Storage and mobilization as antagonistic functional constraints on seed storage globulin evolution.

When seeds germinate nearly all the proteins are degraded in senescing storage tissue cells. All these proteins act as amino acid reserves which are mobilized to nourish the seedling. Nevertheless, the major amount of the seeds' protein reserve consists of a few enzymatically inactive, abundant, genuine storage proteins. In their metabolism the conflicting processes of biosynthesis, protein turnover and breakdown, are temporally separated. No degradation of correctly formed storage proteins was observed at the time of synthesis and accumulation during seed maturation. Breakdown takes place after a (long) period of rest when seeds germinate and seedlings start growing. At that time genuine storage proteins are no longer synthesized. Genuine storage proteins have evolved structural features permitting controlled temporal patterns of protection and proteolysis. The acquisition of inserted sequence stretches as sites accessible to limited proteolysis played a key role in the evolution of this control system and happened in coevolution of genuine storage proteins with specific proteinases. This can be deduced from the results of current research on the mechanisms of limited and unlimited proteolysis of storage globulins and on storage globulin evolution. The evolved system of controlled structure-function interplay between storage globulins and proteinases is part of a syndrome that, in addition, comprises differential compartmentation and gene expression of storage proteins and proteinases for controlling the total spatial and temporal patterns of globulin storage and mobilization in maturing and germinating seeds.

Stored proteinases and the initiation of storage protein mobilization in seeds during germination and seedling growth.

Though endopeptidases and carboxypeptidases are present in protein bodies of dry quiescent seeds the function of these proteases during germination is still a matter of debate. In some plants it was demonstrated that endopeptidases of dry protein bodies degrade storage proteins of these organelles. Other studies describe cases where this did not happen. The role that stored proteinases play in the initiation of storage protein breakdown in germinating seeds thus remains unclear. Numerous reviews state that the initiation of reserve protein mobilization is attributed to de novo formed endopeptidases which together with stored carboxypeptidases degrade the bulk of proteins in storage organs and tissues after seeds have germinated. The evidence that the small amounts of endopeptidases in protein bodies of embryonic axes and cotyledons of dry seeds from dicotyledonous plants play an important role in the initiation of storage protein mobilization during early germination is summarized here.

Stored cysteine proteinases start globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch (Vicia sativa L.) seed germination.

Inhibition of protein synthesis by cycloheximide during vetch seed germination, did not prevent globulin breakdown as indicated by a decrease in vicilin- and legumin-specific immunosignals on Western blots. Protein bodies isolated from embryo axes and cotyledons of dry vetch (Vicia sativa L.) seeds using a non-aqueous method were found to be free of cytoplasmic and organellar contaminations. Lysates of these purified protein bodies were capable of degrading globulins; this process was blocked by the cysteine proteinase (CPR) inhibitor iodoacetic acid. Protein bodies contained the papain-like CPR2 and CPR4, and the legumain-like CPR VsPB2. In vitro assays showed that albumin extracts from protein bodies degraded oligopeptide substrates in the PepTag-Assay and degraded the legumain substrate N-benzoyl-asparaginyl-p-nitroanilide. We conclude that, during germination, globulin mobilization is initiated by stored CPRs in protein bodies of embryonic axes as well as cotyledons, and that de-novo-formed proteolytic enzymes mainly mediate bulk degradation of stored globulin in cotyledons after germination.

Differential tissue-specific expression of cysteine proteinases forms the basis for the fine-tuned mobilization of storage globulin during and after germination in legume seeds.

The temporal and spatial distribution of cysteine proteinases (CPRs) was analyzed immunologically and by in situ hybridization to identify the CPRs involved in the initiation of storage-globulin degradation in embryonic axes and cotyledons of germinating vetch (Vicia sativa L.). At the start of germination several CPRs were found in protein bodies in which they might have been stored in the mature seeds. Cysteine proteinase 1 was predominantly found in organs like the radicle, which first start to grow during germination. Cysteine proteinase 2 was also present at the start of germination but displayed a less-specific histological pattern. Proteinase B was involved in the globulin degradation of vetch cotyledons as well. The histological pattern of CPRs followed the distribution of their corresponding mRNAs. The latter were usually detected earlier than the CPRs but the in situ hybridization signals were histologically not as restricted as the immunosignals. Proteolytic activity started in the radicle of the embryonic axis early during germination. Within 24 h after imbibition it had also spread throughout the whole shoot. At the end of germination, newly synthesized CPRs might have supplemented the early detectable CPRs in the axis. In the cotyledons, only the abaxial epidermis and the procambial strands showed proteinase localization during germination. Both CPR1 and CPR2, as well as the less common proteinase B, might have been present as stored proteinases. Three days after imbibition, proteolytic activity had proceeded from the cotyledonary epidermis towards the vascular strands deeper inside the cotyledons. The histochemical detection of the CPRs was in accordance with the previously described histological pattern of globulin mobilization in germinating vetch [Tiedemann J, et al. (2000)]. A similar link between the distribution of CPRs and globulin degradation was found in germinating seeds of Phaseolus vulgaris L. The coincidence of the histological patterns of globulin breakdown with that of the CPRs indicates that at least CPR1, CPR2 and proteinase B are responsible for bulk globulin mobilization in the seeds of the two legumes.

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.).

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2-3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants.

The families of papain- and legumain-like cysteine proteinases from embryonic axes and cotyledons of Vicia seeds: developmental patterns, intracellular localization and functions in globulin proteolysis.

Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (betaVPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.

Comparison of globulin mobilization and cysteine proteinases in embryonic axes and cotyledons during germination and seedling growth of vetch (Vicia sativa L.).

Vicilin and legumin, the storage globulins of mature dry vetch (Vicia sativa L.) seeds, are found in protein bodies which are present not only in the cotyledons, but also in the radicle, axis and shoot (together, for reasons of simplicity, here called axis). When at 24 h after the start of imbibition (hai) the radicle breaks through the seed coat a major part of the globulins in the axis has already been degraded, whereas in the cotyledons globulin breakdown cannot yet be detected. Globulin mobilization starts with the degradation of vicilin. At 48 hai when globulin mobilization in the cotyledons just begins, the axis is already nearly depleted of globulins. Mobilization of storage globulin is probably brought about by a complex of different cysteine proteinases (CPRs). The papain-like CPR2 and CPR4, and the legumain-like VsPB2, together with their mRNAs, are already present in axes and cotyledons of dry seeds. This means that they must have been formed during seed maturation. Additional papain-like CPRs are formed later during germination and seedling growth. CPR4 and VsPB2 together with their corresponding mRNAs become undetectable as germination and seedling growth proceed. VsPB2 and VsPB2-mRNA are substituted by the homologous legumain-like proteinase B and its mRNA. The composition of stored and newly formed CPRs undergoes developmental changes which differ between axes and cotyledons. It is concluded that storage globulin mobilization in germinating vetch seeds is started by stored CPRs, whereas the mobilization of the bulk of globulin is predominantly mediated by CPRs which are formed de novo.

Deposition of storage proteins.

Plants store amino acids for longer periods in the form of specific storage proteins. These are deposited in seeds, in root and shoot tubers, in the wood and bark parenchyma of trees and in other vegetative organs. Storage proteins are protected against uncontrolled premature degradation by several mechanisms. The major one is to deposit the storage proteins into specialized membrane-bounded storage organelles, called protein bodies (PB). In the endosperm cells of maize and rice prolamins are sequestered into PBs which are derived from the endoplasmic reticulum (ER). Globulins, the typical storage proteins of dicotyledonous plants, and prolamins of some cereals are transported from the ER through the Golgi apparatus and then into protein storage vacuoles (PSV) which later become transformed into PBs. Sorting and targeting of storage proteins begins during their biosynthesis on membrane-bound polysomes where an N-terminal signal peptide mediates their segregation into the lumen of the ER. After cleavage of the signal peptide, the polypeptides are glycosylated and folded with the aid of chaperones. While still in the ER, disulfide bridges are formed which stabilize the structure and several polypeptides are joined to form an oligomer which has the proper conformation to be either deposited in ER-derived PB or to be further transferred to the PSV. At the trans-Golgi cisternae transport vesicles are sequestered which carry the storage proteins to the PSV. Several storage proteins are also processed after arriving in the PSVs in order to generate a conformation that is capable of final deposition. Some storage protein precursors have short N- or C-terminal targeting sequences which are detached after arrival in the PSV. Others have been shown to have internal sequence regions which could act as targeting information. In some cases positive targeting information is known to mediate sorting into the PSV whereas in other cases aggregation and membrane association seem to be major sorting mechanisms.

Genetic engineering for high methionine grain legumes.

Methionine (Met) is the primary limiting essential amino acid in grain legumes. The imbalance in amino acid composition restricts their biological value (BV) to 55 to 75% of that of animal protein. So far improvement of the BV could not be achieved by conventional breeding. Therefore, genetic engineering was employed by several laboratories to resolve the problem. Three strategies have been followed. A) Engineering for increased free Met levels; B) engineering of endogenous storage proteins with increased numbers of Met residues; C) transfer of foreign genes encoding Met-rich proteins, e.g. the Brazil nut 2S albumin (BNA) and its homologue from sunflower, into grain legumes. The latter strategy turned out to be most promising. In all cases the gene was put under the control of a developmentally regulated seed specific promoter and transferred into grain legumes using the bacterial Agrobacterium tumefaciens-system. Integration into and copy numbers in the plant genome as well as Mendelian inheritance and gene dosage effects were verified. After correct precursor processing the mature 2S albumin was intracellularly deposited in protein bodies which are part of the vacuolar compartment. The foreign protein amounted to 5 to 10% of the total seed protein in the best transgenic lines of narbon bean (Vicia narbonensis L., used in the authors' laboratories), lupins (Lupinus angustifolius L., used in CSIRO, Australia), and soybean (Glycine max (L.) Merr., used by Pioneer Hi-Bred, Inc., USA). In the narbon bean the increase of Met was directly related to the amount of 2S albumin in the transgenic seeds, but in soybean it remained below the theoretically expected value. Nevertheless, trangenic soybean reached 100%, whereas narbon bean and lupins reached approximately 80% of the FAO-standard for nutritionally balanced food proteins. These results document that the Met problem of grain legumes can be resolved by genetic engineering.

Evidence for secretion of vacuolar alpha-mannosidase, class I chitinase, and class I beta-1,3-glucanase in suspension cultures of tobacco cells.

We have investigated the possibility that vacuolar proteins can be secreted into the medium of cultured cells of Nicotiana tabacum L. Time-course and balance-sheet experiments showed that a large fraction, up to ca. 19%, of vacuolar alpha-mannosidase (EC 3.2.1.24) and vacuolar class I chitinase (EC 3.2.1.14) in suspension cultures accumulated in the medium within one week after subculturing. This effect was most pronounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D). Under comparable conditions only a small fraction, 1.8-5.1% of the total protein and ca. 1% of malate dehydrogenase (EC 1.1.1.37), which is localized primarily in the mitochondria and cytoplasm, accumulated in the medium. Pulse-chase experiments showed that newly synthesized vacuolar class I isoforms of chitinase and beta-1,3-glucanase (EC 3.2.1.39) were released into the medium. Post-translational processing, but not the release of these proteins, was delayed by the secretion inhibitor brefeldin A. Only forms of the proteins present in the vacuole, i.e. mature chitinase and pro-beta-1,3-glucanase and mature beta-1,3-glucanase, were chased into the medium of tobacco cell-suspension cultures. Our results provide strong evidence that vacuolar alpha-mannosidase, chitinase and beta-1,3-glucanase can be secreted into the medium. They also suggest that secretion of chitinase and beta-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar compartment.

The role of proteolysis in the processing and assembly of 11S seed globulins.

11S seed storage proteins are synthesized as precursors that are cleaved post-translationally in storage vacuoles by an asparaginyl endopeptidase. To study the specificity of the reaction catalyzed by this asparaginyl endopeptidase, we prepared a series of octapeptides and mutant legumin B and G4 glycinin subunits. These contained amino acid mutations in the region surrounding the cleavage site. The endopeptidase had an absolute specificity for Asn on the N-terminal side of the severed peptide bond but exhibited little specificity for amino acids on the C-terminal side. The ability of unmodified and modified subunits to assemble into hexamers after post-translational modification was evaluated. Cleavage of subunits in trimers is required for hexamer assembly in vitro. Products from a mutant gene encoding a noncleavable prolegumin subunit (LeBDeltaN281) accumulated as trimers in seed of transgenic tobacco, but products from the unmodified prolegumin B gene accumulated as hexamers. Therefore, the asparaginyl endopeptidase is required for hexamer assembly.

Does an asparaginyl-specific cysteine endopeptidase trigger phaseolin degradation in cotyledons of kidney bean seedlings?

An asparaginyl-specific cysteine endopeptidase which was named 'legumain-like proteinase' (LLP) and has an apparent molecular mass of 38.1 kDa was isolated from cotyledons of kidney bean (Phaseolus vulgaris L.) seedlings and partially characterized. It is, to our knowledge, the first known proteinase which in vitro extensively degrades native phaseolin, the major storage globulin of this grain legume. Phaseolin that in vitro had been partially degraded by LLP (Pvitro) and phaseolin that was isolated after partial in vivo breakdown 6 days after the start of seed imbibition (Pvivo) showed similar fragment patterns on SDS/polyacrylamide gels. The fragments had identical cleavage sites in Pvitro and Pvivo as determined by partial amino acid sequencing. In both types of partially degraded phaseolin, these cleavage sites have asparagine in the P1 position. Two of the cleavage sites are located in the beta-barrel domain of the C-terminal module and only one cleavage site was found in the beta-barrel domain of the N-terminal module according to the consensus structural model of phaseolin subunits. These results suggest that very likely LLP could in vivo be responsible for the initiation of phaseolin proteolysis. Two different legumain-specific clones named cp6b and p21b were isolated from a cDNA library of germinated bean cotyledons. Cp6b encodes LLP, while p21b encodes a VPE-like enzyme. Southern-blot analysis revealed a single gene copy for Pv-VPE and, presumably, at least two gene copies for LLP in the kidney bean genome. Northern-blot analysis indicated that mRNAs for both clones appear de novo during seed germination. However, the developmental patterns of the transcript levels corresponding to the two clones differed significantly. The temporal pattern of phaseolin degradation and of LLP polypeptide levels agreed well with the suggestion that LLP plays a key role in the mobilization of phaseolin during and after kidney bean germination.

Role of the sulfhydryl redox state and disulfide bonds in processing and assembly of 11S seed globulins.

Seed legumins contain two conserved disulfide bonds: an interchain bond (IE) connecting the acidic and basic chains and an intrachain bond (IA) internal to the acidic chain. Mutant subunits were constructed in which these disulfide bonds were disrupted. Oxidized glutathione stimulated the rate of assembly of trimers with unmodified prolegumin subunits. Stimulation was not detected during assembly of IE mutant subunits and was diminished for the IA mutant. Hexamer assembly with trimers of mature unmodified subunits required oxidizing conditions. Trimers composed of mature IE mutants did not form hexamers. Both mutant and non-mutant subunits accumulated in hexamers when the cDNAs were expressed in tobacco. Hexamer assembly in seeds probably involved trimers with a mixture of mutant and non-mutant subunits. Similarly, mixed trimers that were a mixture of mutant and non-mutant subunits assembled into hexamers in vitro. The results demonstrate the importance of disulfide bonds during the assembly of 11S globulins.

Proteinase A, a storage-globulin-degrading endopeptidase of vetch (Vicia sativa L.) seeds, is not involved in early steps of storage-protein mobilization.

Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25,244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydryl-endopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna mungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination.

Subcellular localization of the 2S globulin narbonin in seeds of Vicia narbonensis.

Narbonin is a 2S protein from the globulin fraction of narbon bean (Vicia narbonensis L.) cotyledons. Its amino acid composition and the pattern of its regulated accumulation in developing seeds led to the suggestion that narbonin could be a storage protein. Therefore, it was expected to be present in protein bodies of the storage tissue cells. Comparison of the cDNA-derived amino acid sequence with a directly determined partial N-terminal sequence revealed that the primary translation product of narbonin mRNA lacks a transient N-terminal signal peptide (V.H. Nong et al., 1995, Plant Mol Biol 28: 61 - 72). Narbonin polypeptides that had been synthesized in a cell-free translation system supplemented with dog pancreas microsomes were not protected against degradation by posttranslationally added proteases (protease protection assay). In accordance with the lack of a signal peptide this indicates that the polypeptide was not cotranslationally sequestered into the microsomes. The protein-body fraction that had been isolated from mature narbon bean cotyledons by a non-aqueous gradient centrifugation procedure was free of narbonin; this was found in the soluble cell fraction. In electron micrographs, narbonin could be localized in the cytoplasm using the immuno gold-labelling technique. Previously, it had already been shown that narbonin is too slowly degraded during narbon bean germination to act as a storage protein. From all these results it has to be concluded that narbonin is a cytoplasmic protein which does not belong to the storage proteins in the restricted sense. Other possible functions are discussed.

Seed-specific immunomodulation of abscisic acid activity induces a developmental switch.

A single-chain Fv antibody (scFv) gene, which has previously been used to immunomodulate abscisic acid (ABA) activity in transgenic tobacco to create a 'wilty' phenotype, was put under control of the seed-specific USP promoter from Vicia faba and used to transform tobacco. Transformants were phenotypically similar to wild-type plants apart from their seeds. Anti-ABA scFv embryo development differed markedly from wild-type embryo development. Seeds which accumulated similar levels of a scFv that binds to oxazolone, a hapten absent from plants, developed like wild-type embryos. Anti-ABA scFv embryos developed green cotyledons containing chloroplasts and accumulated photosynthetic pigments but produced less seed storage protein and oil bodies. Anti-ABA scFv seeds germinated precociously if removed from seed capsules during development but were incapable of germination after drying. Total ABA levels were higher than in wild-type seeds but calculated free ABA levels were near-zero until 21 days after pollination. We show for the first time seed-specific immunomodulation and the resulting switch from the seed maturation programme to a germination programme. We conclude that the immunomodulation of hormones can alter the development programme of target organs, allowing the study of the directly blocked endogenous molecules and manipulation of the system concerned.

Localisation of cardiac GS alpha in transgenic mice overexpressing GS alpha.

The biochemical and physiological effects of GS alpha activation are well known; however, little is known about the anatomical localisation of GS alpha in the myocardium. Knowledge of the localisation might yield insights into G protein function in heart. The utility of immunocytochemistry using immunofluorescent methods is limited in normal hearts because of the low expression of GS alpha. In order to magnify the GS alpha signal, we studied transgenic mice overexpressing myocardial GS alpha. Immunofluorescent techniques with confocal imaging using rabbit antiserum specific for GS alpha were studied in frozen sections of mouse left ventricle. GS alpha labeling appeared to be localised to the T-tubules and intercalated disks in the GS alpha overexpressing mouse hearts, whereas the control mice showed background fluorescence with diffuse faint labeling. The localisation of GS alpha to structures involved in calcium handling and membrane conductance places GS alpha at a focal point in the regulation of these key functions.

Regulation of alpha 1-, beta 1-, and beta 2-adrenergic receptors in rat heart by norepinephrine.

Previous studies suggest that the desensitization and downregulation of beta 1-adrenergic receptors (beta 1-AR) in the failing heart are the result of the elevated plasma catecholamine levels associated with this disease. To examine norepinephrine (NE)-induced regulation of cardiac adrenergic receptors, rats were infused with l-NE (200 micrograms.kg-1.h-1 for 7 days) or vehicle (0.001 N HCl) by implantation of osmotic minipumps. The technique of coverslip autoradiography was used to quantify alpha 1-adrenergic receptors (alpha 1-AR), beta 1-AR, and beta 2-AR in different tissue compartments of rat hearts. For measurement of beta-AR binding, sections were incubated with 70 pM [125I]iodocyanopindolol (ICYP) alone or in the presence of 5 microM dl-propranolol or 5 x 10(-7) M CGP-20712A (a beta 1-antagonist) and then set up for autoradiography. [3H]prazosin (1 nM) with or without phentolamine was used to study alpha-AR binding. Chronic infusion of NE induced a greater downregulation of beta 2-AR compared with beta 1-AR in all regions studied, including atrial and ventricular myocytes, coronary arterioles, and connective tissue. An 18% loss of beta 1-AR was seen only in atrial myocytes; beta 1-AR density actually increased 28% in ventricular myocytes following NE infusion. There was a 15% decrease in alpha 1-AR in ventricular myocytes, whereas no change in alpha 1-AR density was seen in myocardial arterioles. Our study demonstrates that beta 2-AR are more susceptible to NE-induced downregulation than beta 1-AR. Thus other mechanisms may be involved in the selective downregulation of beta 1-AR in certain forms of heart failure.

Limited proteolysis of beta-conglycinin and glycinin, the 7S and 11S storage globulins from soybean [Glycine max (L.) Merr.]. Structural and evolutionary implications.

The G2 (A2B1a) glycinin subunit from soybean (Glycine max L. Merr.) was purified and renatured to the homohexameric holoprotein. This protein along with purified beta-conglycinin were subjected to limited proteolysis by trypsin. The generated polypeptide fragments were separated via SDS/PAGE and the amino acid sequence of the N-terminals was determined. Four cleavage points were detected in the alpha-chain A2 of glycinin as well as in the alpha'-chain of beta-conglycinin. From the known three-dimensional structure of 7S globulin and the hypothetical model of 7S globulin-like 11S globulin structure, it was possible to draw the conclusion that two distinct types of susceptible sites for proteolytic cleavage are characteristic of the subunits of both globulins. The first includes the sequences linking N- and C-terminal domains of both globulins and the sequence of N-terminal extensions of 70-kDa subunits from the vicilin-like 7S globulins. The second type includes the loop between beta-strands E and F of the N-terminal domain of 11S globulins and of the C-terminal domain of 7S globulins. A statistically significant similarity was found between the N-terminal extension of the alpha'-chain of beta-conglycinin and the interdomain linker regions of soybean glycinin and pea legumin. It is proposed that the three sequence regions which form the first type of susceptible sites are of similar structural function and might have evolved from the N-terminal segment of a putative single-domain ancestor.