A Novel Ribonuclease with HIV-1 Reverse Transcriptase Inhibitory Activity Purified from the Field Blewit Mushroom Lepista personata (Agaricomycetes).

A ribonuclease was purified from dry fruiting bodies of the wild edible mushroom Lepista personata (LPR) to 259-fold with a specific activity of 280 U/mg. The purification protocol involved ion-exchange chromatography on DEAE-cellulose, CM-cellulose and SP-sepharose, followed by size exclusion chromatography on Superdex 75. LPR is a homodimeric protein with a molecular weight of 27.8 kDa as determined by SDS-PAGE and by gel filtration. Three inner peptide sequences for LPR were obtained by LC-MS-MS analysis. It demonstrated the optimum pH of 4.0 and temperature optimum of 60°C. The specificity ribonuclease potencies order toward polyhomoribonucleotides was poly C > poly A > poly G > poly U. The ribonuclease inhibited HIV-1 reverse transcriptase with an IC50 of 0.53 µM.

Ageritin from poplar mushrooms: scale-up purification and cytotoxicity towards undifferentiated and differentiated SH-SY5Y cells.

Ageritin is the first reported ribotoxin-like protein from basidiomycetes fungi. It can induce ribosomal integrity damage and translation block, and interferes with mitochondrial redox activity of some glioma and neuroblastoma cell lines. Herein, Ageritin has been investigated as a valuable neurotoxin towards either undifferentiated or retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells showing a selective cell toxicity against undifferentiated cells. MTT and sulforhodamine B (SRB) assays highlighted that Ageritin markedly decreases the mitochondrial redox activity and viability of undifferentiated cells, meanwhile inducing evident morphological changes eliciting neuronal-like appearance in these cells. Data from lactate dehydrogenase release assay, cytofluorimetric analysis and caspase-3 enzymatic activity measurement suggest that Ageritin promotes cell death through a caspase-dependent apoptotic pathway. The Z-VAD-FMK caspase inhibitor was able to prevent this apoptotic pathway activation. Based on the interesting behaviour of Ageritin vs. SH-SY5Y cells, the development of a scale-up procedure to obtain the purified protein in larger amounts (yield 2.5 mg per 100 g) has been optimized.

Purification and characterization of two pathogenesis-related class 10 protein isoforms with ribonuclease activity from the fresh Angelica sinensis roots.

In this study, two pathogenesis-related (PR) class 10 protein isoforms, ASPR-1 and ASPR-2, were purified from fresh roots of the Chinese medicinal plant Angelica sinensis (A. sinensis) using 80% ammonium sulfate precipitation, Sephadex G50 gel filtration chromatography, and DEAE-Sepharose ion-exchange chromatography. The molecular masses of ASPR-1 and ASPR-2 were estimated to be 16.66 kDa and 16.46 kDa, respectively, using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isoforms are both glycoproteins containing glycosyl contents of 1.8% (ASPR-1) and 3.4% (ASPR-2). The two isoforms were predominantly present as monomers, but they partially dimerized in solution. The 15 N-terminal amino acids of ASPR-1 were determined to be GIQKTEVEAPSTVSA, with significant sequence homology to certain PR-10 proteins. ASPR-2 was also identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis to be a PR-10 protein. The isoforms both exhibited ribonuclease (RNase) activity, with ASPR-2 having higher specific activity (128.85 U mg-1) than ASPR-1 (68.67 U mg-1). The isoforms had the same optimal temperature of 50 °C but different optimal pH values of 5.0 (ASPR-1) and 6.0 (ASPR-2). The RNase activities of the isoforms were both stable for 30 min at 50 °C, rapidly decreasing at higher or lower processing temperatures. However, ASPR-1 retained higher residual activity (89.4%-80.9%) than ASPR-2 (74.3%-67.9%) at temperatures from 40 °C to 60 °C. These results provide additional information to enrich the current knowledge of poorly annotated A. sinensis proteins.

Defense-related Proteins from Chelidonium majus L. as Important Components of its Latex.

The aim of this review is to cover most recent research on plant pathogenesis- and defenserelated proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesisrelated (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores.

Isolation and Characterization of a Ubiquitin-Like Ribonuclease from the Cultured Deep Root Mushroom, Oudemansiella radicata (Higher Basidiomycetes).

The isolation of a novel 13.5-kDa ribonuclease, displaying a ubiquitin-like inner peptide sequence, from dried fruiting bodies of the cultured mushroom Oudemansiella radicata (Relhan: Fr.) Singer (=Xerula radicata) is reported. The purification protocol deployed encompassed sequentially, cation/anion exchange chromatography on CM-cellulose, DEAE-cellulose and SP-Sepharose, and FPLC-gel filtration on a Superdex 75 column. The purified enzyme manifested optimum activity at 70 °C and pH 4.6, respectively. The activity of the RNase was inhibited by the majority of metal ions tested, especially Al3+, Hg2+, and Cd2+ ions, but was promoted by K+ ions. It exhibited the highest ribonucleolytic activity toward poly (C), lower activity toward poly (G), and negligible activity toward poly (U) and poly (A). Compared with mushroom ubiquitin-like RNases reported earlier, O. radicata RNase possesses a larger molecular mass, distinctive chromatographic behavior on DEAE-cellulose, a lower optimum pH, and a unique polyhomoribonucleotide specificity.

Isolation of a ribonuclease with antiproliferative and HIV-1 reverse transcriptase inhibitory activities from Japanese large brown buckwheat seeds.

A ribonuclease, with a molecular mass of 22.5 kDa and an N-terminal sequence exhibiting resemblance to previously isolated buckwheat storage proteins and allergens, was isolated from Japanese large brown buckwheat seeds. The ribonuclease was purified using a simple protocol that comprised ion exchange chromatography on Q-Sepharose and DEAE-cellulose and gel filtration on Superdex 75. The ribonuclease exhibited low activity toward poly U, lower activity toward poly C, and very low activity toward poly A and poly G. The enzyme was activated upon exposure to 10 mM of Fe(2+) and Zn(2+) ions but was inhibited by Ca(2+), Mg(2+), and Mn(2+) ions at the same concentration. The optimum pH and optimum temperature for the enzyme were pH 9 and 60 °C, respectively. It inhibited proliferation of HepG2 hepatoma and MCF 7 breast cancer cells, with an IC50 value of 79.2 and 63.8 µM, respectively. It potently inhibited HIV-1 reverse transcriptase activity with an IC50 of 48 µM. However, there were no antifungal and mitogenic activities.

Basic RNases of wild almond (Prunus webbii): cloning and characterization of six new S-RNase and one "non-S RNase" genes.

In order to investigate the S-RNase allele structure of a Prunus webbii population from the Montenegrin region of the Balkans, we analyzed 10 Prunus webbii accessions. We detected 10 different S-RNase allelic variants and obtained the nucleotide sequences for six S-RNases. The BLAST analysis showed that these six sequences were new Prunus webbii S-RNase alleles. It also revealed that one of sequenced alleles, S(9)-RNase, coded for an amino acid sequence identical to that for Prunus dulcis S(14)-RNase, except for a single conservative amino acid replacement in the signal peptide region. Another, S(3)-RNase, was shown to differ by only three amino acid residues from Prunus salicina Se-RNase. The allele S(7)-RNase was found to be inactive by stylar protein isoelectric focusing followed by RNase-specific staining, but the reason for the inactivity was not at the coding sequence level. Further, in five of the 10 analyzed accessions, we detected the presence of one active basic RNase (marked PW(1)) that did not amplify with S-RNase-specific DNA primers. However, it was amplified with primers designed from the PA1 RNase nucleotide sequence (basic "non-S RNase" of Prunus avium) and the obtained sequence showed high homology (80%) with the PA1 allele. Although homologs of PA1 "non-S RNases" have been reported in four other Prunus species, this is the first recorded homolog in Prunus webbii. The evolutionary implications of the data are discussed.

Isolation of a ribonuclease from sanchi ginseng (Panax pseudoginseng) flowers distinct from other ginseng ribonucleases.

A single-chained ribonuclease was isolated from the aqueous extract of sanchi ginseng (Panax pseudoginseng) flowers. It exhibited a molecular mass of 23 kDa, an N-terminal sequence with some similarity to other enzymes involved in RNA metabolism but different from known ribonucleases, and considerably higher activity toward poly U than poly C and only slight activity toward poly A and poly G. The purification protocol entailed ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on carboxymethyl (CM)-cellulose, and gel filtration on Superdex 75. The ribonuclease was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-cellulose. Maximal activity of the ribonuclease was attained at pH 7. On either side of this pH the enzyme activity underwent a drastic decline. The enzyme activity was at its highest at 50 degrees C and dropped to about 20% of the maximal activity when the temperature was decreased to 20 degrees C or elevated to 80 degrees C. The characteristics of sanchi ginseng flower ribonuclease were different from those of the ribonucleases previously purified from sanchi ginseng and Chinese ginseng roots including ribonuclease from Chinese ginseng flowers which are morphologically very similar to sanchi ginseng flowers.

A ribonuclease from Chinese ginseng (Panax ginseng) flowers.

A ribonuclease, with a molecular mass of 23kDa, and much higher activity toward poly(U) than poly(C) and only negligible activity toward poly(A) and poly(G), was isolated from the aqueous extract of Chinese ginseng (Panax ginseng) flowers. The ribonuclease was unadsorbed on diethylaminoethyl-cellulose and adsorbed on Affi-gel blue gel and carboxymethyl-cellulose. High activity of the ribonuclease was maintained at pH 6-7. On either side of this pH range, there was a precipitous drop in enzyme activity. The activity of the enzyme peaked at 50 degrees C and fell to about 20% of the maximal activity when the temperature was lowered to 20 degrees C or raised to 80 degrees C. The characteristics of this ribonuclease were different from those of ribonuclease previously purified from ginseng roots.

Isolation and partial characterization of catalytic antibodies with oligonuclease activity from bovine colostrum.

Catalytic antibodies (abzymes) which hydrolyze RNA and DNA were isolated from bovine colostrum by sequential chromatography on Protein A Sepharose, denaturated DNA-cellulose, Mono Q, and gel permeation chromatography on Superose 12 at pH 2.3 after acidic shock. Metachromatic agar containing toluidine blue and yeast RNA was used to measure RNase activity. Electrophoresis in agarose showed DNase activity on plasmid DNA from Escherichia coli and DNA from calf thymus in fractions from all 4 purification steps. Gel permeation chromatography showed that the abzymes hydrolysed both a single-stranded polyadenylic acid (Poly A) and single-stranded polycitidylic acid (Poly C), while partially purified RNase from the colostrum hydrolysed Poly (C), but not Poly (A). Electrophoresis of purified abzymes under denaturing conditions showed protein bands of molecular mass corresponding to heavy and light chains of IgG. The abzymes immunoreacted with anti-bovine IgG. The RNase activity of the purified abzymes represented 0.022% of total RNase activity in the colostrum; acid shock and gel filtration at low pH reduced the specific RNase activity of abzymes 3.6-fold. The RNase activity of abzymes at pH 6.6 was reduced by 90% by heat treatment at 75 degrees C for 52 min.

Isolation of a novel thermolabile heterodimeric ribonuclease with antifungal and antiproliferative activities from roots of the sanchi ginseng Panax notoginseng.

An isolation procedure, consisting of ion exchange chromatography on CM-Sepharose, affinity chromatography on Affi-gel blue gel, and fast protein liquid chromatography on Mono S, was utilized to purify a base-nonspecific, heterodimeric ribonuclease (RNase) with diverse activities from roots of the sanchi ginseng Panax notoginseng. The RNase is unique in that it consists of two different nonglycoprotein subunits with a molecular weight of 27 and 29 kDa, respectively. The latter subunit is characterized by an N-terminal sequence showing remarkable similarity to that of the bitter gourd RNase. The Panax notoginseng RNase demonstrates potent RNase and translation-inhibitory activities. In addition, it exhibits antiproliferative activity toward leukemia L1210 cells and antifungal activity against Physalospora piricola and Coprinus comatus. Its RNase activity is not heat-resistant, unlike most RNases which are thermostable.

Lagenin, a novel ribosome-inactivating protein with ribonucleolytic activity from bottle gourd (Lagenaria siceraria) seeds.

The seeds of Lagenaria siceraria (Family Cucurbitaceae) were extracted with water and the extract was lyophilized. The lyophilized extract was chromatographed on a DEAE-cellulose column in 10 mM Tris-HCl buffer (pH 7.2). The unadsorbed fraction was applied to an Affi-gel Blue gel column previously equilibrated with the same buffer. After removal of unadsorbed materials, the adsorbed proteins were eluted with 1.5 M NaCl in the Tris-HCl buffer. After dialysis the adsorbed fraction was loaded on a CM-Sepharose CL-6B column which had been equilibrated with and was eluted with the same buffer. After elution of unadsorbed proteins, the column was eluted with a gradient of 0-1 M NaCl in 10 mM Tris-HCl buffer (pH 7.2). The fraction eluting at about 0.55 M NaCl, which represented pure ribosome inactivating protein (RIP), inhibited cell-free translation in a rabbit reticulocyte system with an IC50 of 0.21 nM and exerted ribonuclease activity on yeast tRNA with an activity of 45 U/mg. The RIP was designated lagenin. It possessed a molecular weight of 20 kDa, smaller than the range of 26-32 kDa reported for other RIPs. The N-terminal sequence of lagenin exhibited a lesser extent of similarity to those of other Cucurbitaceae RIPs, characterized by a deletion of the first three amino acid residues and a replacement of the 4th (Phe), 17th (Phe), 18th (Ile) and 22nd (Arg) residues which are invariant in other RIPs.

Quinqueginsin, a novel protein with anti-human immunodeficiency virus, antifungal, ribonuclease and cell-free translation-inhibitory activities from American ginseng roots.

A homodimeric protein designated quinqueginsin, with a molecular weight of 53 kDa, has been isolated from the roots of American ginseng Panax quinquefolium. It was unadsorbed on DEAE cellulose in low ionic strength and neutral pH, and adsorbed on Affigel blue gel and SP-Sepharose under similar conditions. Its N-terminal sequence bore similarity to those of plant ribosome inactivating proteins and fungal ribonucleases. The protein displayed a variety of biological activities. It possessed ribonucleolytic activity toward yeast tRNA and specific activity toward poly C. It inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) of 0.26 nM, and exerted antifungal action against Fusarium oxysporum, Rhizoctonia solani, and Coprinus comatus. An inhibitory action was expressed toward human immunodeficiency virus-1 reverse transcriptase. This action was potentiated after chemical modification with succinic anhydride.

Polyadenylation accelerates the degradation of the mitochondrial mRNA associated with cytoplasmic male sterility in sunflower.

In sunflower, PET1-cytoplasmic male sterility is correlated with the presence of a novel mitochondrial gene (orf522) located 3' to the atpA gene. The dicistronic atpA-orf522 transcripts are preferentially destabilized in male florets of 'restored to fertility' plants as compared with sterile plants. In this report, we show that atpA-orf522 transcripts may be polyadenylated in vivo at their 3' termini and that a tissue-specific increase in the level of polyadenylated atpA-orf522 transcripts correlates with the tissue-specific instability of atpA-orf522 mRNAs in male florets of the restored hybrid plants. In addition, we have identified two distinct ribonuclease activities in sunflower mitochondria, one of which preferentially degrades polyadenylated as compared with non-polyadenylated RNA substrates corresponding to the 3' UTR of atpA-orf522 transcripts. These in vivo and in vitro results show that polyadenylation is involved in the degradation pathway of the mitochondrial atpA-orf522 transcripts and that polyadenylation can be developmentally regulated by a nuclear gene(s) upon restoration of fertility.

Crystallization and preliminary diffraction data of a major pollen allergen. Crystal growth separates a low molecular weight form with elevated biological activity.

Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients. In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein. The allergen was expressed in Escherichia coli and subsequently purified. Spontaneous conversion of these preparations to a mixture of various forms with molecular sizes between 10 and 29 kDa was consistently observed. Surprisingly, crystals could be grown from this heterogenous preparation. Single crystals, redissolved and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot, yielded one distinct low molecular weight protein, which was identified by amino acid sequencing as the C-terminal 13-kDa portion of the allergen. Histamine release assays with single crystal solutions using basophils of an allergic patient demonstrated allergenicity comparable with that of the holo-allergen. By contrast, RNase activity of the crystallized C-terminal form was 23 times higher than that of the full-length parent allergen. Crystals were used to collect preliminary diffraction data; the space group was evaluated to I4122 with cell dimensions of a = 87.7 A, b = 87.7 A, and c = 59.6 A. We conclude that preferential crystal growth of the 13-kDa form is indicative of a compact conformation of this particular C-terminal portion of the allergen. Thus, we show here that protein crystallization is not only a prerequisite for structural analyses, but it also can provide a unique separation technique to localize the functional domain of a major allergen.

Self-incompatibility (S) alleles of the Rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily.

Stylar ribonucleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus x domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.

The major birch pollen allergen, Bet v 1, shows ribonuclease activity.

The major birch (Betula alba L.) pollen allergen, Bet v 1, has been shown to be homologous to pathogenesis-related proteins in a number of plants. Recently, it was demonstrated that a ginseng protein with high homology to an intracellular pathogenesis-related protein of parsley and to Bet v 1 is a ribonuclease (RNase). Birch pollen extract was separated in an RNase activity gel. Four major RNase bands were excised from the gel, reseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting with a specific Bet v 1 monoclonal antibody and patient's serum. Thus the monomer and the dimer of Bet v 1 showed RNase activity. Purified recombinant Bet v 1 was shown to degrade plant RNA. The RNase activity of recombinant Bet v 1 was 180 units.mg-1.

Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self-pollen.

S proteins, pistil-specific ribonucleases that cosegregate with S alleles, have previously been shown to control rejection of self-pollen in Petunia inflata and Nicotiana alata, two solanaceous species that display gametophytic self-incompatibility. The ribonuclease activity of S proteins was thought to degrade RNA of self-pollen tubes, resulting in the arrest of their growth in the style. However, to date no direct evidence has been obtained. Here, the ribonuclease activity of S3 protein of P. inflata was abolished, and the effect on the pistil's ability to reject S3 pollen was examined. The S3 gene was mutagenized by replacing the codon for His-93, which has been implicated in ribonuclease activity, with a codon for asparagine, and the mutant S3 gene was introduced into P. inflata plants of S1S2 genotype. Two transgenic plants produced a level of mutant S3 protein comparable to that of the S3 protein produced in self-incompatible S1S3 and S2S3 plants, yet they failed to reject S3 pollen. The mutant S3 protein produced in these two transgenic plants did not exhibit any detectable ribonuclease activity. We have previously shown that transgenic plants (S1S2 plants transformed with the wild-type S3 gene) producing a normal level of wild-type S3 protein acquired the ability to reject S3 pollen completely. Thus, the results reported here provide direct evidence that the biochemical mechanism of gametophytic self-incompatibility in P. inflata involves the ribonuclease activity of S proteins.

Expression, purification, and crystallization of natural and selenomethionyl recombinant ribonuclease H from Escherichia coli.

Ribonuclease H (RNase H) from Escherichia coli is an endonuclease that specifically degrades the RNAs of RNA:DNA hybrids. The enzyme is a single polypeptide chain of 155 amino acid residues, of which 4 are methionines. To solve the crystallographic three-dimensional structure of E. coli RNase H by the multi-wavelength anomalous diffraction technique, we have constructed methionine auxotrophic strains of E. coli that overexpress selenomethionyl RNase H. MIC88 yields about 10 mg of selenomethionyl RNase H per liter of culture, which is comparable to the overexpression of the natural recombinant protein. We have purified both proteins to homogeneity and crystallized them isomorphously in the presence of sulfate. These are Type I crystals of space group P2(1)2(1)2(1) with the cell parameters a = 41.8 A, b = 86.4 A, c = 36.4 A, one monomer per asymmetric unit, and approximately 36% (v/v) solvent. Crystals of both proteins diffract to beyond 2-A Bragg spacings and are relatively durable in an x-ray beam. On replacement of sulfate with NaCl, crystals of natural RNase H grow as Type I' (very similar to Type I) at pH between 7.0 and 8.0; at pH 8.8, crystals of Type II are obtained in space group P2(1)2(1)2(1) with a = 44.3 A, b = 87.3 A, and c = 35.7 A. Type II crystals can be converted to Type I by soaking in phosphate buffer. RNase H crystals of Type II have also been reported by Kanaya et al. (Kanaya, S., Kohara, A., Miyakawa, M., Matsuzaki, T., Morikawa, K., and Ikehara, M. (1989) J. Biol. Chem. 264, 11546-11549).

Biochemical properties and hormonal regulation of barley nuclease.

The amino acid composition and NH2-terminal amino acid sequence of barley nuclease (EC 3.1.30.2) were determined. The amino acid composition is similar to that of mung bean nuclease, and therefore the biochemical properties of barley nuclease were characterized and compared with those of mung bean and other plant nucleases. The 3'-nucleotidase activity of barley nuclease is greater for purine than for pyrimidine ribonucleotides. The enzyme has little activity towards ribonucleoside 2' and 5'-monophosphates, and deoxyribonucleoside 3' and 5'-monophosphates, and is also inactive towards the 3'-phosphoester linkage of nucleoside cyclic 2',3' and 3',5'-monophosphates. The enzyme hydrolyzes dinucleoside monophosphates, showing strong preference for purine nucleosides as the 5' residues. Barley nuclease shows significant base preference for homoribonucleic acids, catalyzing the hydrolysis of polycytidylic acid greater than polyuridylic acid greater than polyadenylic acid much greater than polyguanylic acid. The enzyme also has preference for single-stranded nucleic acids. Hydrolysis of nucleic acids is primarily endonucleolytic, whereas the products of digestion possess 5'-phosphomonoester groups. Nuclease activity is inhibited by ethylenediaminetetraacetic acid and zinc is required for reactivation. Secretion of nuclease from barley aleurone layers is dependent on the hormone gibberellic acid [Brown, P.H. and Ho, T.-h. D. (1986) Plant Physiol. 82, 801-806]. Consistent with these results, gibberellic acid induces up to an eight-fold increase in the de novo synthesis of nuclease in aleurone layers. The secreted enzyme is a glycoprotein having an apparent molecular mass of 35 kDa. It consists of a single polypeptide having an asparagine-linked, high-mannose oligosaccharide. The protein portion of the molecule has a molecular mass of 33 kDa.