimic mutant (spl1) reveals tightly localized probenazole-induced protein (PBZ1) in cells undergoing programmed cell death

Numerous reports have predicted/hypothesized a role for probenazole-induced protein (PBZ1) as a molecular marker in rice self-defense mechanism. However, the precise function of PBZ1 remains unknown. In the present study, we examined PBZ1 as a putative cell death marker in rice. For this, we focused our attention on a rice lesion mimic mutant (LMM), spotted leaf 1 ( spl1), which has been used to study the programmed cell death (PCD) phenomenon during lesion development in leaf. Using two-dimensional gel electrophoresis (2-DGE), 18 colloidal Coomassie brilliant blue stained protein spots were found to be differentially expressed in the leaves of spl1 mutant. After analysis of these spots by MALDI-TOF-MS, we identified the PBZ1 protein to be highly inducible in spl1. On the basis of these results, we proceeded to verify whether PBZ1 is highly expressed in the tissues undergoing PCD in rice. To do so, we performed immunoblot analysis and immunolocalization and used transgenic lines carrying the PBZ1 promoter fused with GFP. Results demonstrated that the expression levels and localizations of PBZ1 dramatically coincided with tissues undergoing PCD, namely, during leaf senescence, root aerenchyma formation, coleoptiles senescence, root cap, and seed aleurone layer. Furthermore, localization of the PBZ1 protein was also tightly correlated with TUNEL signal in the seed aleurone layer. As DNA fragmentation is a hallmark of PCD, this result clearly indicates a role for PBZ1 in rice tissues undergoing PCD. In conclusion, our results provide strong support for the hypothesis that PBZ1 is a molecular marker in rice defense response, and can serve as a novel potential marker for cell death/PCD in rice.     

Selective cleavage of human ACTH, beta-lipotropin, and the N-terminal glycopeptide at pairs of basic residues by IRCM-serine protease 1. Subcellular localization in small and large vesicles

We present a study of the cleavage specificity of IRCM-serine protease 1 from frozen porcine pituitary neurointermediate lobes using polypeptide substrates representing different segments of human pro-opiomelanocortin. Using 125I-labeled ACTH(11-24) and a 125I-labeled model beta-lipotropin (beta-LPH) peptide, the preference of this protease for cleavage C-terminal to the pairs of basic residues Lys-Arg and Lys-Lys was clearly seen. This study was extended to larger unlabeled natural human polypeptides including ACTH(1-39), beta-LPH(1-89), and the N-terminal glycopeptide (1-76), which are known to serve as substrates for further cleavage in vivo. In these substrates IRCM-serine protease 1 cleaved C-terminal to all pairs of basic residues known to be cleaved in vivo. In addition, the enzyme cleaved between two pairs of basic amino acids found in NT(1-76) which are also known to be cleaved in vivo. Many potential "tryptic-like" cleavage sites were not cleaved by the enzyme. However, IRCM-serine protease 1 cleaved C-terminal to Phe-Arg in the three melanocyte-stimulating hormone sequences of pro-opiomelanocortin. In order to better understand the physiological role of IRCM-serine protease 1, differential centrifugation was used to study the subcellular distribution of the enzyme from porcine pituitary anterior lobe homogenates. We present evidence that the active enzyme form, isolated from the subcellular fractions, possesses a similar molecular architecture as the enzyme isolated from frozen tissue (Mr 38,000 catalytic domain linked via disulfide bridge(s) to another polypeptide chain(s) to form an Mr 88,000 monomeric structure). The majority of IRCM-serine protease activity is found to be associated with small vesicles (150,000 X g for 5 h) of as yet undetermined nature. In addition, a latent activity was found to be associated with a 27,000 X g (15 min) pellet containing the majority of mature secretory granules. If IRCM-serine protease 1 participates in prohormone maturation in vivo, we propose a model in which this protease is present in an enzymatically active form in small vesicles, possibly within clathrin-coated structures (prosecretory granules) which are then transformed to mature secretory granules by a process which would also inactivate most of the enzyme.     

Okadaic acid, a protein phosphatase inhibitor, blocks calcium changes, gene expression, and cell death induced by gibberellin in wheat aleurone cells.

The cereal aleurone functions during germination by secreting hydrolases, mainly alpha-amylase, into the starchy endosperm. Multiple signal transduction pathways exist in cereal aleurone cells that enable them to modulate hydrolase production in response to both hormonal and environmental stimuli. Gibberellic acid (GA) promotes hydrolase production, whereas abscisic acid (ABA), hypoxia, and osmotic stress reduce amylase production. In an effort to identify the components of transduction pathways in aleurone cells, we have investigated the effect of okadaic acid (OA), a protein phosphatase inhibitor, on stimulus-response coupling for GA, ABA, and hypoxia. We found that OA (100 nM) completely inhibited all the GA responses that we measured, from rapid changes in cytosolic Ca2+ through changes in gene expression and accelerated cell death. OA (100 nM) partially inhibited ABA responses, as measured by changes in the level of PHAV1, a cDNA for an ABA-induced mRNA in barley. In contrast, OA had no effect on the response to hypoxia, as measured by changes in cytosolic Ca2+ and by changes in enzyme activity and RNA levels of alcohol dehydrogenase. Our data indicate that OA-sensitive protein phosphatases act early in the transduction pathway of GA but are not involved in the response to hypoxia. These data provide a basis for a model of multiple transduction pathways in which the level of cytosolic Ca2+ is a key point of convergence controlling changes in stimulus-response coupling.     

A single limit dextrinase gene is expressed both in the developing endosperm and in germinated grains of barley

The single gene encoding limit dextrinase (pullulan 6-glucanohydrolase; EC 3.2.1.41) in barley (Hordeum vulgare) has 26 introns that range in size from 93 to 822 base pairs. The mature polypeptide encoded by the gene has 884 amino acid residues and a calculated molecular mass of 97,417 D. Limit dextrinase mRNA is abundant in gibberellic acid-treated aleurone layers and in germinated grain. Gibberellic acid response elements were found in the promoter region of the gene. These observations suggest that the enzyme participates in starch hydrolysis during endosperm mobilization in germinated grain. The mRNA encoding the enzyme is present at lower levels in the developing endosperm of immature grain, a location consistent with a role for limit dextrinase in starch synthesis. Enzyme activity was also detected in developing grain. The limit dextrinase has a presequence typical of transit peptides that target nascent polypeptides to amyloplasts, but this would not be expected to direct secretion of the mature enzyme from aleurone cells in germinated grain. It remains to be discovered how the enzyme is released from the aleurone and whether another enzyme, possibly of the isoamylase group, might be equally important for starch hydrolysis in germinated grain.     

Hydrogen sulfide delays GA-triggered programmed cell death in wheat aleurone layers by the modulation of glutathione homeostasis and heme oxygenase-1 expression

Hydrogen sulfide (H₂S) is considered as a cellular signaling intermediate in higher plants, but corresponding molecular mechanisms and signal transduction pathways in plant biology are still limited. In the present study, a combination of pharmacological and biochemical approaches was used to study the effect of H₂S on the alleviation of GA-induced programmed cell death (PCD) in wheat aleurone cells. The results showed that in contrast with the responses of ABA, GA brought about a gradual decrease of l-cysteine desulfhydrase (LCD) activity and H₂S production, and thereafter PCD occurred. Exogenous H₂S donor sodium hydrosulfide (NaHS) not only effectively blocked the decrease of endogenous H₂S release, but also alleviated GA-triggered PCD in wheat aleurone cells. These responses were sensitive to hypotaurine (HT), a H₂S scavenger, suggesting that this effect of NaHS was in an H₂S-dependent fashion. Further experiment confirmed that H₂S, rather than other sodium- or sulphur-containing compounds derived from the decomposing of NaHS, was attributed to the rescuing response. Importantly, the reversing effect was associated with glutathione (GSH) because the NaHS triggered increases of endogenous GSH content and the ratio of GSH/oxidized GSH (GSSG) in GA-treated layers, and the NaHS-mediated alleviation of PCD was markedly eliminated by l-buthionine-sulfoximine (BSO, a selective inhibitor of GSH biosynthesis). The inducible effect of NaHS was also ascribed to the modulation of heme oxygenase-1 (HO-1), because the specific inhibitor of HO-1 zinc protoporphyrin IX (ZnPP) significantly suppressed the NaHS-related responses. By contrast, the above inhibitory effects were reversed partially when carbon monoxide (CO) aqueous solution or bilirubin (BR), two of the by-products of HO-1, was added, respectively. NaHS-triggered HO-1 gene expression in GA-treated layers was also confirmed. Together, the above results clearly suggested that the H₂S-delayed PCD in GA-treated wheat aleurone cells was associated with the modulation of GSH homeostasis and HO-1 gene expression.     

High concentration active enzyme centrifugation studies with pig kidney phosphofructokinase. Detection of 9.8 S, 25 S, and 53 S active polymeric forms

This laboratory has carried out the first detailed studies of the active polymeric forms of phosphofructokinases over the concentration region of 1 to 1200 micrograms/ml. This includes the concentration range in which the enzymes exist in vivo and the concentration range in which their association-dissociation equilibria shift to yield various polymeric forms. Previously, active enzyme centrifugation experiments were limited to the concentration range below a few micrograms per ml. The present experiments were made possible by the recent development in this laboratory of a new technique called high concentration active enzyme centrifugation (Wei, G. J., and Deal, W. C., Jr. (1979) Biochemistry 18, 1129). We report here three new active polymeric forms of pig kidney phosphofructokinase which have been observed in high concentration active enzyme centrifugation experiments. These include: 1) a 9.8 S form (Mr = 2.6 X 10(5)); 2) a 25 S form (Mr = 1.01 X 10(6)); and 3) a 53 S form (too asymmetric to estimate Mr). In addition, a 5.4 S form is predicted from the Mr (8.8 X 10(4)) of the polypeptide chain obtained from sodium dodecyl sulfate gel electrophoresis; it is not known whether or not it is active. The 9.8 S value is the limiting sedimentation coefficient value observed in active enzyme centrifugation experiments. The 25 S form is indicated by a plateau in the 50 to 200 micrograms/ml region of the s versus c curve. The 53 S form is observed as a plateau in the 600 to 1000 micrograms/ml region of the s versus c curve.