Diacetyl production by different strains of Lactococcus lactis subsp. lactis var. diacetylactis and Leuconostoc spp.

Summary Several strains of Lactococcus lactis subsp. lactis var. diacetylactis and Leuconostoc spp. were compared for product formation from citrate in milk cultures. Most strains produced acetoin and butanediol. Some strains derived from buffer starter cultures produced, in addition, -acetolactate. Lactococcus lactis strain C17, which produced acetoin and butanediol but no -acetolactate in culture, was compared physiologically with L. lactis strain Ru4, which produced only -acetolactate. Activities of enzymes involved in citrate metabolism were almost identical in both strains, with the exception of -acetolactate decarboxylase, which was missing in strain Ru4. The formation of -acetolactate, acetoin and diacetyl was further analysed in cell-free extracts. -Acetolactate synthase activity saturated at a high pyruvate concentration (100 mm). This is in agreement with the observed accumulation of pyruvate externally, and probably internally, during -acetolactate, acetoin and butanediol production by L. lactis cells.Correspondence to: J. Hugenholtz     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary The cell wall proteinases of Lactococcus lactis subsp. lactis NCDO 763 and L. lactis subsp. cremoris AC1 hydrolyse -casein with a similar specificity even though some quantitative differences can be observed for a few degradation products analysed by reverse phase HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The main peptides soluble in 1.1% trifluoroacetic acid and liberated by the two proteinases were identified and have been found to be the same for the two enzymes. They are located in two areas of the -casein sequence (53–93 and the C-terminal part: 129–209) and they include bitter tasting or physiologically active fragments. No narrow specificity was observed for these proteinases. However, glutamine and serine residues are more frequently encountered in position P1 and P1 of the sensitive peptide bond and the close environment (position P2 to P4 and P2 to P4) of the cleaved bond is mainly hydrophobic.     

Comparison of α-acetolactate synthase and α-acetolactate decarboxylase in Lactococcus spp. and Leuconostoc spp.

Summary Cell-free extracts of Leuconostoc and Lactococcus species were tested for their -acetolactate synthase and -acetolactate decarboxylase activities. In Leuconostoc mesenteroides subsp. cremoris, Leuconostoc mesenteroides subsp. mesenteroides and Leuconostoc lactis, the Km of -acetolactate synthase for pyruvate was close to 10 mM whereas it was 30 mM in Lactococcus lactis subsp. lactis biovar. diacetylactis. The Km of -acetolactate decarboxylase for -acetolactic acid was very low (0.3 mM) in Leuconostoc species in comparison to Lactococcus lactis subsp. lactis biovar. diacetylactis (60 mM). In the latter bacterium, -acetolactate decarboxylase showed a sigmoidal dependance upon -acetolactic acid and was activated by the three branchedchain amino acids: leucine, isoleucine and valine.     

Purification of an X-prolyl-dipeptidyl aminopeptidase from the cell wall proteolytic system of Lactococcus lactis subsp. cremoris

Summary The -casein specific cell wall proteolytic system of Lactococcus lactis subsp. cremoris P8-2-47 contains a metal-independent X-prolyl-dipeptidyl-aminopeptidase. Suitable substrates for its assay are Gly-Pro-nitroanilide and Ala-Pronitroanilide. It is suggested that the function of the enzyme is to cleave the proline-rich sequences of -casein, as shown by the degradation of -casomorphin. It is a serine proteinase with a monomer molecular mass of about 90 000 daltons, a temperature optimum of 45°–50°C, and a pH optimum of about 7.     

Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Manα3R β2-N-acetylglucosaminyl-transferase I using synthetic substrate analogues

UDP-GlcNAc: Man3R 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M _r 42,000). TheV _max for the pure enzyme with [Man6(Man3)Man6](Man3)Man4GlcNAc4GlcNAc-Asn as substrate was 4.6 µmol min^–1 mg^–1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds anN-acetylglucosamine (GlcNAc) residue in 1–2 linkage to the Man3Man-terminus of the substrate. Several derivatives of Man6(Man3)Man-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the -linked mannose of Man6(Man3)Man-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the 3-linked mannose (Man) of the substrate increases theK _M 20-fold. Modifications on the 6-linked mannose or on the core structure affect mainly theK _M and to a lesser degree theV _max, e.g., substitutions of the Man6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower theK _M, whereas various other substitutions at the 3-position increase theK _M slightly. Man6(Man3)4-O-methyl-Man4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.Abbreviations BSA Bovine serum albumin - Bn benzyl - Fuc, F l-fucose - Gal, G d-galactose - GalNAc, GA N-acetyl-d-galactosamine - Glc d-glucose - GlcNAc, Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man, M d-mannose - mco 8-methoxycarbonyl-octyl, (CH₂)₈ COOOCH₃ - Me methyl - MES 2-(N-morpholino)ethanesulfonate - NMR nuclear magnetic resonance - PMSF phenylmethylsulfonylfluoride - pnp p-nitrophenyl - SDS sodium dodecyl sulfate - T transferase - Tal d-talose - Xyl d-xylose; - {0, 2 + F} Man6 (GlcNAc2Man3) Man4GlcNAc4 (Fuc6) GlcNAc - {2, 2} GlcNAc2Man6 (GlcNAc2Man3) Man4GlcNAc4GlcNAc; M₅-glycopeptide, Man6 (Man3) Man6 (Man3) Man4 GlcNAc4GlcNAc-Asn Enzymes: GlcNAc-transferase I, EC 2.4.1.101; GlcNAc-transferase II, EC 2.4.1.143; GlcNAc-transferase III, EC 2.4.1.144; GlcNAc-transferase IV, EC 2.4.1.145; GlcNAc-transferase V, UDP-GlcNAc: GlcNAc2 Man6-R (GlcNAc to Man) 6-GlcNAc-transferase; GlcNAc-transferase VI, UDP-GlcNAc: GlcNAc6(GlcNAc2) Man6-R (GlcNAc to Man) 4-GlcNAc-transferase; Core 1 3-Gal-transferase, EC 2.4.1.122; 4-Gal-transferase, EC 2.4.1.38; 3-Gal-transferase, UDP-Gal: GlcNAc-R 3-Gal-transferase; blood group i 3-GlcNAc-transferase, EC 2.4.1.149; blood group I 6-GlcNAc-transferase, UDP-GlcNAc: GlcNAc3Gal-R (GlcNAc to Gal) 6-GlcNAc-transferase.     

Heat shock-induced protein synthesis inLactococcus lactis subsp.lactis

Heat shock inLactococcus lactis subsp.lactis may induce as many as 16 proteins after a temperature shift from 30° to 40°C. Five induced proteins were found to be immunologically related to theEscherichia coli GroEL, DnaK, DnaJ, and GrpE proteins, and to theBacillus subtilis ⁴³ factor. From these initial studies we conclude that, inL. lactis subsp.lactis, a heat shock response similar to that known to occur in other prokaryotes might exist.     

A descriptive model for citrate utilization by Lactococcus lactis ssp lactis bv diacetylactis

A model for the use of citrate by Lactococcus lactis ssp lactis bv diacetylactis CNRZ 125 is proposed. Citrate metabolism by this strain leads to the production of acetate, CO₂ and C4 compounds (diacetyl, acetoin, 2,3-butylene glycol). The model furnishes correct simulations, consistent with published results on the pathways used and on lactose-citrate co-metabolism. Citric acid is incorporated independently of growth. The production of flavoring compounds is a complex process, depending on the rate of citrate utilization, on the proportion of pyruvate arising from citrate and which condenses to form -acetolactate and CO₂, on the rate of transformation of -acetolactate to diacetyl and acetoin, as well as on the rate of reduction of these compounds to 2,3-butylene glycol.     

The polypeptide structure and assembly of Ly-2/3 heterodimers

Mild reduction of mature, thymic Ly-2/3 heterodimers of M _r 67 000 resulted in dissociation into three individual polypeptide chains, , , and , of respective M _r values 38000, 35000, and 30000. The and chains were both immunoprecipitated by a monoclonal antibody directed to the Ly-2.1 epitope whereas the Ly-3.1 antibody bound only the chain. The possibility that the and chains of each heterodimer established their interchain links within a labile precursor protein in which a and segments were fused was considered but discounted by the finding that in mice heterozygous for both Ly-2 and Ly-3 loci, the Ly-2 product of one chromosome was not exclusively joined to Ly-3 structures coded by the same chromosome. By utilizing ionic detergents which selectively alter the charge of intrinsic membrane proteins, both Ly-2 and Ly-3 polypeptides were shown to have membrane insertion sites. It is suggested that as a consequence of their likely synthesis on membrane-bound polysomes, newly synthesized Ly-2 and Ly-3 structures accumulate within the same subcellular compartment — the membranes of the rough endoplasmic reticulum. Their elevated concentration within this space may facilitate a low affinity binding interaction between Ly-2 and Ly-3 which is later stabilized by interchain disulfide bond formation.Abbreviations used in this paper BSA bovine serum albumin - DOC sodium deoxycholate - DTT dithiothreitol - HA hemagglutinin - HTAB hexadecyltrimethylammonium bromide - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TX100 Triton X-100     

Interaction of alpha-lactalbumin with mini-alphaA-crystallin

A-Crystallin can function like a molecular chaperone. We have recently shown that residues 71-88 in A-crystallin represent the chaperone active site of the protein. A peptide containing the sequence of A-crystallin sequence DFVIFLDVKHFSPEDLTVK (mini A-crystallin) by itself displays the antiaggregation property of A-crystallin. We have prepared a complex of reduced -lactalbumin and mini-A-crystallin and investigated the nature, conformation, and properties of the complex by dynamic light scattering, HPLC analysis, CD spectroscopy, and fluorescence studies. Although mini-A was able to prevent the precipitation of reduced -lactalbumin, large aggregates (50-500 nm) of the complex were formed during the assay. Amino acid composition estimation revealed that -lactalbumin and mini-A-crystallin were present in 1:2 ratio in the aggregates. During our study significant red shift in the Trp fluorescence emission maximum and an increase in Bis-ANS binding to the mini A-crystallin-bound -lacatalbumin were observed. The CD spectra of the complex showed a significant loss of -helical content but the -sheet content appeared to be less affected, indicating the molten-globule state of the reduced lactalbumin in the complex. These data show that the active site of A-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form.     

Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Lacticin 3147 favours isoleucine transamination by Lactococcus lactis IFPL359 in a cheese-model system

The bacteriocin, lacticin 3147, increased isoleucine transamination by Lactococcus lactis IFPL359 in a cheese model system. The formation of -keto--methyl-n-valeric acid and 2-hydroxy-3-methyl-valeric acid increased by three times in cheese slurries at 12 °C and cheese aroma intensity increased as well, which corresponded with a higher 2-methylbutanal formation.     

X-ray microanalysis of calcium binding sites in Paramecium

Summary In Paramecium cells Ca⁺⁺-stimulated triggering of the exocytosis of secretory vesicles (trichocysts) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some resting trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the inner lamellar sheath from where deposits seemed to radiate into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those resting trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca⁺⁺-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca⁺⁺ and then fixed with OsO₄ plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including ciliary granule plaques) also contained Ca, P and S. Cells contain osmiophilic calcium-storing vacuoles which were selectively rich in Ca and S but devoid of P.     

A new mouse T-cell receptorα chain variable region family

Sequence analysis of the rearranged T-cell receptor a chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribedV gene family. We have designated this familyV 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously describedV haplotypes:V ^a ,V ^b , andV ^c .     

Expression pattern of voltage-dependent calcium channel α1 and β subunits in adrenal gland of N-type Ca2+ channel α1B subunit gene-deficient mice

The Ca²⁺ channel _1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although N-type Ca²⁺ channel plays a role in a variety of neuronal functions, _1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca²⁺ channel ₁ or subunit. In this study, we studied the levels of _1A, _1C, _1D, _1E, ₁, ₂, ₃ and ₄ mRNAs in adrenal gland of _1B-deficient mice. The _1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of _1C, _1D, _1E, ₁, ₂, ₃ and ₄ was found among wild, heterozygous and homozygous mice. The protein level of _1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of _1A gene, we examined lacZ expression in _1B-deficient × _1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of _1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous _1B-deficient × _1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of _1B-deficient mice is that compensation by _1A subunit occurs and that 6.3-kb 5-upstream region of _1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: a consequence of the formation of "native-like conformation".

The influence of n-propanol on the overall -helical conformation of -globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of -helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of -helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of -globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The -helical conformation induced into - and -globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the -helical conformation of both - and -chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced -helical conformation of globins and the -helical conformation of - and -chains. A cross-correlation of the n-propanol induced increase in the fluorescence of -globin with the corresponding increase in the -helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the -helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a native-like structure. The induction of -helical conformation into these proteins in the presence of n-propanol and the consequent generation of native-like conformation is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an -helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume -helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high -helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of -helical proteins. Consistent with this concept, the induction of -helical conformation into shorter polypeptide fragments of 30 residues, (e.g., _1-30, which exists in an -helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Linkage of the β-Like ω-Globin Gene to α-Like Globin Genes in an Australian Marsupial Supports the Chromosome Duplication Model for Separation of Globin Gene Clusters

The structure, function, and evolutionary history of globin genes have been the subject of extensive investigation over a period of more than 40 years, yet new globin genes with highly specialized functions are still being discovered and much remains uncertain about their evolutionary history. Here we investigate the molecular evolution of the -globin gene family in a marsupial species, the tammar wallaby, Macropus eugenii. We report the complete DNA sequences of two -like globin genes and show by phylogenetic analyses that one of these genes is orthologous to embryonically expressed -globin genes of marsupials and eutherians and the other is orthologous to adult expressed -globin genes of marsupials and eutherians. We show that the tammar wallaby contains a third functional -like globin gene, -globin, which forms part of the -globin gene cluster. The position of -globin on the 3 side of the -globin cluster and its ancient phylogenetic history fit the criteria, originally proposed by Jeffreys et al. (1980), of a fossil -globin gene and suggest that an ancient chromosome or genome duplication preceded the evolution of unlinked clusters of - and -globin genes in mammals and avians. In eutherian mammals, such as humans and mice, -globin has been silenced or translocated away from the -globin locus, while in marsupials -globin is coordinately expressed with the adult -globin gene just prior to birth to produce a functional hemoglobin (_{2 2}).     

Comparative Analysis of the Mechanisms Underlying Nifedipine-Induced Blockade of Three Subtypes of T-Type Ca2+ Channels

We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca²⁺ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC₅₀ = 98 and 243 M, maximum blocking intensity A_max = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC₅₀ = 5 M and A_max = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca²⁺ channels in native cells.     

Variability in the 11S globulin fraction of seed storage proteins ofHelianthus (Asteraceae)

The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (₁₁ and ₂₂). The subunit corresponding to the ₁₁ pair is present inH. petiolaris and in the three populations ofH. annuus studied. The _2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific _2a2 and ₄₄ pairs andH. annuus a specific ₃₃ pair. InH. argophyllus _{11 33} or ₄₄ are never observed but are replaced by ₁₃ and ₃₁ pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.