Subunit structure and functional properties of the predominant globulin of perilla (Perilla frutescens var. frutescens) seeds

Approximately 40% of defatted perilla seeds consists of proteins which are primarily composed of globulin (84%). The amino acid profile of perilla proteins demonstrated balanced amounts of all essential amino acids, except for lysine. The molecular mass of the predominant globulin was estimated to be 340 kDa by gel filtration. This globulin was separated into three intermediary subunits (54, 57 and 59 kDa) by SDS-PAGE. It is suggested from these results that the globulin exists as a hexamer. A treatment with 50 mM dithiothreitol enabled the intermediary subunits to be separated into three acidic subunits (31-34 kDa) and four basic subunits (23-25 kDa). It is interesting that this subunit structure is the same as that of sesame α-globulin, despite them coming from different families. Compared to sesame α-globulin, the heat-induced gel of perilla globulin had better water-holding ability, despite it displaying the same degree of gel hardness.     

Network structure and forces involved in perilla globulin gelation: comparison with sesame globulin

Scanning electron micrographs show that perilla globulin gel had a finer network structure than sesame α-globulin gel. The effects of various reagents on the gel formation and solubility of perilla and sesame gels were compared. The contribution of disulfide bonds to the formation and stability of perilla gel was greater than to sesame gel, despite having the same subunit structure.     

Network Structure and Forces Involved in Perilla Globulin Gelation: Comparison with Sesame Globulin

Scanning electron micrographs show that perilla globulin gel had a finer network structure than sesame α-globulin gel. The effects of various reagents on the gel formation and solubility of perilla and sesame gels were compared. The contribution of disulfide bonds to the formation and stability of perilla gel was greater than to sesame gel, despite having the same subunit structure.     

Subunit Composition of the Zinc Proteins α- and β-Lipovitellin from Chicken

Chicken α- and β-lipovitellin are derived from parent vitellogenin proteins and contain four subunits (125, 80, 40, and 30 kDa) and two subunits (125 and 30 kDa), respectively. Metal analyses demonstrate both are zinc proteins containing 2.1 ± 0.2 mol of zinc/275 kDa per α-lipovitellin and 1.4 ± 0.2 mol of zinc/155 kDa per β-lipovitellin, respectively. The subunits of β-lipovitellin, Lv 1 (MW 125 kDa) and Lv 2 (MW 30 kDa), are separated by gel exclusion chromatography in the presence of zwittergent 3–16. Zinc elutes with Lv 1, suggesting that this subunit binds zinc in the absence of Lv 2. The subunits of α- and β-lipovitellin were separated by SDS-PAGE, digested with trypsin, and mapped by reverse-phase HPLC. The peptide maps of the 125-kDa subunits from α- and β-lipovitellin are essentially identical. Similar results are obtained for the 30-kDa subunits of both lipovitellins. The sequences of five and four peptides of the 125-kDa subunit of α- and β-Lv, respectively, and two peptides of the 30-kDa subunit of α- and β-lipovitellin were determined and match those predicted from the gene for vitellogenin II, Vtg II. Comparison of the amino acid composition of the 125- and 30-kDa subunits of α- and β-lipovitellin support the conclusion that they originate from the same gene. The sequences of peptides from the 80- and 40-kDa subunits of α-lipovitellin have not been found in the NCBI nonredundant data bank. The 27-amino acid N-terminal sequence of the 40-kDa protein is 56% similar to the last third of the Lv 1-coding region of the Vtg II gene, suggesting it may come from an analogous region of the Vtg I gene. We propose a scheme for the precursor—product relationship of Vtg I.     

Subunit Structure of Sesame 13S Globulin

On SDS-polyacrylamide gel electrophoresis, sesame seed 13S globulin was separated into three intermediary subunits termed IS₁ IS₂ and IS₃. Following a treatment with 0.2M 2-mercaptoethanol, the globulin was separated into three acidic subunits termed AS₁ AS₂ and AS₃, and four basic subunits termed BS₁ BS₂, BS₃ and BS₄. Two dimensional SDS-gel electrophoresis before and after treatment with 0.2 M 2-mercaptoethanol revealed that IS₁ was composed of two combinations of acidic and basic subunits, these being S₁ and BS₂, and AS₂ and BS₂. IS₂ was found to be composed of AS₃ and BS₁, and IS₃ was composed of AS₂ and BS₃, and AS₂ and BS₄. These combinations were consistent with the reactivity of each subunit to a fluorescent thiol reagent. The amino acid compositions were similar among the three acidic subunits and also among the four basic subunits. However, between the acidic and basic subunits, there were great differences in the amino acid composition, especially in the amount of glutamic acid.     

Inter-laboratory optimization of protein extraction,separation, and fluorescent detection of endogenous rice allergens

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52–63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14–16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.     

Interaction Involving Disulfide Bridges between Subunits of Soybean Seed Globulin and between Subunits of Soybean and Sesame Seed Globulins

Native subunit proteins of glycinin, the acidic and the basic subunits designated as AS₁₊₂, AS₂₊₃, AS₄, AS₅, and AS₆ and BS, respectively, were isolated by DEAE-Sephadex A-50 column chromatography in the presence of 6 m urea and 0.2 m 2-mercaptoethanol.

Reconstitution of intermediary subunits involving a disulfide bridge from native acidic and basic subunits was investigated. Formation of the intermediary subunit was observed in combinations between BS and each acidic subunit except AS₆. The yields of the reconstituted intermediary subunits differed from one another.

Further, formation of the intermediary complexes was observed when native acidic and basic subunits of soybean glycinin and sesame 13 S globulin, respectively (or reverse combinations), were mixed under reductively denatured condition and subjected to the reconstitution procedure. Considerring the overall evidence, we may conclude that the complexes are probably a hybrid intermediary subunit.     

Immunological properties of isolated protein fractions from Artemia ribosomes

Acid-soluble ribosomal proteins from cysts of Artemia salina were separated by high-resolution polyacrylamide gel electrophoresis at pH 4.3. Three distinct protein bands, occurring in different parts of the electrophoretic pattern, were used for immunization in rabbits, and the γ-globulin fractions of the antisera were prepared. These preparations produced precipitation lines in agarose gel with protein extracted from whole 80S ribosomes and from 60S and 40S ribosomal subunits. With γ-globulin preparations from non-immune or anti-ovalbumin sera no reactions were obtained.     

Properties of a major α-globulin of rice endosperm

Globulin was isolated from milled rice (Oryza sativa, line IR480-5-9) by 5% NACl extraction and was precipitated by (NH₄)₂SO₂ or by dialysis against water. The extract was purified by repeated isoelectric precipitation at pH 4.5. The major globulin fraction (40%) exhibited one band by electrophoresis at pH 4.5 but two bands at pH 8.3. Similarly, one sharp peak was shown by sedimentation corresponding to 1.41S (α-globulin) in acetic acid (pH 2) and NaOH (pH 11.7) but a broad asymmetric peak was obtained at pH 6.7, 8.3 and 8.9. Gel filtration of the α-globulin at pH 6.5 exhibited 2 proteins with MW 20 000 and 98 000. The results suggest a pH dependent aggregation phenomenon. The two proteins could not be separated by DEAE-cellulose chromatography. SDS-polyacrylamide electrophoresis of α-globulin revealed one subunit with MW 18 000. This α-globulin is poorer in lysine and histidine but richer in cystine, methionine, arginine, tyrosine and glutamic acid than whole milled rice proteinfa]Taken part from the M.S. thesis of AAP from the University of the Philippine at Los Baños (1977).     

Subunit Structure of Soybean 11S Globulin

The acidic and the basic subunits were shown to be present in equimolar amounts in the 11S globulin molecule by the densitometric scanning of the SDS gel and the molecular weight consideration. The four acidic subunits (A₁, A₂, A₃ and A₄) were found to be present in the approximate molar ratio of 1:1:2:2. Four basic subunits separated and designated as B₁, B₂, B₃ and B₄ based on the relative mobilities in the acidic gel in 7 m urea were found to be present in the approximate molar ratio of 1:1:2:2. The four basic subunits were fractionated in approximately same amounts into three different peaks, peak I (B₁ and B₂), peak II (B₃) and peak III (B₄) by CM-Sephadex C–50 column chromatography in the presence of 6 m urea. Three kinds of intermediary subunits of 11S globulin were fractionated with DEAE-Sephadex A–50 in the absence of reducing agents in 6 m urea, and disulfide bonds appeared to participate in the binding between the acidic and the basic subunits in the molar ratio of 1: 1 with the following combinations; A₁ and A₂ combined with B₃, A₃ with B₁ and B₂, and A₄ with B₄. In view of the above results and molecular weight consideration, a new model of subunit structure was proposed for 11S globulin.     

A novel acid phosphatase excreted by Penicillium funiculosum that hydrolyzes both phosphodiesters and phosphomonoesters with aryl leaving groups

An acid phosphatase has been purified from the culture broth of Penicillium funiculosum by procedures including SP-Sephadex column chromatography and Sephacryl S-200 gel filtration. The phosphatase appears to be a 76 kDa heterodimer composed of 51 and 26 kDa subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzes both phosphodiesters and phosphomonoesters, but only those with aryl leaving groups. At the early phase of degradation of bis-p-nitrophenyl phosphate by the enzyme, inorganic phosphate and the intermediary product, p-nitrophenyl phosphate, are liberated at approximately the same rate. This indicates that the intermediary phosphomonoester produced on the enzyme is further hydrolyzed in situ or dissociates into the medium at approximately the same probability.     

The occurrence of a premature form of egg-specific protein in vitellogenic follicles ofBombyx mori

Summary Analysis of yolk proteins of the silkworm,Bombyx mori, by SDS-polyacrylamide gel electrophoresis and immunoblotting showed that there was a developmental change in subunit composition of egg-specific protein; egg-specific protein consisting of 72 kDa subunits alone (premature form) was found in vitellogenic follicles, whereas the protein in mature eggs was composed of 72 kDa and 64 kDa subunits (mature form). The premature form of egg-specific protein was purified from young ovaries to homogeneity using a high performance liquid chromatography system. The purified protein had an apparent molecular mass of 225 kDa which could not be distinguished from that of the mature form. By circular dichroism analysis, both egg-specific proteins were estimated to have about 30% -helix and 20% -sheet, but the mature form showed a relatively rigid conformation in the aromatic region. The premature egg-specific protein purified from vitellogenic ovaries, consisted of three 72 kDa subunits, whereas mature egg-specific protein was composed of two 72 kDa subunits and one 64 kDa subunit. All of these subunits showed the same immunoreactivity towards antiserum raised against the mature form. An identical NH₂-terminal amino acid sequence was found in both 72 kDa polypeptides and 64 kDa polypeptide for the initial 10 amino acids.Abbreviations SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - ESP egg-specific protein - Vtn vitellin     

Subunit constitution of carbonic anhydrase from Chlamydomonas reinhardtii

Carbonic anhydrase purified from the cell surface of Chlamydomonas reinhardtii was inactivated by treatment with dithiothreitol. This treatment caused dissociation of the holoenzyme into 35-kDa (A) and 4-kDa (B) subunits as revealed by SDS/PAGE. The 35-kDa subunit was further separated into two components A1 (35 kDa) and A2 (36.5 kDa) by SDS/PAGE using a gradient gel. These two components have the same amino acid sequence up to at least the 10th amino acid from the N-terminus. The molecular masses were estimated at 76 kDa and 35 kDa for the holoenzyme and the large subunit, respectively, and the molar ratio of the former to the latter at 1:2, by using the techniques of low-angle laser light-scattering photometry and precision differential refractometry combined with gel-filtration HPLC. The molar ratio of the 35-kDa/4-kDa subunits was estimated at 1:1 the gel-filtration HPLC monitored with precision differential refractometry. Atomic-absorption spectrophotometry revealed that the holoenzyme contains two atoms of zinc. These results suggest that the holoenzyme is a heterotetramer composed of two large subunits (A1 and A2) and two small subunits (B).     

Characterization of vitellin,egg-specific protein and 30 kDa protein from Bombyx eggs,and their fates during oogenesis and embryogenesis

Three major yolk proteins, vitellin, egg-specific protein and 30 kDa proteins, were purified from the same extracts of Bombyx mori eggs by high-performance liquid chromatography on a molecular sieving column. Each preparation was judged to be homogeneous by polyacrylamide gel electrophoresis. The subunit structure was estimated to be as follows: vitellin is a tetramer with a molecular mass of 420 kDa, consisting of two heavy subunits (178 kDa) and two light subunits (43 kDa); egg-specific protein is a trimer (225 kDa) of two heavy subunits (72 kDa) and one light subunit (64 kDa); 30 kDa proteins are a mixture of three monomers (1, 2 and 3) consisting of respective subunit molecular masses of 32.0, 31.0 and 29.5 kDa. The three yolk proteins contained the usual amino acids together with various lipids and carbohydrates. Antisera to each protein did not cross-react. The titration of vitellin, egg-specific protein and 30 kDa proteins on rocket immunoelectrophoresis showed a differential accumulation pattern during the course of oogenesis. In newly laid eggs, vitellin, egg-specific protein and 30 kDa proteins accounted for approx. 40%, 25% and 35%, respectively, in weight. The eggs developed in male hosts after implantation of ovary discs were deficient in vitellin but contained egg-specific protein and 30 kDa proteins at comparable levels to the normal female eggs. During embryogenesis, egg-specific protein was rapidly and completely utilized. Approx. 35% vitellin and 50% 30 kDa proteins remained unused and were carried over to the hatched larvae. Such accumulation and utilization of yolk proteins are correlated with the fates of the proteins during oogenesis and embryogenesis of B. mori.     

Participation of a metalloprotease in the fertilization-associated conversion of the egg envelope (chorion) of the fish, Oryzias latipes

The unfertilized egg envelope of medaka ( Oryzias latipes ) consists of two major groups of subunits, ZI-1,2 (74–76 kDa) and ZI-3 (49kDa). During egg envelope hardening after egg activation, both subunit groups decreased in amount, new protein bands of 57–65, 110 and 125 kDa appeared and, finally, no bands were detectable on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The 110 and 125 kDa bands are intermediates formed by polymerization of such subunit groups. In contrast, treatment with iodoacetamide, an inhibitor of polymerization, revealed that the 57–65 kDa intermediates originated from ZI-1,2 by limited hydrolysis. ZI-1,2 comprises at least three distinct proteins of quite similar structure with their N -termini undetectable by Edman degradation, while the 57–65 kDa intermediates also consist of at least three proteins with the same N -terminal amino acid sequence: DGKPSNPQQPQVPQYPSK-. This fact strongly suggests a participation of a protease in the conversion of ZI-1,2 into 57–65 kDa proteins. EDTA and 1,10-phenanthrolinium inhibited the conversion and both Ca²⁺ and Zn²⁺ recovered the inhibition. These results suggest that the assumed protease is a metalloprotease.     

Purification and characterization of prophenoloxidase from the haemolymph of Locusta migratoria

Prophenoloxidase (proPO) was purified from plasma of the locust, Locusta migratoria. This was achieved in three steps (gel filtration on 5300, anion exchange on QMA Memsep, and affinity chromatography on blue Trisacryl) without the use of anticoagulant buffer or inhibitors. The native protein had an apparent molecular mass of 250 kDa as determined by gel filtration and was likely composed of three non-covalently associated subunits of 81 kDa. Its amino acid composition was found to be very similar to that of Bombyx mori proPO. Purified locust proPO could be converted into phenoloxidase (PO) by α-chymotrypsin. Using L-dopa as substrate, K_in and V_max were determined to be 1.5 mM and 5 μM/s, respectively. © 1996 Wiley-Liss, Inc.     

Reevaluation of the Subunit Molecular Weights of Soybean 11S Globulin

The acidic and basic subunits are the main constituents of soybean 11S globulin. Each of these two subunits consists of three major polypeptides of similar size. The molecular weights of the acidic and basic subunits have been previously estimated to be 37,000 and 22,000, respectively, by SDS-polyacrylamide gel electrophoresis (Catsimpoolas et al, J. Set Food. Agric., 22, 448 (1971)). Reevaluation of the molecular weights by 6 m guanidine gel chromatography gave the values of 28,000 and 18,000, respectively. These are supported by results of equilibrium sedimentation in the same solvent. The previously reported values seem to have been overestimated, especially for the acidic subunits. The overestimations seem to be related to the high percentage of acidic amino acids, which causes the conformation of the SDS-protein polypeptide complexes of these subunits to deviate from those of proteins usually employed as standards for molecular weight estimations.     

Structural homology among the major 7s globulin subunits of soybean seed storage proteins

The soybean seed 7S globulin subunits, i.e. α, α′, β and γ-subunits of β-conglycinin, the γ-conglycinin subunit and the HI/HII and LII subunits of basic 7S globulin were purified and the NH₂-terminal amino acid sequences of all these subunits except the γ-subunit of β-conglycinin were determined. Only the NH₂-terminal regions of the α and α′-subunits showed high sequence homology. However, sequencing of tryptic peptides from the seven subunits revealed that internal region sequences were highly homologous among the four subunits of β-conglycinin. In contrast to the β-conglycinin subunits, no sequence homology was found among the other subunits. On the basis of these results, the major 7S globulin fraction is considered more heterogeneous in primary structure than another major globulin fraction, 11S globulin (glycinin), in soybean seeds.     

Effect of Trifluoroethanol on the Structural and Functional Properties of α-Crystallin

Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of β- and γ-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native α-crystallin and its two constituent subunits. A conformational change occurs from the characteristic β-sheet to the α-helix structure in both native α-crystallin and its subunits with the increase in TFE levels. Among the two subunits, αA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of αB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of αA-crystallin to bind and prevent the aggregation of αB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of αA-crystallin exposed to TFE and the inability of peptide chaperone—the functional site of αA-crystallin—to stabilize αB-crystallin at 20–30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct.     

Peptide Mapping Reveals Considerable Sequence Homology among the Three Polypeptide Subunits of G1 Storage Protein from French Bean Seed

The major storage protein, G1 globulin, of bean (cv. Tendergreen) seeds was subjected to limited proteolysis with trypsin, chymotrypsin, papain, proteinase K, and protease V8 and to cleavage with cyanogen bromide and 2-(2-nitrophenylsulfanyl)-3-methyl-3′bromoindolenine. Mapping of peptides separated from each of the three G1 subunits by polyacrylamide gel electrophoresis revealed that many proteolytic cleavage sites were present at similar positions on the subunits. Evidence was adduced that the G1 subunits are homologous in amino acid sequence for about 61% of their length. The remaining region (possibly COOH-terminal) of the subunits appears to be heterologous, with the α subunit bearing an additional methionine residue.