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The gaseous plant hormone ethylene modulates a wide range of biological processes, including fruit ripening. It is synthesized by the ascorbate-dependent oxidation of 1-aminocyclopropyl-1-carboxylate (ACC), a reaction catalyzed by ACC oxidase. Recombinant avocado (Persea americana) ACC oxidase was expressed in Escherichia coli and purified in milligram quantities, resulting in high levels of ACC oxidase protein and enzyme activity. An optimized assay for the purified enzyme was developed that takes into account the inherent complexities of the assay system. Fe(II) and ascorbic acid form a binary complex that is not the true substrate for the reaction and enhances the degree of ascorbic acid substrate inhibition. The K(d) value for Fe(II) (40 nM, free species) and the K(m)'s for ascorbic acid (2.1 mM), ACC (62 microM), and O(2) (4 microM) were determined. Fe(II) and ACC exhibit substrate inhibition, and a second metal binding site is suggested. Initial velocity measurements and inhibitor studies were used to resolve the kinetic mechanism through the final substrate binding step. Fe(II) binding is followed by either ascorbate or ACC binding, with ascorbate being preferred. This is followed by the ordered addition of molecular oxygen and the last substrate, leading to the formation of the catalytically competent complex. Both Fe(II) and O(2) are in thermodynamic equilibrium with their enzyme forms. The binding of a second molecule of ascorbic acid or ACC leads to significant substrate inhibition. ACC and ascorbate analogues were used to confirm the kinetic mechanism and to identify important determinants of substrate binding.

The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). In addition to ACC, Fe(II), O2, CO2, and ascorbate are required for in vitro enzyme activity. Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme.(substrate or cofactor).NO complexes. These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O2 complexes. It is shown here that the ternary Fe(II)ACCO.ACC.NO complex is readily formed, but no Fe(II)ACCO.ascorbate.NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site. The binding modes of ACC and the structural analog alanine specifically labeled with 15N or 17O were examined by using Q-band electron nuclear double resonance (ENDOR). The data indicate that these molecules bind directly to the iron through both the alpha-amino and alpha-carboxylate groups. These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O2 bind to the iron as a step in O2 activation. We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O2, thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis.

During ripening of avocado (Persea americana), the CYP71A1 mRNA and protein accumulate to relatively high levels. Although the CYP71A1 gene was the first plant P450 to be cloned and sequenced, the functional role of this P450 remains obscure. Substrate studies have shown that CYP71A1 will metabolize various monoterpenes (nerol and geraniol), although these have not been detected in ripening fruit. Using DNA from a conserved domain of the CYP71A1 gene, we have explored the scope of the CYP71 (or related) gene family in avocado using low stringency DNA hybridization. This analysis suggests that there are approximately 10-12 genes in the CYP71 family. An alternative approach using PCR gave essentially identical results.

A cytochrome P-450 present in ripening avocado (Persea americana) fruit mesocarp (CYP71A1) had previously been shown to metabolize the monoterpenoids nerol and geraniol (Hallahan et al. (1992) Plant Physiol. 98, 1290-1297). Using DNA encoding CYP71A1 as a hybridization probe, we have shown by Southern analysis that a related gene is present in the catmint, Nepeta racemosa. RNA blot analysis, together with Western analysis of catmint leaf polypeptides using avocado cyt P-450 antiserum, showed that a closely related gene is expressed in catmint leaves. Cytochrome P-450 in catmint microsomes catalysed the specific hydroxylation of nerol and geraniol at C-10, whereas avocado CYP71A1, in either avocado microsomes or heterologously expressed in yeast, catalysed 2,3- or 6,7-epoxidation of these substrates. These results suggest that orthologous genes of the CYP71 family are expressed in these two plant species, but catalyse dissimilar reactions with monoterpenoid substrates.

One of the mRNAs that accumulates during the ripening of avocado (Persea americana Mill. cv Hass) has been previously identified as a cytochrome P450 (P450) monooxygenase and the corresponding gene designated CYP71A1. In this report we demonstrate that during ripening the accumulation of antigenically detected CYP71A1 gene product (CYP71A1) correlates with increases in total P450 and two P450-dependent enzyme activities: para-chloro-N-methylaniline demethylase, and trans-cinnamic acid hydroxylase (tCAH). To determine whether both of these activities are derived from CYP71A1, we have expressed this protein in yeast (Saccharomyces cerevisiae) using a galactose-inducible yeast promoter. Following induction, the microsomal fraction of transformed yeast cells undergoes a large increase in P450 level, attributable almost exclusively to the plant CYP71A1 protein. These membranes exhibit NADPH-dependent para-chloro-N-methylaniline demethylase activity at a rate comparable to that in avocado microsomes but have no detectable tCAH. These results demonstrate both that the CYP71A1 protein is not a tCAH and that a plant P450 is fully functional upon heterologous expression in yeast. These findings also indicate that the heterologous P450 protein can interact with the yeast NADPH:P450 reductase to produce a functional complex.

Biosynthesis of the phytohormone ethylene in higher plants proceeds via the following pathway: S-adenosylmethionine----1-aminocyclopropane-1-carboxylic acid (ACC)----ethylene. Ethylene-forming enzyme (EFE), the enzyme responsible for the oxidation of ACC to ethylene, has been only partially characterized in vitro. We have obtained authentic EFE activity in vitro from extracts of avocado fruit (Persea americana Mill. cv Hass). Ammonium sulfate fractionation revealed the presence of two EFE activities, which we designate as EFE1 and EFE2. EFE1 activity utilizes ACC and O2 as substrates and requires Fe(II) and ascorbate as cofactors. The enzyme has a relatively low Km (32 microM) for ACC, discriminates diastereomers of 1-amino-2-ethyl-cyclopropane-1-carboxylic acid, and is inhibited competitively by 2-aminoisobutyric acid, thus confirming its identity with authentic EFE. Activity is retained in a 100,000 x g supernatant and has a pH optimum of 7.5-8.0, suggesting a cytosolic localization.

Fruit ripening involves a series of changes in gene expression regulated by the phytohormone ethylene. AVOe3, a ripening-related gene in avocado fruit (Persea americana Mill. cv Hass), was characterized with regard to its ethylene-regulated expression. The AVOe3 mRNA and immunopositive protein were induced in mature fruit within 12 hours of propylene treatment. The AVOe3 mRNA levels reached a maximum 1 to 2 days before the ethylene climacteric, whereas the immunopositive protein continued to accumulate. RNA selected by the pAVOe3 cDNA clone encoded a polypeptide with molecular mass of 34 kilodaltons, corresponding to the molecular mass of the AVOe3 protein determined by immunoblots. The protein was soluble, remaining in solution at 100,000 gravity and eluted as a monomer on gel filtration. Because of its pattern of induction and relationship to an ethylene-related gene of tomato, the possible involvement of AVOe3 in ethylene biosynthesis is discussed.

Mesocarp protoplasts were isolated from mature avocado fruits (Persea americana cv. Hass) at varying stages of propylene-induced ripening. Qualitative changes in the pattern of radiolabel incorporation into polypeptides were observed in cells derived from fruit at the different stages. Many of these differences correlate with those observed during radiolabeling of polypeptides from fresh tissue slices prepared from unripe and ripe fruit. Protoplasts isolated from fruit treated with propylene for one day or more were shown to synthesize cellulase (endo-ß-1,4-glucanase) antigen, similar to the intact propylene-treated fruit. These results suggest that the isolated protoplasts retain at least some biochemical characteristics of the parent tissue. The cells may also be used in transient gene expression assays. Protoplasts isolated from preclimacteric and climacteric fruit were equally competent in expressing a chimeric test gene, composed of the CaMV 35S RNA promoter fused to the bacterial chloramphenicol acetyltransferase gene, which was introduced by electroporation.

We present in this paper the structural analysis of two members of a small cellulase gene family, designated cel1 and cel2, from avocado. These genes were isolated by screening a lambda EMBL3 genomic library with a ripening-induced cellulase cDNA. Restriction endonuclease and Southern blot analyses showed that the cel1 gene is highly homologous to the cellulase cDNA and thus represents a ripening-related cellulase gene. The other cellulase gene, cel2, is closely related to cel1, but is divergent at its 5' end. The nucleotide sequence of a 5 kb region encompassing the cel1 gene was determined. Four previously characterized cellulase cDNAs from ripe fruit are identical to the eight exons of the cel1 gene. RNase protection and primer extension analyses were used to define the transcription start site of cel1 and to quantitate cel1 transcripts in ripening fruit. The cel1 mRNA was present at a low level in unripe fruit and increased 37-fold during ripening. Partial DNA sequence analysis of cel2 and comparison to the cel1 sequence revealed a high degree of similarity both at the DNA and deduced amino acid sequence levels. No characterized cellulase cDNAs derived from ripe fruit represent cel2 transcripts. These data suggest that the cel1 gene is responsible for a major portion, if not all, of the cellulase transcripts in ripe fruit. The DNA sequence of 1.4 kb of 5' flanking DNA of the cel1 gene was compared to the upstream sequence of other ethylene-regulated genes. Several interesting upstream sequence motifs were identified and are discussed.

Protein molecular weight standards were biotinylated by reaction with biotinyl-N-hydroxysuccinimide ester. The biotinylated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose paper. The resolved protein bands were detected by formation of a streptavidin-biotin/horseradish peroxidase complex and reaction with 4-chloro-1-naphthol and hydrogen peroxide. The biotinylated proteins are easy to prepare and are useful as molecular weight standards with most procedures employing immunodetection of proteins following transfer to nitrocellulose paper.

The poly(A) (+)RNA populations from avocado fruit (Persea americana Mill cv. Hass) at four stages of ripening were isolated by two cycles of oligo-dT-cellulose chromatography and examined by invitro translation, using the rabbit reticulocyte lysate system, followed by two-dimensional gel electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of the resulting translation products. Three mRNAs increased dramatically with the climacteric rise in respiration and ethylene production. The molecular weights of the corresponding translation products from the ripening-related mRNAs are 80,000, 36,000, and 16,500. These results indicate that ripening may be linked to the expression of specific genes.

Hartree-Fock self-consistent field calculations using the ab initio molecular fragment technique have been performed on some phenyloxamic and m-phenylenedioxamic acids, which exhibit markedly different activities in the rat passive cutaneous anaphylaxis (PCA) assay. Attention is focused upon structural features that are most likely to affect the drug-receptor interactions, such as the preferred molecular geometry, the electronic charge distribution, and the nature of the higher occupied (HOMO) and lower unoccupied (LUMO) molecular orbitals. Judging from the regions of high density in HOMOs and LUMOs, the benzene ring would preferably act as an electron acceptor, while the oxamic acid moiety would serve best as an electron donor. Factors affecting the relative PCA activities of oxamic and dioxamic acids are discussed.

The biosynthesis and processing of cellulase from ripening avocado fruit was studied. The mature protein is a glycoprotein, as judged by concanavalin A binding, with a molecular weight of 54,200. Upon complete deglycosylation by treatment with trifluoromethane sulfonic acid the mature protein has a molecular weight of 52,800 whereas the immunoprecipitated in vitro translation product has a molecular weight of 54,000. This result indicates that cellulase is synthesized as a large molecular weight precursor, which presumably possesses a short-lived signal peptide. A membrane-associated and heavily glycosylated form of the protein was also identified. This putative secretory precursor was enzymically active and the carbohydrate side chains were sensitive to endoglycosidase H cleavage. Results of partial endoglycosidase H digestion suggest that this precursor form of the mature glycoprotein possesses two high-mannose oligosaccharide side chains. The oligosaccharide chains of the mature protein were insensitive to endoglycosidase H cleavage, indicating that transport of the membrane-associated cellulase to the cell wall was accompanied by modification of the oligosaccharide side chains. The presence of a large pool of endoglycosidase H-sensitive membrane-associated cellulase (relative to an endoglycosidase H-insensitive form) suggest that transit of this protein through the Golgi is rapid relative to transit through the endoplasmic reticulum.

The respiration of carrot (Daucus carota L.) roots is stimulated by ethylene. Polyribosomes were shown to proliferate concomitantly with the increase in respiration, and the extent of polyribosome augmentation was closely correlated to the amount of respiratory stimulation. In addition to the increase in quantity, ethylene caused a 2-fold increase in the average polyribosome size, suggesting tighter packing of ribosomes on RNA. In vitro translation of carrot polyadenylylated RNA with the rabbit reticulocyte lysate system followed by electrophoresis of the resulting translation products showed that ethylene treatment results in the appearance of new mRNAs.

A cDNA library was constructed from poly(A)(+)RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)(+)-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.

A nonlinear regression model has been employed to investigate the activity of a series of 51 drugs in the rat passive cutaneous anaphylaxis assay. Although several classes of molecules are represented in the sample, there are certain common structural features that enable detailed geometric and electronic comparisons to be made. Ab initio Hartree-Fock SCF calculations were performed by using the molecular fragment approach to characterize the electronic structure and preferred conformations of the molecules. The statistical results establish the importance of nine structural factors in determining the potency as inhibitors of histamine release. Both the conformation of a drug and its capacity to act effectively as an electron acceptor in charge-transfer interactions are shown to be critical for high activity.

The respiration of potato tubers (Solanum tuberosum var. Russet Burbank) which have been kept at room temperature for 10 days is stimulated upon subsequent treatment with C(2)H(4) (10 microliters per liter) and O(2). The respiratory rise reaches a peak in 24 to 30 hours and thereafter declines. Coincident with the rise in tuber respiration is an increase in the respiratory rates of fresh slices and isolated mitochondria. Slices and mitochondria from C(2)H(4)- and O(2)-treated tubers also display substantial resistance to CN, and the resistant respiration is inhibited by hydroxamates.The longer the tubers are stored after harvest, the less effective is C(2)H(4) in causing CN resistance in slices and mitochondria from treated tubers. Addition of 10% CO(2) to the C(2)H(4)-O(2) mixture, however, causes extensive CN resistance to develop, even in slices and mitochondria from old tubers. The results show that C(2)H(4), O(2), and CO(2) act synergistically to induce alternative path development in potatoes.

Ab initio calculations on di-, tri-, tetra-, and pentapeptides of glycine in various conformations are reported. Hydrogen bonding is observed as an important stabilizing force in alpha-helical conformations. These studies on polypeptides of glycine of reasonable size show for the first time that the forces acting to stabilize polypeptide conformations can be extracted directly from theoretical studies, without prior postulation of their existence or need for concern that neglect or approximation of various integrals may have biased the results.