Import of glyoxysomal malate dehydrogenase precursor into glyoxysomes: A heterologous in-vitro system

A heterologous in-vitro system is described for the import of the precursor to glyoxysomal malate dehydrogenase from watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) cotyledons into glyoxysomes from castor-bean (Ricinus communis L.) endosperm. The 41-kDa precursor is posttranslationally sequestered and correctly processed to the mature 33-kDa subunit by a crude glyoxysomal fraction or by glyoxysomes purified on a sucrose gradient. The import and the cleavage of the extrasequence is not inhibited by metal chelators such as 1,10-phenanthroline and ethylenediaminetetraacetic acid. Uncouplers (carbonylcyanide m-chlorophenylhydrazone), ionophores (valinomycin), or inhibitors of oxidative phosphorylation (oligomycin) and ATP-ADP translocation (carboxyatractyloside) do not interfere, thus indicating the independence of the process of import by the organelle from the energization of the glyoxysomal membrane.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - EDTA ethylenediaminetetraacctic acid - gMDH glyoxysomal malate dehydrogenase - PMSF phenylmethylsulfonyl fluoride     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

Sequence homologies between glyoxysomal and mitochondrial malate dehydrogenase

The comparison of mitochondrial and glyoxysomal malate dehydrogenase (EC 1.1.1.37) from cotyledons of germinating watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) by means of serological methods and peptide patterns revealed a high degree of homology. The N-terminal sequence analysis yielded a distinct presequence of eight or nine amino-acid residues, respectively, which is followed by an almost identical stretch of at least 20 amino-acid residues. A very similar domain has been recognized for mitochondrial malate dehydrogenase from porcine heart and yeast, and for Escherichia coli malate dehydrogenase.Abbreviations gMDH glyoxysomal malate dehydrogenase - mMDH mitochondrial malate dehydrogenase - SDS sodium dodecyl sulfate     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

Fluorescence immunohistochemical localization of malate dehydrogenase isoenzymes in watermelon cotyledons : a developmental study of glyoxysomes and mitochondria

Monospecific antibodies to glyoxysomal, mitochondrial, and cytosolic I malate dehydrogenase were used for the fluorescence immunohistochemical localization of these isoenzymes in dark-grown watermelon (Citrullus vulgaris Schrad.) cotyledons. It was demonstrated that, with cell organelles isolated by sucrose density gradient centrifugation, antibodies to glyoxysomal malate dehydrogenase were specific markers for glyoxysomes, and similarly, antibodies to mitochondrial malate dehydrogenase were markers for mitochondria. The time course of the glyoxysomal malate dehydrogenase appearance and decline was not synchronous for the individual tissues and differed completely from that of the mitochondria. The cytosolic malate dehydrogenase I was confined to restricted regions of the lower epidermis. The activity which was definitively localized outside the cell organelles decreased during the first days of germination.     

Organelle and translocatable forms of glyoxysomal malate dehydrogenase. The effect of the N-terminal presequence

Many organelle enzymes coded for by nuclear genes have N-terminal sequences, which directs them into the organelle (precursor) and are removed upon import (mature). The experiments described below characterize the differences between the precursor and mature forms of watermelon glyoxysomal malate dehydrogenase. Using recombinant protein methods, the precursor (p-gMDH) and mature (gMDH) forms were purified to homogeneity using Ni2+-NTA affinity chromatography. Gel filtration and dynamic light scattering have shown both gMDH and p-gMDH to be dimers in solution with p-gMDH having a correspondingly higher molecular weight. p-gMDH also exhibited a smaller translational diffusion coefficient (D(t)) at temperatures between 4 and 32 degrees C resulting from the extra amino acids on the N-terminal. Differential scanning calorimetry described marked differences in the unfolding properties of the two proteins with p-gMDH showing additional temperature dependent transitions. In addition, some differences were found in the steady state kinetic constants and the pH dependence of the K(m) for oxaloacetate. Both the organelle-precursor and the mature form of this glyoxysomal enzyme were crystallized under identical conditions. The crystal structure of p-gMDH, the first structure of a cleavable and translocatable protein, was solved to a resolution of 2.55 A. GMDH is the first glyoxysomal MDH structure and was solved to a resolution of 2.50 A. A comparison of the two structures shows that there are few visible tertiary or quaternary structural differences between corresponding elements of p-gMDH, gMDH and other MDHs. Maps from both the mature and translocatable proteins lack significant electron density prior to G44. While no portion of the translocation sequences from either monomer in the biological dimer was visible, all of the other solution properties indicated measurable effects of the additional residues at the N-terminal.     

Glyoxysomal malate dehydrogenase and malate synthase from soybean cotyledons (Glycine max L.): enzyme association,antibody production and cDNA cloning

In order to investigate a possible association between soybean malate synthase (MS; l-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD⁺ oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionicstrength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M_r) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl₂, with the elution of MS as an octamer of M_r 510000 and of gMDH as a dimer of M_r 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X₆)HL motif also found in plant and mammalian thiolases.The nucleotide sequence data reported in this paper have been submitted to Genbank and assigned the accession numbers LOI628 for gMDH and L01629 for MS.     

Glyoxysomal Malate Dehydrogenase in Pumpkin: Cloning of a cDNA and Functional Analysis of Its Presequence

Glyoxysomal malate dehydrogenase (gMDH) is an enzyme of theglyoxylate cycle that participates in degradation of storageoil. We have cloned a cDNA for gMDH from etiolated pumpkin cotyledonsthat encodes a polypep-tide consisting of 356 amino acid residues.The nucleotide and N-terminal amino acid sequences revealedthat gMDH is synthesized as a precursor with an N-terminal extrapeptide.The N-terminal presequence of 36 amino acid residues containstwo regions homologous to those of other micro-body proteins,which are also synthesized as large precursors. To investigatethe functions of the N-terminal presequence of gMDH, we generatedtransgenic Arabidopsis that expressed a chimeric protein consistingof rß-glucuroni-dase and the N-terminal region ofgMDH. Immunologi-cal and immunocytochemical studies revealedthat the chimeric protein was imported into microbodies suchas gly-oxysomes and leaf peroxisomes and was then subsequentlyprocessed. Site-directed mutagenesis studies showed that theconserved amino acids in the N-terminal presequence, Arg-10and His-17, function as recognition sites for the targetingto plant microbodies, and Cys-36 in the presequence is responsiblefor its processing. These results correspond to those from theanalyses of glyoxysomal citrate synthase (gCS), which was alsosynthesized as a large precursor, suggesting that common mechanismsthat can recognize the targeting or the processing of gMDH andgCS function in higher plant cells. (Received July 10, 1997; Accepted November 22, 1997)     

Thiolase mRNA translated in vitro yields a peptide with a putative N-terminal presequence

Thiolase is part of the fatty acid oxidation machinery which in plants is located within glyoxysomes or peroxisomes. In cucumber cotyledons, proteolytic modification of thiolase takes place during the transfer of the cytosolic precursor into glyoxysomes prior to the intraorganellar assembly of the mature enzyme. This was shown by size comparison of the in vitro synthesized precursor and the 45 kDa subunit of the homodimeric glyoxysomal form. We isolated a full-length cDNA clone encoding the 48 539 Da precursor of thiolase. This plant protein displayed 40% and 47% identity with the precursor of fungal peroxisomal thiolase and human peroxisomal thiolase, respectively. Compared to bacterial thiolases, the precursor of the plant enzyme was distinguished by an N-terminal extension of 34 amino acid residues. This putative targeting sequence of cucumber thiolase shows similarities with the cleavable presequences of rat peroxisomal thiolase and plant peroxisomal malate dehydrogenase.     

Oxidation of NADH in Glyoxysomes by a Malate-Aspartate Shuttle

Glyoxysomes isolated from germinating castor bean endosperm accumulate NADH by β-oxidation of fatty acids. By utilizing the glutamate: oxaloacetate aminotransferase and malate dehydrogenase present in glyoxysomes and mitochondria, reducing equivalents could be transferred between the organelles by a malate-aspartate shuttle. The addition of aspartate plus α-ketoglutarate to purified glyoxysomes brought about a rapid oxidation of accumulated NADH, and the oxidation was prevented by aminooxyacetate, an inhibitor of aminotransferase activity. Citrate synthetase activity in purified glyoxysomes could be coupled readily to glutamate: oxaloacetate aminotransferase activity as a source of oxaloacetate, but coupling to malate dehydrogenase and malate resulted in low rates of citrate formation. Glyoxysomes purified in sucrose or Percoll gradients were permeable to low molecular weight compounds. No evidence was obtained for specific transport mechanisms for the proposed shuttle intermediates. The results support a revised model of gluconeogenic metabolism incorporating a malate-aspartate shuttle in the glyoxysomal pathway.     

Organelle-bound malate dehydrogenase isoenzymes are synthesized as higher molecular weight precursors

Biosynthesis of malate dehydrogenase isoenzymes was studied in cotyledons of watermelons (Citrullus vulgaris Schrad., var. Stone Mountain). The glyoxysomal and mitochondrial isoenzymes are synthesized as higher molecular weight precursors which can be immunoprecipitated by mono-specific antibodies from the products of in vitro translation in reticulocyte lysates programed with cotyledonary mRNA and with the same size from enzyme extracts of pulse-labeled cotyledons. During translocation from the cytosol into the organelles, processing takes place. An 8 kilodalton extra sequence is cleaved from the glyoxysomal precursor and a 3.3 kilodalton extra sequence from the mitochondrial precursor producing the native subunits of 33 and 38 kilodaltons, respectively. The data support a post-translational translocation of the organelle-destined malate dehydrogenase isoenzymes. The in vitro translation of the cytosolic malate dehydrogenase I yields a product which has the same molecular weight as the subunit of the native isoenzyme (39.5 kilodaltons).     

Functional Analysis of the N-Terminal Prepeptides of Watermelon Mitochondrial and Glyoxysomal Malate Dehydrogenases*

Mitochondrial and glyoxysomal malate dehydrogenase (mMDH; gMDH; L-malate: NAD⁺ oxidoreductase; EC 1.1.1.37) of watermelon (Citrullus vulgaris) cotyledons are synthesized with N-terminal cleavable presequences which are shown to specify sorting of the two proteins. The two presequences differ in length (27 or 37 amino acids) and primary structure. Precursor proteins of the two isoenzymes with site-directed mutations in their presequences and hybrid precursor proteins with reciprocally exchanged presequences were analyzed for proper import using two approaches, namely in vitro using isolated watermelon organelles or in vivo after synthesis in the heterologous host, Hansenula polymorpha. The mitochondrial presequence is essential and sufficient to target the mature glyoxysomal isoenzyme into mitochondria (Gietl et al., 1994). As to the function of the mitochondrial presequence a substitution of ^?3R (considered important for one step precursor cleavage in yeast and mammals) with ^?3L permitted import into mitochondria but cleavage of the transit peptide and conversion into active mature enzyme was impeded. Substitution of ^?13R^?12S (in a sequence reminiscent of the octapeptide motif serving as a substrate for the mammalian and yeast intermediate peptidase) into ^?13L¹²F permitted mitochondrial import and processing like the wild type transit peptide. Purified rat mitochondrial processing protease, which can effect single step cleavage of mitochondrial protein precursors, cleaves in vitro translated watermelon mMDH precursor into its mature form. The glyoxysomal presequence is essential and sufficient to target the mature mitochondrial isoenzyme into peroxisomes of Hansenula polymorpha, but these peroxisomes lack a processing enzyme to cleave the presequence (Gietl et al., 1994). We here show that isolated watermelon organelles also import the hybrid proteins in vitro and process the glyoxysomal presequence. Site directed mutations within the conserved RI-X₅-HL-motif impede efficiency of import and cleavage by watermelon organelles.     

Recovery of plastids from photooxidative damage: Significance of a plastidic factor

Glyoxysomal citrate synthase (gCS) was purified from crude extracts of watermelon (Citrullus vulgaris Schrad.) cotyledons, yielding a homogenous protein with a subunit MW of 48 kDa. The enzyme was selectively inhibited by 5,5-dithiobis-(2-nitrobenzoic acid), allowing quantification in the presence of the mitochondrial isoenzyme (mCS). Differences were also observed with respect to inhibition by ATP (k _i=2.6 mmol · l^-1 for gCS, k _i=0.33 mmol · l^-1 for mCS). The antibodies prepared against gCS did not cross-react with mCS. The immunocytochemical localization of gCS by the indirect protein A-gold procedure was restricted to the glyoxysomal membrane or the peripheral matrix of glyoxysomes. Other compartments, e.g. the endoplasmic reticulum, were not labeled. Xenopus oocytes were used for the translation of watermelon polyadenylated RNA (poly(A)⁺RNA). A translation product with a MW of 51 kDa was immunoprecipitated by the anti-gCS antibodies. It was absent in controls without poly(A)⁺RNA or with preimmune serum. A similar translation product was also immunoprecipitated after cell-free synthesis of watermelon poly(A)⁺RNA in a reticulocyte system, in contrast to the in-vivo labeled gCS (48 kDa). It was concluded that gCS is synthesized as a higher-molecular-weight precursor.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - gCS glyoxysomal citrate synthase - gMDH glyoxysomal malate dehydrogenase - k _i inhibitor constant - mCS mitochondrial citrate synthase - OAA oxaloacetate - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Biosynthesis of a microbody matrix enzyme in greening cotyledons

Earlier work on microbody biosynthesis has shown that glyoxysomal and liver peroxisomal proteins synthesized in the cytosol are made as precursors which are then transferred into the organelles and processed. Here, it is demonstrated that the unprecessed precursor detected in the cytosol after protein synthesis in vivo for an enzyme at the transition stage between glyoxysomes and leaf peroxisomes is indistinguishable from the product of translation in vitro. It is assumed that the transfer of extraorganellarly made precursor across the glyoxysomal membranes is followed by processing of the precursor and oligomerization to the tetrameric or 16-meric form of the enzyme. Oligomerization was, however, also observed in a portion of the cytosolic form.     

Characterization of glyoxysomes from castor bean endosperm

Electron micrographs are presented which establish the identity of the components of the 3 major bands observed after sucrose density centrifugation of the crude particulate fraction from the endosperm of germinating castor bean seedlings. These are: mitochondria (density 1.19 g/cc), proplastids (density 1.23 g/cc) and glyoxysomes (density 1.25 g/cc). Further evidence is provided on the enzymatic composition of the glyoxysomes. Essentially all of the particulate malate synthetase, isocitrate lyase, catalase, and glycolic oxidase is present in these organelles. The distribution of glyoxysomal enzymes on sucrose density gradients is contrasted with that of the strictly mitochondrial enzymes fumarase, NADH oxidase, and succinoxidase. Malate dehydrogenase and citrate synthetase are present in both organelles. The functional role of glyoxysomes and their relationship to cytosomes from other tissues is discussed.     

Lecithin synthesis during microbody biogenesis in watermelon cotyledons

During the normal development of watermelon seedlings, leaf peroxisomes succeed glyoxysomes as the major microbody component in the cotyledons. The possibility has thus been raised that the two organelles are ontogenetically related; that leaf peroxisomes are derived from glyoxysomes. The behavior of lecithin, an important constituent of the membranes of both kinds of organelle was examined in this study. Using labeled choline as a precursor of lecithin, its incorporation into various membrane fractions was followed during the period when glyoxysomal activity was declining and that of leaf peroxisomes increasing after exposure to light. The results showed that glyoxysomal membrane was selectively destroyed during this period. Furthermore, from double-labeling experiments using [¹⁴C]- and [³H]choline it was shown that newly synthesized lecithin was incorporated into the membranes of the developing leaf peroxisomes. These results support the thesis that leaf peroxisomes are not derived from glyoxysomes and instead represent two distinct microbody populations.     

A Pisum sativum glyoxysomal malate dehydrogenase induced by cadmium exposure.

The glyoxysomal malate dehydrogenase (gMDH) catalyses the formation of oxaloacetate from malate during beta-oxidation of fatty acids in the glyoxysome. A partial Pisum sativum L. (cv. Greenfeast) cDNA was first isolated from a suppression subtractive hybridisation cDNA library obtained from heavy metal stressed plants. The full length cDNA was then isolated by rapid amplification of cDNA ends. The translated sequence showed strong similarity to Cucumis sativus and Citrullus lanatus gMDH including a typical glyoxysome-targeting presequence comprising the PTS2 motif and a cleavage site for a cystein-directed protease. Exposure of pea plants to Cd2+ induced expression of the gMDH gene in mature pea leaves indicating that the enzyme is under environmental control in addition to the normal developmental regulation pattern.     

Evidence that glyoxysomal malate synthase is segregated by the endoplasmic reticulum

At the onset of castor bean (Ricinus communis) germination, 76% of the cellular malate synthase activity of the endosperm tissue was located in the microsomal fraction, with the remainder in the glyoxysomal fraction. During later developmental stages, when rapid malate synthase synthesis was occurring, an increasing proportion of the enzyme was recovered in glyoxysomes. The kinetics of [³⁵S]methionine incorporation into microsomal and glyoxysomal malate synthase in 2-day-old endosperm tissue was followed by employing antiserum raised against glyoxysomal malate synthase to precipitate specifically the enzyme from KCl extracts of these organelle fractions. This experiment showed that microsomal malate synthase was labeled before the glyoxysomal enzyme. When such kinetic experiments were interrupted by the addition of an excess of unlabeled methionine, ³⁵S-labeled malate synthase was rapidly lost from the microsomal fraction and was quantitatively recovered in the glyoxysomal fraction.

Free cytoplasmic ribosomes were separated from bound ribosomes (rough microsomes) using endosperm tissue labeled with [³⁵S]methionine or ¹⁴C-amino-acids. Nascent polypeptide chains were released from polysome fractions using a puromycin-high salt treatment, and radioactive malate synthase was shown to be exclusively associated with bound polysomes.

Together these data establish that malate synthase is synthesized on bound ribosomes and vectorially discharged into the endoplasmic reticulum cisternae prior to its ultimate sequestration in glyoxysomes.

     

A cysteine endopeptidase isolated from castor bean endosperm microbodies processes the glyoxysomal malate dehydrogenase precursor protein.

A plant cysteine endopeptidase with a molecular mass of 35 kD was purified from microbodies of germinating castor bean (Ricinus communis) endosperm by virtue of its capacity to specifically process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro. Processing of the glyoxysomal malate dehydrogenase precursor occurs sequentially in three steps, the first intermediate resulting from cleavage after arginine-13 within the presequence and the second from cleavage after arginine-33. The endopeptidase is unable to remove the presequences of prethiolases from rape (Brassica napus) glyoxysomes and rat peroxisomes at the expected cleavage site. Protein sequence analysis of N-terminal and internal peptides revealed high identity to the mature papain-type cysteine endopeptidases from cotyledons of germinating mung bean (Vigna mungo) and French bean (Phaseolus vulgaris) seeds. These endopeptidases are synthesized with an extended pre-/prosequence at the N terminus and have been considered to be processed in the endoplasmic reticulum and targeted to protein-storing vacuoles.     

Purification and Characterization of Glyoxysomal Enzymes from Germinating Pumpkin Cotyledons

Four glyoxysomal enzymes, malate synthase, malate dehydrogenase,3-hydroxyacyl-CoA dehydrogenase and citrate synthase, were purifiedfrom glyoxysomes of germinating pumpkin cotyledons. Molecularweights of their subunits were as follows: malate synthase,60,000; malate dehydrogenase, 33,000; 3-hydroxyacyl-CoA dehydrogenase,72,000 and citrate synthase, 45,000. Malate synthase and 3-hydroxyacyl-CoAdehydrogenase activities were exclusively localized in glyoxysomes,whereas malate dehydrogenase and citrate synthase activitieswere found in both glyoxysomes and mitochondria. Monospecificantibodies against malate dehydrogenase and citrate synthaseinhibited their activities present in glyoxysomes but in mitochondria.Immunocytochemical analysis using the protein A-gold techniquecombined with Lowicryl K4M embedding showed that the antigenicsites for these enzymes were found exclusively in glyoxysomes.These data indicates that malate dehydrogenase and citrate synthasepresent in glyoxysomes are immunologically different from thosein mitochondria, respectively. ¹ This is paper No. 9 in the series "Analytical Studies on MicrobodyTransition". ³ Present address: Meiji Institute of Health Science, Naruta,Odawara, Kanagawa 250, Japan. ⁵ Present address: Department of Biology, Faculty of Science,Kobe University, Rokkoudai, Nada, Kobe 657, Japan. (Received December 23, 1987; Accepted January 27, 1988)