Tissue-Specific Isoforms of Catalase Subunits in Castor Bean Seedlings

Catalases purified from endosperm glyoxysomes and non-specializedmicrobodies from hypocotyls of castor bean seedlings differedin their specific activity [90–164 and 0.89–4.9kunits (mg protein)^–1, respectively] and in their constituentsubunits [two subunits of 54 and 56 kDa for the endosperm enzymeand only one of 56 kDa for the hypocotyl enzyme]. Immunoblotanalysis also showed that particulate fractions from the endospermsand from etiolated and green cotyledons contained two catalasesubunits of 54 and 56 kDa, whereas such fractions from the hypocotylsand roots contained only the 56-kDa subunit. Leaf peroxisomesfrom green leaves had two catalase subunits of around 55 kDaeach. Results of translation in vitro indicated that the 54-and 56-kDa subunits were translated from distinct mRNAs andlevels of both mRNAs increased in the endosperms during germination,prior to increases in levels of catalase proteins. In the hypocotyls,the 56-kDa subunit seemed to be synthesized constitutively. ¹Present addresses: YO, Toyota Central Institute, 31-9 Musashizuka,Nagabuchi, Nagakute, Aichi 480-11, Japan     

Distribution of 59- and 55-kDa Catalase in Dark- and Light-grown Pumpkin and Various Other Plant Tissues

The catalase molecule in germinating pumpkin cotyledons is synthesizedas a precursor (59-kDa) form, whose relative molecular massis larger than the mature enzyme (55-kDa). Although both typesof molecules are localized in the microbodies, the 59-kDa specieshas been shown to be present predominantly in the leaf peroxisomesisolated from green cotyledons, while the 55-kDa species ispredominantly in the glyoxysomes from etiolated cotyledons [Yamaguchiet al. (1984) Proc. Natl. Acad. Sci. USA, 81: 4809]. We examinedthe distribution of the 59- and 55-kDa catalase molecules indark- and light-grown tissues of pumpkin seedlings as well asin other plant species, using the immunoblotting technique.The ratios of the 59- and 55-kDa catalase species differed inthe pumpkin tissues examined. Light interferes with the conversionof the 59-kDa precursor to the 55-kDa form, especially in thecotyledons. The effect of light was less pronounced in the rootsand hypocotyls, indicating that the light regulation of theconversion is tissue-specific. Dark- and light-grown cotyledonsfrom cucumber and watermelon seedlings showed a similar lightregulation, suggesting that cucurbitaceous plants possess similarlight-regulatory mechanism. From the analysis of catalase proteinfrom various plant tissues, a limited correlation between molecularforms of catalase and different microbody populations was observed. (Received September 6, 1986; Accepted December 4, 1986)     

Identification of porin-like polypeptide(s) in the boundary membrane of oilseed glyoxysomes

A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaf-type peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.     

Two temporally synthesized charge subunits interact to form the five isoforms of cottonseed (Gossypium hirsutum) catalase.

Five charge isoforms of tetrameric catalase were isolated from cotyledons of germinated cotton (Gossypium hirsutum L.) seedlings. Denaturing isoelectric focusing of the individual isoforms in polyacrylamide gels indicated that isoforms A (most anodic) and E (most cathodic) consisted of one subunit of different charge, whereas isoforms B, C and D each consisted of a mixture of these two subunits. Thus the five isoforms apparently were formed through combinations of two subunits in different ratios. Labelling cotyledons in vivo with [35S]methionine at three daily intervals in the dark, and translation in vivo of polyadenylated RNA isolated from cotyledons at the same ages, revealed synthesis of two different subunits. One of the subunits was synthesized in cotyledons at all ages studied (days 1-3), whereas the other subunit was detected only at days 2 and 3. This differential expression of two catalase subunits helped explain previous results from this laboratory showing that the two anodic forms (A and B) found in maturing seeds were supplemented with three cathodic forms (C-E) after the seeds germinated. These subunit data also helped clarify our new findings that proteins of isoforms A, B and C (most active isoforms) accumulated in cotyledons of plants kept in the dark for 3 days, then gradually disappeared during the next several days, whereas isoforms D and E (least active isoforms) remained in the cells. This shift in isoform pattern occurred whether seedlings were kept in the dark or exposed to continuous light after day 3, although exposure to light enhanced this process. These sequential molecular events were responsible for the characteristic developmental changes (rise and fall) in total catalase activity. We believe that the isoform changeover is physiologically related to the changeover in glyoxysome to leaf-type-peroxisome metabolism.     

Identification of Peroxisome Membrane Proteins (PMPs) in Sunflower (Helianthus annuus L.) Cotyledons and Influence of Light on the PMP Developmental Pattern

Boundary membranes were recovered from glyoxysomes, transition peroxisomes, and leaf-type peroxisomes purified from cotyledons of sunflower (Helianthus annuus L.) at three stages of postgerminative growth. After membranes were washed in 100 mM Na2CO3 (pH 11.5), integral peroxisome membrane proteins (PMPs) were solubilized in buffered aminocaproic acid/dodecyl maltoside (0.63 M/1.5%) and analyzed by nondenaturing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Six prominent nondenatured PMP complexes and 10 prominent SDS-denatured polypeptides were identified in the membranes of the three types of peroxisomes. A nondenatured complex of approximately 140 kD, composed mainly of 24.5-kD polypeptides, decreased temporally, independently of seedling exposure to white, blue, or red light; only far-red light seemed to prevent its decrease. PMP complexes of approximately 120 and 70 kD, in contrast, were present at all stages and changed in polypeptide content. It remains to be determined whether these data reflect changes within in vivo complexes or within complexes formed following/during detergent solubilization. Conversion of glyoxysomes to leaf-type peroxisomes in white or red light after a 2-d dark period was accompanied by the appearance of three SDS-denatured PMPs: 27.5, 28, and 47 kD. The former two became part of the PMP120 and 70 complexes, as well as part of a new PMP130 complex that also possessed the PMP47. Growth of seedlings in blue or far-red light did not promote the appearance of PMPs 27.5 or 28. Blue light promoted the appearance of PMP47, and far-red light seemed to prevent its appearance. Chlorophyll likely is not the photoreceptor involved in accumulation of PMPs because the PMP composition is distinctly different in seedlings irradiated with red or blue light of comparable fluence rates. Several lines of evidence indicate that the synthesis and acquisition of membrane and all matrix proteins are not coupled. The data provide evidence for a change in PMP composition when sunflower or any other oilseed glyoxysomes are converted to leaf-type peroxisomes and suggest that the change is regulated by both photobiological and temporal mechanisms.     

Catalase Synthesis and Turnover during Peroxisome Transition in the Cotyledons of Helianthus annuus L

Based on measurements of total catalase hematin and the degradation constants of catalase hematin, zero order rate constants for the synthesis of catalase were determined during the development of sunflower cotyledons (Helianthus annuus L.). Catalase synthesis reached a sharp maximum of about 400 picomoles hematin per day per cotyledon at day 1.5 during the elaboration of glyoxysomes in the dark. During the transition of glyoxysomes to leaf peroxisomes (greening cotyledons, day 2.5 to 5) catalase synthesis was constant at a level of about 30 to 40 picomoles hematin per day per cotyledon. In the cotyledons of seedlings kept in the dark (day 2.5 to 5) catalase synthesis did not exceed 10 picomoles hematin per day per cotyledon. During the peroxisome transition in the light, total catalase hematin was maintained at a high level, whereas total catalase activity rapidly decreased. In continuous darkness, total catalase hematin decreased considerably from a peak at day 2. The results show that both catalase synthesis and catalase degradation are regulated by light. The turnover characteristics of catalase are in accordance with the concept that glyoxysomes are transformed to leaf peroxisomes as described by the one population model and contradict the two population model and the enzyme synthesis changeover model which both postulate de novo formation of the leaf peroxisome population and degradation of the glyoxysome population.     

A correlative ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds

Summary Sunflower, cucumber, and tomato cotyledons, which contain microbodies in both the early lipid-degrading and the later photosynthetic stages of post-germinative growth, were processed for electron microscopy according to conventional procedures and examined 1, 4 and 7 days after germination. Homogenates of sunflower cotyledons were assayed for enzymes characteristic of glyoxysomes and leaf peroxisomes (both of which are defined morphologically as microbodies) at stages corresponding to the fixations for electron microscopy. The particulate nature of these enzymes was demonstrated by differential and equilibrium density centrifugation, making it possible to relate them to the microbodies seen in situ.One day after germination, the microbodies are present as small organelles among large numbers of protein and lipid storage bodies; the cell homogenate contains catalase but no detectable isocitrate lyase (characteristic of glyoxysomes) or glycolic acid oxidase (characteristic of leaf peroxisomes). 4 days after germination, numerous microbodies (glyoxysomes) are in extensive and frequent contact with lipid bodies. The microbodies often have cytoplasmic invaginations. At this stage the cells are rapidly converting lipids to carbohydrates, and the homogenate has high isocitrate lyase activity. 7 days after germination, microbodies (peroxisomes) are appressed to chloroplasts and frequently squeezed between them in the green photosynthetic cells. The homogenate at this stage has substantial glycolic acid oxidase activity but a reduced level of isocitrate lyase. It is yet to be determined whether the peroxisomes present at day 7 are derived from preexisting glyoxysomes or arise as a separate population of organelles.     

Rhizomelic chondrodysplasia punctata: biochemical studies of peroxisomes isolated from cultured skin fibroblasts.

Peroxisomes isolated from cultured skin fibroblasts of two patients with rhizomelic chondrodysplasia punctata (RCDP) and two controls were compared for biochemical studies. These experiments provided the following results: (1) peroxisomes isolated from RCDP-cultured skin fibroblasts had the same density (1.175 g/ml) as control peroxisomes; (2) dihydroxyacetone phosphate acyltransferase activity, the first enzyme in the synthesis of plasmalogens, was deficient (0.5% of control) in RCDP peroxisomes and this activity was not observed in any other region of the gradient; (3) the rate of activation (lignoceroyl-CoA ligase) and oxidation of lignoceric acid was normal in RCDP peroxisomes; and (4) peroxisomes from RCDP contained 3-ketoacyl-CoA thiolase in the unprocessed form (44-kDa protein), whereas control peroxisomes had both processed (41-kDa protein) and unprocessed forms of 3-ketoacyl-CoA thiolase. The presence of both processed and unprocessed 3-ketoacyl-CoA thiolase in control peroxisomes and the unprocessed form in RCDP peroxisomes suggests that processing of 3-ketoacyl-CoA thiolase takes place in peroxisomes. Although the specific activity and percentage of activity of 3-ketoacyl-CoA thiolase in RCDP peroxisomes was only 22-26% of control, the normal oxidation of lignoceric acid in RCDP peroxisomes indicates that unprocessed 3-ketoacyl-CoA thiolase is active. The remaining peroxisomal 3-ketoacyl-CoA thiolase activity in RCDP was observed in a protein fraction (peroxisome ghosts) lighter than peroxisomes. The normal oxidation of fatty acids in peroxisomes and the absence of such activity in peroxisome ghosts (d = 1.12 g/ml) containing peroxisomal proteins in RCDP suggest that RCDP has only one population of functional peroxisomes (d = 1.175 g/ml).     

Purification of glyoxysomal catalase and immunochemical comparison of glyoxysomal and leaf peroxisomal catalase in germinating pumpkin cotyledons

As a step to study the mechanism of the microbody transition (glyoxysomes to leaf peroxisomes) in pumpkin (Cucurbita sp. Amakuri Nankin) cotyledons, catalase was purified from glyoxysomes. The molecular weight of the purified catalase was determined to be 230,000 to 250,000 daltons. The enzyme was judged to consist of four identical pieces of the monomeric subunit with molecular weight of 55,000 daltons. Absorption spectrum of the catalase molecule gave two major peaks at 280 and 405 nanometers, showing that the pumpkin enzyme contains heme. The ratio of absorption at 405 and 280 nanometers was 1.0, the value being lower than that obtained for catalase from other plant sources. These results indicate that the pumpkin glyoxysomal catalase contains the higher content of heme in comparison with other plant catalase.

The immunochemical resemblance between glyoxysomal and leaf peroxisomal catalase was examined by using the antiserum specific against the purified enzyme preparation from pumpkin glyoxysomes. Ouchterlony double diffusion and immunoelectrophoretic analysis demonstrated that catalase from both types of microbodies cross-reacted completely whereas the immunotitration analysis showed that the specific activity of the glyoxysomal catalase was 2.5-fold higher than that of leaf peroxisomal catalase. Single radial immunodiffusion analysis showed that the specific activity of catalase decreased during the greening of pumpkin cotyledons.

     

Functional transformation of plant peroxisomes

Peroxisomes in higher plant cells are known to differentiate into at least three different classes, namely, glyoxysomes, leaf peroxisomes, and unspecialalized peroxisomes, dependending on the cell types. In germinating fatty seedlings, glyoxysomes that first appear in the etiolated cotyledonary cells are functionally transformed into leaf peroxisomes during greening. Subsequently, the organelles are transformed back into glyoxysomes during senescence of the cotyledons. Flexibility of function is a distinct feature of plant peroxisomes. This article briefly describes recent studies of the regulatory mechanisms involved in the changes of the function of plant peroxisomes.     

Intraorganellar distribution of superoxide dismutase in plant peroxisomes (glyoxysomes and leaf peroxisomes)

The intraorganellar distribution of superoxide dismutase (SOD) (EC 1.15.1.1) in two types of plant peroxisomes (glyoxysomes and leaf peroxisomes) was studied by determinations of SOD latency in intact organelles and by solubilization assays with 0.2 molar KCl. Glyoxysomes were purified from watermelon (Citrullus vulgaris Schrad.) cotyledons, and their integrity, calculated on the basis of glyoxysomal marker enzymes, was about 60%. Under the same conditions, the latency of SOD activity determined in glyoxysomes was 40%. The difference between glyoxysomal intactness and SOD latency was very close to the percentage of isozyme Mn-SOD previously determined in glyoxysomes (LM Sandalio, LA Del Río 1987 J Plant Physiol 127: 395-409). In matrix and membrane fractions of glyoxysomes, SOD exhibited a solubilization pattern very similar to catalase, a typical soluble enzyme of glyoxysomes. The analysis of the distribution of individual SOD isozymes in glyoxysomal fractions treated with KCl showed that Cu,Zn-SOD II, the major SOD isozyme in glyoxysomes, was present in the soluble fraction of these organelles, whereas Mn-SOD was bound to the glyoxysomal membrane. These data in conjunction with those of latency of SOD activity in intact glyoxysomes suggest that Mn-SOD is bound to the external side of the membrane of glyoxysomes. On the other hand, in intact leaf peroxisomes where only a Mn-containing SOD is present (LM Sandalio, JM Palma, LA Del Río 1987 Plant Sci 51: 1-8), this isozyme was found in the peroxisomal matrix. The physiological meaning of SOD localization in matrix and membrane fractions of glyoxysomes and the possibility of new roles for plant peroxisomes in cellular metabolism related to activated oxygen species is discussed.     

Leaf peroxisomes are directly transformed to glyoxysomes during senescence of pumpkin cotyledons

Summary After the functional transition of glyoxysomes to leaf peroxisomes during the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Immunocytochemical labeling with protein A-gold was performed to analyze the reverse microbody transition using antibodies against a leaf-peroxisomal enzyme, glycolate oxidase, and against two glyoxysomal enzymes, namely, malate synthase and isocitrate lyase. The intensity of labeling for glycolate oxidase decreased in the microbodies during senescence whereas in the case of malate synthase and isocitrate lyase intensities increased strikingly. Double labeling experiments with protein A-gold particles of different sizes showed that the leaf-peroxisomal enzymes and the glyoxysomal enzymes coexist in the microbodies of senescing pumpkin cotyledons, indicating that leaf peroxisomes are directly transformed to glyoxysomes during senescence.     

cDNA cloning and differential gene expression of three catalases in pumpkin

Three cDNA clones (cat1, cat2, cat3) for catalase (EC 1.11.1.6) were isolated from a cDNA library of pumpkin (Cucurbita sp.) cotyledons. In northern blotting using the cDNA-specific probe, the cat1 mRNA levels were high in seeds and early seedlings of pumpkin. The expression pattern of cat1 was similar to that of malate synthase, a characteristic enzyme of glyoxysomes. These data suggest that cat1 might encode a catalase associated with glyoxysomal functions. Furthermore, immunocytochemical analysis using cat1-specific anti-peptide antibody directly showed that cat1 encoding catalase is located in glyoxysomes. The cat2 mRNA was present at high levels in green cotyledons, mature leaf, stem and green hypocotyl of light-grown pumpkin plant, and correlated with chlorophyll content in the tissues. The tissue-specific expression of cat2 had a strong resemblance to that of glycolate oxidase, a characteristic enzyme of leaf peroxisomes. During germination of pumpkin seeds, cat2 mRNA levels increased in response to light, although the increase in cat2 mRNA by light was less than that of glycolate oxidase. cat3 mRNA was abundant in green cotyledons, etiolated cotyledons, green hypocotyl and root, but not in young leaf. cat3 mRNA expression was not dependent on light, but was constitutive in mature tissues. Interestingly, cat1 mRNA levels increased during senescence of pumpkin cotyledons, whereas cat2 and cat3 mRNAs disappeared during senescence, suggesting that cat1 encoding catalase may be involved in the senescence process. Thus, in pumpkin, three catalase genes are differentially regulated and may exhibit different functions.     

Role of glycosylation in the processing of newly translated insulin proreceptor in 3T3-L1 adipocytes

A procedure was developed for the immunoprecipitation of glycosylated and nonglycosylated forms of the insulin receptor and its precursors without prior purification using lectins. 3T3-L1 adipocytes were labeled with [35S]methionine after which 35S-labeled receptor polypeptides were specifically immunoprecipitated and characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The first 35S-polypeptide detected was a 190-kDa glycosylated proreceptor which was rapidly (t1/2 approximately equal to 15 min) processed to a 210-kDa intermediate. The latter precursor was more slowly (t1/2 approximately equal to 2 h) proteolytically processed to 125-kDa (alpha') and 83-kDa (beta') precursors of the mature alpha- and beta-receptor subunits. Immediately prior to insertion into the plasma membrane, i.e. about 3 h after translation, the alpha'- and beta'-precursor polypeptides were converted to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits. The characteristics of the oligosaccharide moieties of the receptor precursors and products were investigated. The 210-kDa precursor and its two products, the 125-kDa alpha'- and 83-kDa beta'-species, and the mature alpha- and beta-receptor subunits bind tightly to wheat germ lectin, whereas the 190-kDa proreceptor species is not bound. Upon incubation with endoglycosidase H, both the 210- and 190-kDa species are converted to a 180-kDa species. The 125-kDa alpha'- and 83-kDa beta'-species are also cleaved by endoglycosidase H, being reduced in size to 97 and 79 kDa, respectively. Based on their sensitivity to endoglycosidase H and insensitivity to neuraminidase, the oligosaccharide chains of the receptor precursors (190, 210, 125, and 83 kDa) do not contain terminal sialic acid (or other capping sugars). However, near the time of insertion into the plasma membrane, capping of the alpha'- and beta'-species by sialic acid occurs, giving rise to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits, which are partially endoglycosidase H-resistant and neuraminidase-sensitive. When 3T3-L1 adipocytes are treated with tunicamycin, a 180-kDa proreceptor aglycopolypeptide is synthesized which is incapable of undergoing further processing and proteolytic cleavage to the alpha- and beta (or alpha'- and beta'-)-subunits. The 180-kDa species, which appears to be the aglyco-form of hte 190-kDa proreceptor generated by endoglycosidase H, is resistant to trypsin in the intact cell and apparently has not reached the cell surface. Thus, the oligosaccharide moieties of the insulin receptor precursor are crucial for proper processing, intracellular translocation, and formation of functionally competent insulin re     

Ontogeny of microbodies (glyoxysomes) in cotyledons of dark-grown watermelon (Citrullus vulgaris Schrad.) seedlings

The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.Abbreviations CAT catalase - DAB 3,3 diaminobenzidine tetrahydrochloride - ER endoplasmic reticulum - GOX glycolate oxidase - HPR hydroxypyruvate reductase - HVEM high-voltage electron microscopy - ICL isocitrate lyase - MS malate synthase - RER rough endoplasmic reticulum In the figures bars represent 0.1 m (if not stated otherwise)     

NADPH is a specific inhibitor of protein import into glyoxysomes

We have studied the import of proteins into glyoxysomes in vitro and show that this process is specifically inhibited by NADPH. NADPH affects both binding and translocation of proteins into glyoxysomes, and inhibition is determined by the ratio of NADP⁺ to NADPH. The site of action of NADPH is most likely within the glyoxysome because (1) pretreatment of glyoxysomes with NADPH, followed by re-isolation of the organelles prior to the import assay, resulted in inhibition of import that could be restored by the addition of NADP⁺; (2) low concentrations of NADPH inhibited binding of proteins to broken glyoxysome membranes. The sensitivity of protein import to inhibition by NADPH declines as glyoxysomes are converted to leaf-type peroxisomes. A model is proposed that speculates on a possible role for NADPH in regulating protein import into plant peroxisomes.     

Apparent Catalase Synthesis in Sunflower Cotyledons during the Change in Microbody Function: A Mathematical Approach for the Quantitative Evaluation of Density-labeling Data

Density-labeling with 10 mm K¹⁵NO₃/70% ²H₂O has been used to investigate catalase synthesis in different developmental stages of sunflower (Helianthus annuus L.) cotyledons. A mathematical approach is introduced for the quantitative evaluation of the density-labeling data. The method allows, in the presence of preexisting enzyme activity, calculation of this synthesized activity (apparent enzyme synthesis) which results from the balance between actual enzyme synthesis and the degradation of newly synthesized enzyme at a given time. During greening of the cotyledons, when the catalase activity declines and the population of leaf peroxisomes is formed, the apparent catalase synthesis is lower than, or at best equal to, that occurring during a developmental stage when the leaf peroxisome population is established and catalase synthesis and degradation of total catalase are in equilibrium. This result suggests a formation, in fatty cotyledons, of the leaf peroxisomes by transformation of the glyoxysomes rather than by de novo synthesis.