Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: Comparison to the barley amylase/subtilisin inhibitor

We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known -amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for -amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to -amylase. PAPI protein is secreted from aleurone layers into the incubation medium.Abbreviations ABA abscisic acid - ASI barley amylase/subtilisin inhibitor - bp nucleotide base pairs - Da dalton - dpa days post anthesis - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor - poly(A)RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Differential synthesis in vitro of barley aleurone and starchy endosperm proteins

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA₃. B and C hordein polypeptides and the salt-soluble proteins β-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the α-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, β-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of α-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for α-amylase, these hormones have the opposite effect on ASI mRNA levels.     

Lipid transfer protein genes specifically expressed in barley leaves and coleoptiles

Genes/cDNAs encoding so-called lipid-transfer proteins (LTPs) have been isolated from a variety of tissues from different plants, but the in-vivo function of the LTP proteins is not yet known. In barley (Hordeum vulgare L.), the LTP1 gene (encoding a probable amylase/ protease inhibitor, Mundy and Rogers 1986, Planta 169, 51–63) is active in aleurone tissue, and in this paper two LTP-encoding cDNAs isolated from green leaves are described. The encoded proteins start with signal sequences, they are 75% homologous to each other, 60–63% homologous to rice aleurone LTP and maize seed/ coleoptile LTP, but only 48% homologous to barley aleurone LTP. Northern hybridization experiments established that the two seedling-specific genes are both highly expressed in leaves and coleoptiles whereas the LTP1 gene is inactive in seedlings. No LTP gene expression was detected in roots using either seedling or aleurone cDNA clones as probes. Tissue-print hybridization indicates that the LTP genes are first expressed in young epidermal cells in leaves and coleoptiles, and subsequently expressed in the vascular strands. Genomic Southern analysis indicates that the barley LTP gene family has four to six members.Abbreviation LTP lipid transfer protein I thank Dr. J. Mundy, Carlsberg Research Laboratory, Copenhagen, Denmark for the PAPI cDNA clone and R. Barkardottir, Department of Molceular Biology, University of Aarhus, Denmark for providing RNA for some of the Northern analyses. I also thank I. Bjørndal and L. Kjeldbjerg for excellent technical assistance. This work was supported by the The Danish Biotechnology Programme.     

Production of a recombinant full-length collagen type I α-1 and of a 45-kDa collagen type I α-1 fragment in barley seeds

Recombinant DNA technology can be used to design and express collagen and gelatin-related proteins with predetermined composition and structure. Barley seed was chosen as a production host for a recombinant full-length collagen type I α1 (rCIa1) and a related 45-kDa rCIa1 fragment. The transgenic barley seeds were shown to accumulate both the rCIa1 and the 45-kDa rCIa1 fragment. Even when the amount of the rCIa1 was just above the detection threshold, this work using rCIa1 as a model demonstrated for the first time that barley seed can be used as a production system for collagen-related structural proteins. The 45-kDa rCI1a fragment expression, targeted to the endoplasmic reticulum, was controlled by three different promoters (a constitutive maize ubiquitin , seed endosperm-specific rice glutelin and germination-specific barley α - amylase fusion) to compare their effects on rCIa1 accumulation. Highest accumulation of the 45-kDa rCIa1 was obtained with the glutelin promoter (140 mg/kg seed), whereas the lowest accumulation was obtained with the α - amylase promoter. To induce homozygosity for stable 45-kDa rCIa1 production in the transgenic lines, doubled haploid (DH) progeny was generated through microspore culture. The 45-kDa rCIa1 expression levels achieved from the best DH lines were 13 mg/kg dry seeds under the ubiquitin promoter and 45 mg/kg dry seeds under the glutelin promoter. Mass spectroscopy and amino acid composition analysis of the purified 45-kDa rCIa1 fragment revealed that a small percent of prolines were hydroxylated with no additional detectable post-translational modifications.     

A cysteine protease from maize isolated in a complex with cystatin

We recently purified a latent but SDS-activated protease complex (40, 15- or 13-kDa proteins) from maize [Yamada et al. (1998) Plant Cell Physiol. 39: 106]. Here, we revealed that the complex was composed of a cysteine protease (40 kDa) and a cystatin, cysteine protease inhibitor (15- or 13-kDa). This is the first report on the isolation of a complex consisting of a cystatin and a target cysteine protease from plants. Cloning of the cysteine protease revealed that it had low homology (25-30%) to other maize cysteine proteases cloned to date but was highly homologous to other plant cysteine proteases such as rice oryzain alpha (84%) and the homologs (50-80%). The cysteine protease expressed in Escherichia coli showed the same substrate and inhibitor specificities as the protease of the complex, demonstrating that the isolated cDNA clone exactly encodes the protease of the complex. The protease expressed in E. coli itself was active but not latent, probably because it was not bound to cystatin. It is most likely that in vitro activation of the protease complex by SDS is caused by the release of bound cystatin. The mRNA of protease was expressed in various tissues except for seeds.     

The rice nucellin gene ortholog OsAsp1 encodes an active aspartic protease without a plant-specific insert and is strongly expressed in early embryo

The barley nucellin gene was reported to be nucellus specific in its expression and was hypothesized to play a role in the programmed cell death of the nucellus as an aspartic protease. Here we provide direct evidence that the rice ortholog encodes an active aspartic protease, but we prefer the name aspartic protease1 (OsAsp1) to nucellin after a detailed analysis of its expression pattern in rice and barley. Northern blots, RT-PCR and RNA in situ hybridization showed that OsAsp1 is expressed most abundantly in zygotic embryos 1-2 d after fertilization. It is also expressed in pollen, nucellus, ovary wall, shoot and root meristem, coleoptiles of immature seeds, and somatic embryos. A parallel study in barley showed that the barley nucellin gene was expressed not only in the nucellus but also strongly in embryos. Recombinant protein proOsAsp1 expressed in the bacterium Escherichia coli refolded and autolysed at acidic pH 3.5 in vitro, and the mature peptide displayed protease activity. Nucellin has three close homologs in rice on chromosomes 11 and 12 and in Arabidopsis on chromosomes 1 and 4. They lack the plant-specific insert that distinguishes the typical plant aspartic protease from aspartic proteases of other organisms. They constitute a new class of aspartic protease that is present in both monocots and dicots but whose function remains to be explored further.     

Isolation and characterization of a novel endogenous inhibitor of the proteasome

A novel endogenous inhibitor of the proteasome (high molecular weight multicatalytic protease) has been isolated and characterized from human erythrocytes. After purification by ion-exchange and sizing chromatography, the inhibitor displayed a native molecular mass of approximately 200 kDa and contained a single subunit of 50 kDa with an isoelectric point of 6.9. Although the inhibitor noncompetitively blocks proteolysis of [methyl-14C]-alpha-casein (Ki = 7.1 x 10(-8) M) and inhibits hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC, it did not affect hydrolysis of other peptide substrates, such as MeOSuc-Phe-Leu-Phe-MNA and Z-Ala-Arg-Arg-MNA. To further characterize the 50-kDa inhibitor, a monoclonal antibody (MI-8) was generated that showed specific binding upon Western blot analysis of both native PAGE and SDS-PAGE. Immunoprecipitation with MI-8 specifically removed inhibitor activity against the proteasome. The 50-kDa inhibitor is distinct from a previously described 40-kDa inhibitor of the proteasome (Murakami, K., & Etlinger, J.D. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7588-7592) on the basis of lack of cross-reactivity with MI-8 and dissimilar peptide digest patterns. It is suggested that these endogenous inhibitors may have a role in ATP/ubiquitin-dependent proteolysis and/or other cellular functions involving this protease.     

Comparison of a major heat-stable microtubule-associated protein in HeLa cells and 190-kDa microtubule-associated protein in bovine adrenal cortex

A heat-stable microtubule-associated protein (MAP) with a molecular weight of 190,000, termed 190-kDa MAP, has been purified from bovine adrenal cortex (Murofushi, H. et al. (1986) J. Cell Biol. 103, 1911-1919). Immunoblotting experiments using an antibody against this MAP revealed that several kinds of culture cells derived from human tissues contain proteins with an apparent molecular weight of 180,000 reacting with the antibody. Indirect immunofluorescence microscopic observation of HeLa cells showed that the immunoreactive protein co-exists with microtubules, indicating that the protein is one of the HeLa MAPs. A heat-stable MAP with a molecular weight of 180,000, termed here HeLa 180-kDa MAP, was purified by the taxol-dependent procedure (Vallee, R.B. (1982) J. Cell Biol. 92, 435-442) and successive co-polymerization with brain tubulin. This protein was the most abundant MAP in HeLa cells, suggesting that the MAP is identical to the major HeLa MAP previously reported by Bulinski and Borisy (Bulinski, J.C. & Borisy, G.G. (1980) J. Biol. Chem. 255, 11570-11576) and Weatherbee et al. [1980) Biochemistry 19, 4116-4123). It was shown that, like bovine adrenal 190-kDa MAP, yet distinct from brain MAP2 and tau, purified HeLa 180-kDa MAP does not interact with actin filaments. This common characteristic of the two MAPs along with the same heat-stability strongly suggests that they are members of the same group of MAPs. The fact that HeLa 180-kDa MAP reacts with an antibody against bovine adrenal 190-kDa MAP means that they share common epitopes, in other words, common local amino acid sequences. However, the limited proteolytic patterns of the two MAPs with S. aureus V8 protease and chymotrypsin were distinct from each other, suggesting the presence of large differences in the overall primary structures between bovine adrenal 190-kDa MAP and HeLa 180-kDa MAP.     

Rice Allergenic Protein and Molecular-genetic Approach for Hypoallergenic Rice

Allergenic proteins with a molecular mass of about 14 to 16 kDa were isolated from a rice salt-soluble fraction based on the reactivity with IgE antibodies from patients allergic to rice. cDNA clones encoding these allergenic proteins were isolated from a cDNA library of maturing rice seeds, and the deduced amino acid sequences showed considerable similarity to wheat and barley α-amylase/trypsin inhibitors, which have recently been identified as major allergens associated with baker’s asthma. An antisense RNA strategy was applied to repress the allergen gene expression in maturing rice seeds. Immunoblotting and ELISA analyses of the seeds using a monoclonal antibody to a 16-kDa allergen showed that allergen content of seeds from several transgenic rice plants was markedly lower than that of the seeds from parental wild type rice.     

Purification and characterization of proteolytic fragments of lipocortin I that inhibit phospholipase A2

Human lipocortin I is a 38.5-kDa phospholipase A2 inhibitor that has been produced in Escherichia coli in large quantities by recombinant DNA technology (Wallner, B.P., Mattaliano, R.J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L.K., Foeller, C., Chow, E.P., Browning, J.L., Ramachandran, K.L., and Pepinsky, R.B. (1986) Nature 320, 77-80). To localize the region within the protein responsible for its inhibitory activity, we generated a series of fragments of the recombinant product by limited proteolysis with elastase and characterized their structure by sequencing and peptide mapping. Five active fragments have been analyzed in detail. The smallest is an 18-kDa fragment derived from the amino-terminal half of lipocortin. Three of the larger fragments contain this region. The fifth fragment is missing 83 amino acids from the amino terminus. A region common to all the active fragments (amino acid residues 97-178) is 70% homologous with the corresponding region from a second member of the lipocortin family which recently was cloned (Huang, K-S., Wallner, B.P., Mattaliano, R.J., Tizard, R., Burne, C., Frey, A., Hession, C., McGray, P., Sinclair, L.K., Chow, E.P., Browning, J.L., Ramachandran, K.L., Tang, J., Smart, J.E., and Pepinsky, R.B. (1986) Cell 46, 191-199) and thus presumably is important for activity. In addition to inhibitory fragments, we have isolated a 3-kDa proteolytic fragment from the amino terminus of lipocortin I that contains the known phosphorylation site for protein-tyrosine kinases. Because of sequence homology of the 3-kDa fragment with biologically active synthetic peptides from pp60v-src and middle T antigen, its release by proteases may represent an important part of the activity of lipocortin.     

Molecular cloning and characterization of a cysteine-rich 16.6-kDa prolamin in rice seeds

An alcohol-soluble storage protein, a 16.6-kDa prolamin found in rice seeds, was purified from both the total protein body and purified type I protein body fractions. The partial amino acid sequences of three tryptic peptides generated from the purified polypeptide were analyzed. A part of the 16.6-kDa prolamin cDNA was amplified from developing seed mRNA by the reverse transcribed polymerase chain reaction using an oligo (dT) primer and a primer which was synthesized based on the partial amino acid sequence. The amplified product was used to isolate the full-length cDNA clone (lambda RP16) from a developing seed cDNA library. The cDNA has an open reading frame encoding a hydrophobic polypeptide of 149 amino acids. The polypeptide was rich in glutamine (20.0%), cysteine (10.0%), and methionine (6.9%). The cysteine content was higher than those of most other rice storage proteins. Messenger RNA of the 16.6-kDa prolamin was detected in seeds, but not in other aerial tissues.     

The amino acid sequence of a cereal Bowman-Birk type trypsin inhibitor from seeds of Jobs' tears (Coix lachryma-jobi L.)

The major trypsin inhibitor from seeds of Jobs' tears (Coix lachryma-jobi) was purified by heat treatment, fractional precipitation with (NH4)2SO4, ion-exchange chromatography on DEAE-Sepharose, gel-filtration on Sephadex G-75 and preparative reverse-phase HPLC. The complete amino acid sequence was determined by analysis of peptides derived from the reduced and S-carboxymethylated protein by digestion with trypsin, chymotrypsin and the S. aureus V8 protease. The polypeptide contained 64 amino acids with a high content of cysteine. The sequence exhibited strong homology with a number of Bowman-Birk inhibitors from legume seeds and similar proteins recently isolated from wheat and rice.     

Purification and amino acid sequence of a bitter gourd inhibitor against an acidic amino acid-specific endopeptidase of Streptomyces griseus.

An inhibitor (BGIA) against an acidic amino acid-specific endopeptidase of Streptomyces griseus (Glu S. griseus protease) was isolated from seeds of the bitter gourd Momordica charantia L., and its amino acid sequence was determined. The molecular weight of BGIA based on the amino acid sequence was calculated to be 7419. BGIA competitively inhibited Glu S. griseus protease with an inhibition constant (Ki) of 70 nM, and gel filtration analyses suggested that BGIA forms a 1:1 complex with this protease. However, two other acidic amino acid-specific endopeptidases, protease V8 from Staphylococcus aureus and Bacillus subtilis proteinase (Glu B. subtilis protease), were not inhibited by BGIA. BGIA had no inhibitory activity against chymotrypsin, trypsin, porcine pancreatic elastase, and papain, although subtilisin Carlsberg was strongly inhibited. The amino acid sequence of BGIA shows similarity to potato chymotrypsin inhibitor, barley subtilisin-chymotrypsin inhibitor CI-1 and CI-2, and leech eglin C, especially around the reactive site. Although the residue at the putative reactive site of these inhibitors is leucine or methionine, the corresponding amino acid in BGIA is alanine.     

Modification of the Bacillus sphaericus 51- and 42-kilodalton mosquitocidal proteins: effects of internal deletions, duplications, and formation of hybrid proteins

The 51- and 42-kDa proteins which constitute the binary mosquitocidal toxin of Bacillus sphaericus 2362 have a low overall sequence similarity but share several regions of near identity (L. Baumann, A. H. Broadwell, and P. Baumann, J. Bacteriol. 170:2045-2050, 1988). By using site-directed mutagenesis, deletions of 6 to 16 amino acids in three of these regions of the 51- and 42-kDa proteins were made, and the modified proteins were expressed in Bacillus subtilis. Deletions in both of these proteins resulted in a loss of toxicity for mosquito larvae. Hybrid proteins containing exchanged fragments of the 51- and 42-kDa proteins were inactive when tested in a variety of combinations, thereby indicating that potentially analogous fragments of these two proteins were not functionally equivalent. An internal duplication of 73 amino acids in the 51-kDa protein and 72 amino acids in the 42-kDa protein resulted in a major reduction in toxicity. These results indicate that the conserved regions of the 51- and 42-kDa proteins are necessary for toxicity to larvae and that the 51- and 42-kDa proteins, despite their sequence similarity, are unique, differing from each other by at least one essential attribute.     

The Processing of a 57-kDa Precursor Peptide to Subunits of Rice Glutelin

The processing of a 57-kDa peptide into 37- and 22-kDa subunitsof glutelin, a major storage protein of rice, was confirmedby the immunological compatibility between the precursor andglutelin subunits. The 57-kDa peptide reacted with the antiseraraised against purified 37-kDa and 22-kDa subunits of glutelin.The processing was further confirmed by alteration of an invivo protein synthesis by monensin, a sodium ionophore whichinhibits the intracellular transport of secretory and membraneproteins. Infusion of monensin into developing rice grains resultedin suppressed formation of mature glutelin subunits with concomitantaccumulation of the 57-kDa peptide. The present results indicatethat both subunits of rice glutelin were produced by post-translationalcleavage of the 57-kDa peptide. (Received July 9, 1986; Accepted October 1, 1986)     

Modification of the Bacillus sphaericus 51- and 42-kilodalton mosquitocidal proteins: effects of internal deletions, duplications, and formation of hybrid proteins.

The 51- and 42-kDa proteins which constitute the binary mosquitocidal toxin of Bacillus sphaericus 2362 have a low overall sequence similarity but share several regions of near identity (L. Baumann, A. H. Broadwell, and P. Baumann, J. Bacteriol. 170:2045-2050, 1988). By using site-directed mutagenesis, deletions of 6 to 16 amino acids in three of these regions of the 51- and 42-kDa proteins were made, and the modified proteins were expressed in Bacillus subtilis. Deletions in both of these proteins resulted in a loss of toxicity for mosquito larvae. Hybrid proteins containing exchanged fragments of the 51- and 42-kDa proteins were inactive when tested in a variety of combinations, thereby indicating that potentially analogous fragments of these two proteins were not functionally equivalent. An internal duplication of 73 amino acids in the 51-kDa protein and 72 amino acids in the 42-kDa protein resulted in a major reduction in toxicity. These results indicate that the conserved regions of the 51- and 42-kDa proteins are necessary for toxicity to larvae and that the 51- and 42-kDa proteins, despite their sequence similarity, are unique, differing from each other by at least one essential attribute.     

Expression and analysis of the human cytomegalovirus UL80-encoded protease: identification of autoproteolytic sites.

The 45-kDa assembly protein of human cytomegalovirus is encoded by the C-terminal portion of the UL80 open reading frame (ORF). For herpes simplex virus, packaging of DNA is accompanied by cleavage of its assembly protein precursor at a site near its C terminus, by a protease encoded by the N-terminal region of the same ORF (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). By analogy with herpes simplex virus, we investigated whether a protease is contained within the N-terminal portion of the human cytomegalovirus UL80 ORF. The entire UL80 ORF was expressed in Escherichia coli, under the control of the phage T7 promoter. UL80 should encode a protein of 85 kDa. Instead, the wild-type construct produces a set of proteins with molecular masses of 50, 30, 16, 13, and 5 kDa. In contrast, when mutant UL80 is deleted of the first 14 amino acids, it produces only an 85-kDa protein. These results suggest that the UL80 polyprotein undergoes autoproteolysis. We demonstrate by deletional analysis and by N-terminal sequencing that the 30-kDa protein is the protease and that it originates from the N terminus of UL80. The UL80 polyprotein is cleaved at the following three sites: (i) at the C terminus of the assembly protein domain, (ii) between the 30- and 50-kDa proteins, and (iii) within the 30-kDa protease itself, which yields the 16- and 13-kDa proteins and may be a mechanism to inactivate the protease.     

The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran [correction of brain] of rice (Oryza sativa L.) seeds.

A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the alpha-amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Cellulose and Toyopearl CM-650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S-alkylated protein, and by manual DABITC/PITC micro-sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, elastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds.     

The 21-kDa protein is a transformation-sensitive metalloproteinase inhibitor of chicken fibroblasts.

We report the electrophoretic purification and characterization of the 21-kDa protein, an extracellular matrix component synthesized during the early stages of transformation of chicken embryo fibroblasts infected with Rous sarcoma virus (Blenis, J., and Hawkes, S. P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 770-774; Blenis, J., and Hawkes, S. P. (1984) J. Biol. Chem. 259, 11563-11570). The NH2-terminal amino acid sequence of the protein is greater than 60% identical to a consensus sequence of mammalian tissue inhibitor of metalloproteinases (TIMP). It shares several biochemical properties with other metalloproteinase inhibitors, including evidence of intrachain disulfide bonds and resistance to cleavage by trypsin. An electrophoretic assay employing a metal ion-dependent gelatinase from conditioned cell culture medium demonstrates inhibitor activity for purified 21-kDa protein. The 21-kDa protein is the major inhibitor in the extracellular matrix and appears unique in solubility properties among inhibitors with a TIMP-like sequence. Statistical analysis of amino acid composition data for these inhibitors defines two distinct groups (TIMP and TIMP-2) and supports a close relationship for the 21-kDa protein with the TIMP group. However, the apparent size and lack of glycosylation align it more closely with the TIMP-2 group of proteins. Therefore, it is possible that the 21-kDa protein is a variant of TIMP or, alternatively, represents a third protein within the metalloproteinase inhibitor family. This report provides the first evidence that avian metalloproteinase inhibitors are similar in sequence to their mammalian counterparts.     

Primary Structure of a Cysteine Proteinase Inhibitor from the Fruit of Avocado (Persea americana Mill)

The complete amino acid sequence of a proteinaceous cysteine proteinase inhibitor from the fruit of avocado (avocado cystatin) is presented. The protein consists of 100 amino acid residues and has a molecular mass of 11,300 Da. Comparison of this sequence with sequences of plant cysteine proteinase inhibitors (phytocystatins), including oryzacystatins I and II from rice seeds, cowpea cystatin, and corn cystatin, showed that the avocado cystatin molecule has 60% and 54% residues identical with the two forms of the rice seed proteins, oryzacystatins I and II, respectively, and 64% and 63% with the cowpea and corn proteins, respectively. The totally conserved sequence, Gln-Val-Val-Ala-Gly, among several of the animal cystatins as well as phytocystatins, is at positions 47-51 in the avocado cystatin molecule.