Crystal structure of Plasmodium falciparum phosphoglycerate kinase: evidence for anion binding in the basic patch

3-Phosphoglycerate kinase (EC 2.7.2.3) is a key enzyme in the glycolytic pathway and catalyzes an important phosphorylation step leading to the production of ATP. The crystal structure of Plasmodium falciparum phosphoglycerate kinase (PfPGK) in the open conformation is presented in two different groups, namely I222 and P6₁22. The structure in I222 space group is solved using MAD and refined at 3 Å whereas that in P6₁22A is solved using MR and refined at 2.7 Å. I222 form has three monomers in asymmetric unit whereas P6₁22 form has two monomers in the asymmetric unit. In both crystal forms a sulphate ion is located at the active site where ATP binds, but no Mg²⁺ ion is observed. For the first time another sulphate ion is found at the basic patch where the 3-phosphate of 1,3-biphosphoglycerate normally binds. This was found in both chains of P6₁22 form but only in chain A of I222 form.     

Characterization of a tetrameric G4 form of acetylcholinesterase from bovine brain: a comparison with the dimeric G2 form of the electric organ

Globular forms (G forms) of acetylcholinesterase (AChE) are formed by monomers, dimers and tetramers of the catalytic subunits (G₁, G₂ and G₄). In this work the hydrophobic G₂ and G₄ AChE forms were purified to homogeneity from Discopyge electric organ and bovine caudate nucleus and studied from different points of view, including: velocity sedimentation, affinity to lectins and SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions. The polypeptide composition of Discopyge electric organ G₂ is similar to Torpedo, however the pattern of the brain G₄ AChE is much complex. Under non-reducing conditions the catalytic subunit possesses a molecular weight of 65 kDa, however this value increases to 68 kDa after reduction, suggesting that intrachain-disulfide bonds are important in the folding of the catalytic subunits of the AChE. Also it was found that after mild proteolysis; the (¹²⁵I)-TID-20 kDa fragment decreased its molecular weight to approximately 10 kDa with little loss of AChE activity. Finally, we suggest a model for the organization of the different domains of the hydrophobic anchor fragment of the G₄ form.     

Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines

Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed inEscherichia coli as periplasmic inclusion bodies. Their variable heavy (V_H) and light (V_L) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II ofDrosophila melanogaster and rat MAb Yol1/34, specific for pig brain α-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv′)₂ was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv′)₂ have binding constants quite close to those of the parental MAbs and fourfold higher than scFv′ monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.     

Short communication: Purification and properties of acid phosphatase (EC 3.1.3.2) secreted by strain 74A of the mould Neurospora crassa

Both P_i-repressible acid phosphatases, IIb (mycelial) and IIc (extracellular), synthesized by Neurospora crassa and purified to apparent homogeneity by 7.5% PAGE, are monomers, are inhibited by 2 mm ZnCl₂ and are non-specifically stimulated by salts. However, the IIc form is activated by p-nitrophenylphosphate (in a negative co-operativity effect with a K _0.5 of 2.5 mm) whereas form IIb shows Michaelis kinetics, with a K _m of 0.5 mm. Thus, since both enzymatic forms may be expressed by the same gene (pho-3), it is possible that post-translational modifications lead to the excretion of an enzymatic form with altered Michaelis kinetics compared with the enzymatic form retained by the mycelium.     

Reactivation and refolding of reassociated dimers of rabbit muscle creatine kinase

Creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) is a good model for studying dissociation and reassociation during unfolding and refolding. This study compares self-reassociated CK dimers and CK dimers that contain hybrid dimers under proper conditions. Creatine kinase forms a monomer when denatured in 6 M urea for 1 h which will very quickly form a dimer when the denaturant is diluted under suitable conditions. After modification by DTNB, CK was denatured in 6 M urea to form a modified CK monomer. Dimerization of this modified subunit of CK occurred upon dilution into a suitable buffer containing DTT. Therefore, three different types of reassociated CK dimers including a hybrid dimer can be made from two different CK monomers in the proper conditions. The CK monomers are from a urea-denatured monomer of DTNB-modified CK and from an unmodified urea dissociated monomer. Equal enzyme concentration ratios of these two monomers were mixed in the presence of urea, then diluted into the proper buffer to form the three types of reassociated CK dimers including the hybrid dimer. Reassociated CK dimers including all three different types recover about 75% activity following a two-phase course (k ₁ = 4.88 × 10^–3 s^–1, k ₂ = 0.68 × 10^–3 s^–1). Intrinsic fluorescence spectra of the three different CK monomers which were dissociated in 6 M urea, dissociated in 6 M urea after DTNB modification, and a mixture of the first two dissociated enzymes were studied in the presence of the denaturant urea. The three monomers had different fluorescence intensities and emission maxima. The intrinsic fluorescence maximum intensity changes of the reassociated CK dimers were also studied. The refolding processes also follow biphasic kinetics (k ₁ = 3.28 × 10^–3 s^–1, k ₂ = 0.11 × 10^–3 s ^–1) after dilution in the proper solutions. Tsou's method [Tsou (1988), Adv. Enzymol. Rel. Areas Mol. Biol. 61, 381–436] was also used to measure the kinetic reactivation rate constants for the different three types of reassociated CK dimers, with different kinetic reactivation rate constants observed for each type. CK dissociation and reassociation schemes are suggested based on the results.     

Catalytically Active Monomers of E. coli Glyceraldehyde-3-Phosphate Dehydrogenase

Monomeric forms of E. coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting K _M values close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.     

Genetical control and linkage relationships of isozyme markers in sugar beet (B. vulgaris L.)

Summary Five isozyme systems were genetically investigated. The different separation techniques, the developmental expression and the use as marker system in sugar beet genetics and breeding is discussed. Isocitrate dehydrogenase was controlled by two genes. The gene products form inter- as well as intralocus dimers, even with the gene products of the Icd gene in B. procumbens and B. patellaris. Adenylate kinase was controlled by one gene. Three different allelic forms were detected, which were active as monomeric proteins. Glucose phosphate isomerase showed two zones of activity. One zone was polymorphic. Three allelic variants, active as dimers, were found. Phosphoglucomutase also showed two major zones of activity. One zone was polymorphic and coded for monomeric enzymes. Two allelic forms were found in the accessions studied. The cathodal peroxidase system was controlled by two independent genes, of which only one was polymorphic. The gene products are active as monomers. Linkage was found between red hypocotyl color (R) and Icd ₂. Pgm ₁, Gpi ₂, Ak ₁ and the Icd ₂-R linkage group segregated independently.     

Antirestriction activity of the monomeric and dimeric forms of T7 Ocr

The Ocr antirestriction protein, which is encoded by bacteriophage T7 0.3 (ocr), specifically inhibits type I restriction-modification enzymes. Ocr belongs to a family of DNA-mimicking proteins. Native Ocr forms homodimers both in solution and in crystal. Ocr mutants with two amino acid substitutions (Orc F53D A57E and Ocr F53R V77D) were constructed to occur as monomers in solution. The dissociation constant K _d for the Ocr complex with EcoKI (R₂M₂S) proved to differ by three orders of magnitude between the (Ocr)₂ dimer and Ocr F53D A57E and Ocr F53R V77D monomers (10^?10 M vs. 10^?7 M). Antimodification activity was substantially lower in the Ocr monomers. The dimeric form found to be essential for high inhibitory activity of Ocr.     

Cuticle lipids on heteromorphic leaves of Populus euphratica Oliv. growing in riparian habitats differing in available soil moisture

Populus euphratica is an important native tree found in arid regions from North Africa and South Europe to China, and is known to tolerate many forms of environmental stress, including drought. We describe cuticle waxes, cutin and cuticle permeability for the heteromorphic leaves of P. euphratica growing in two riparian habitats that differ in available soil moisture. Scanning electron microscopy revealed variation in epicuticular wax crystallization associated with leaf type and site. P. euphratica leaves are dominated by cuticular wax alkanes, primary‐alcohols and fatty acids. The major cutin monomers were 10,16‐diOH C_16:0 acids. Broad‐ovate leaves (associated with adult phase growth) produced 1.3‐ and 1.6‐fold more waxes, and 2.1‐ and 0.9‐fold more cutin monomers, than lanceolate leaves (associated with juvenile phase growth) at the wetter site and drier site, respectively. The alkane‐synthesis‐associated ECERIFERUM1 (CER1), as well as ABC transporter‐ and elongase‐associated genes, were expressed at much higher levels at the drier than wetter sites, indicating their potential function in elevating leaf cuticle lipids in the dry site conditions. Higher cuticle lipid amounts were closely associated with lower cuticle permeability (both chlorophyll efflux and water loss). Our results implicate cuticle lipids as among the xeromorphic traits associated with P. euphratica adult‐phase broad‐ovate leaves. Results here provide useful information for protecting natural populations of P. euphratica and their associated ecosystems, and shed new light on the functional interaction of cuticle and leaf heterophylly in adaptation to more arid, limited‐moisture environments.     

RCN1/OsABCG5, an ATP‐binding cassette (ABC) transporter,is required for hypodermal suberization of roots in rice (Oryza sativa)

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP‐binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP‐RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C₂₈ and C₃₀ fatty acids or ω‐OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.     

Purification and properties of the RuvA and RuvB proteins of Escherichia coli

Summary The RuvA and RuvB proteins of Escherichia coli play important roles in the post-replicational repair of damaged DNA, genetic recombination and cell division. In this paper, we describe the construction of over expression vectors for RuvA and RuvB and detail simple purification schemes for each protein. The purified 22 kDa RuvA polypeptide forms a tetrameric protein (M_r ca. 100000) as observed by gel filtration. The tetramer is stabilised by strong disulphide bridges that resist denaturation during SDS-PAGE (in the absence of boiling and -mercaptoethanol). In contrast, purified RuvB polypeptides (37 kDa) weakly associate to form a dimeric protein (M_r ca. 85000). At low protein concentrations, the RuvB dimer dissociates into monomers. The multimeric forms of each protein may be covalently linked by the bifunctional cross-linking reagent dimethyl suberimidate. Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction was found to stimulate the rate of strand exchange leading to the rapid formation of heteroduplex DNA.     

The effect of lipids on the enzymatic activity of 6-phosphofructo-1-kinase from B. stearothermophilus

6-Phosphofructo-1-kinase (PFK-1), a major regulatory enzyme in the glycolysis pathway, is a cytoplasmic enzyme with complicated allosteric kinetics. Here we investigate the effects of lipids on the activity of PFK from Bacillus stearothermophilus (BsPFK), to determine whether BsPFK shares any of the membrane binding or lipid binding properties reported for some mammalian PFKs. Our results show that large unilamellar vesicles (LUVs) composed of either the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or of 1:1 (mole ratio) DOPC and the fatty acid, oleic acid (OA), cause a three-fold increase in V_max, depending on the lipid concentration and vesicle composition, but no change in K_m. Further studies show lipids do not reverse the allosteric inhibitory effects of phosphoenolpyruvate (PEP) on BsPFK. SDS/PAGE studies do not show significant binding of the BsPFK tetramer to the surface of the phospholipid vesicles, suggesting that modulation of catalytic activity is due to binding of lipid monomers. By simulating the kinetics of BsPFK interaction with vesicles and lipid monomers we conclude that the change in BsPFK catalytic activity with respect to lipid concentration is consistent with monomer abstraction from vesicles rather than direct uptake of lipid monomers from solution.     

Isolation and characterization of octopus hepatopancreatic glutathione S-transferase. Comparison of digestive gland enzyme with lens S-crystallin

Glutathione S-transferase fromOctopus vulgaris hepatopancreas was purified to apparent homogeneity by single glutathione-Sepharose-4B affinity chromatography with overall yield 46% and purification 249-fold. The enzyme was a homodimer with subunitM _r 24,000, which was smaller than that of the octopus lens S-crystallin (M _r 27,000) with glutathione-S-transferase-like structure. Both proteins showed substrate specificities similar to/-type isozyme of glutathione S-transferase. Under native conditions, both proteins exhibited multiple forms upon polyacrylamide gel electrophoresis or isoelectric focusing, albeit with distinct mobilities; however, only one kind of N-terminal amino acid sequence was determined for the multiple forms of each protein. The hepatopancreatic GST, withpI value 6.6–7.3, dissociated into two monomers in an acidic or alkaline environment. Two amino acid residues, withpK _a values 5.69±0.14 and 9.03±0.11 were involved in the subunit interactions of the hepatopancreatic enzyme.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - IEF isoelectric focusing - GSH glutathione - GST glutathione S-transferase - CDNB 1-chloro-2,4-dinitrobenzene - EA ethacrynic acid [2,3-dichloro-4-(2-methylenebutyryl) phenoxy)acetic acid]     

Interaction of dimeric and monomeric enkephalins with NG108-15 hybrid cells

The binding of the enkephalin dimer [d-Ala², Leu⁵-NH-CH₂-]₂ (DPE₂) is characterized by (1) its high affinity for receptors on NG108-15 hybrid cells, the affinity constantK=4.7×10⁹ M^–1 is up to 8-fold that of monomers (0.6 to 1.0×10⁹ M^–1), and (2) a maximal binding capacity equal to one half that of the monomers. Kinetic studies showed that DPE₂ binds with a 2-fold higher rate, k₁=6.3×10⁷ M^–1min^–1, than monomers (2.4 to 3.8×10⁷ M^–1min^–1), and dissociates at a slower rate than monomers. Dissociation of DPE₂ was consistently bi- or multiphasic but increased about 12% only after 3 hr of dissociation in the presence of a large excess of unlabeled enkephalin. The dissociation kinetics of monomers varied with enkephalin and experimental conditions used. Consistent with the value for the maximal binding capacity, the kinetic studies are interpreted in support of the hypothesis that DPE₂ binds by cross-linking two subunits of one receptor.     

Stereoselective Degradation of alpha‐Cypermethrin and Its Enantiomers in Rat Liver Microsomes

Alpha‐cypermethrin (α‐CP), [(RS)‐a‐cyano‐3‐phenoxy benzyl (1RS)‐cis‐3‐(2, 2‐dichlorovinyl)‐2, 2‐dimethylcyclopropanecarboxylate], comprises a diastereoisomer pair of cypermethrin, which are (+)‐(1R‐cis‐αS)–CP (insecticidal) and (?)‐(1S‐cis‐αR)–CP (inactive). In this experiment, the stereoselective degradation of α‐CP was investigated in rat liver microsomes by high‐performance liquid chromatography (HPLC) with a cellulose‐tris‐ (3, 5‐dimethylphenylcarbamate)‐based chiral stationary phase. The results revealed that the degradation of (?)‐(1S‐cis‐αR)‐CP was much faster than (+)‐(1R‐cis‐αS)‐CP both in enantiomer monomers and rac‐α‐CP. As for the enzyme kinetic parameters, there were some variances between rac‐α‐CP and the enantiomer monomers. In rac‐α‐CP, the V_max and CL_int of (+)‐(1R‐cis‐αS)–CP (5105.22 ± 326.26 nM/min/mg protein and 189.64 mL/min/mg protein) were about one‐half of those of (?)‐(1S‐cis‐αR)–CP (9308.57 ± 772.24 nM/min/mg protein and 352.19 mL/min/mg protein), while the K_m of the two α‐CP enantiomers were similar. However, in the enantiomer monomers of α‐CP, the V_max and K_m of (+)‐(1R‐cis‐αS) ‐CP were 2‐fold and 5‐fold of (?)‐(1S‐cis‐αR)‐CP, respectively, which showed a significant difference with rac‐α‐CP. The CL_int of (+)‐(1R‐cis‐αS)–CP (140.97 mL/min/mg protein) was still about one‐half of (?)‐(1S‐cis‐αR)–CP (325.72 mL/min/mg protein) in enantiomer monomers. The interaction of enantiomers of α‐CP in rat liver microsomes was researched and the results showed that there were different interactions between the IC₅₀ of (?)‐ to (+)‐(1R‐cis‐αS)‐CP and (+)‐ to (?)‐(1S‐cis‐αR)‐CP(IC_50(?)/(+) / IC_50(+)/(?) = 0.61). Chirality 28:58–64, 2016. © 2015 Wiley Periodicals, Inc.     

Palmitoylation participates in G protein coupled signal transduction by affecting its oligomerization

Much in vivo and in vitro evidence has shown that the α subunits of heterotrimeric GTP-binding proteins (G proteins) exist as oligomers in their base state and disaggregate when being activated. In this article, the influence of palmitoylation modification of Gα_o on its oligomerization was explored extensively. Gα_o protein was expressed and purified from Escherichia coli strain JM109 cotransformed with pQE60(Gα_o) and pBB131(N-myristoyltransferase). Non-denaturing gel electrophoresis analysis revealed that Gα_o existed to a small extent as monomers but mostly as oligomers including dimers, trimers, tetramers and pentamers which could disaggregate completely into monomers by GTPγS stimulation. Palmitoylated Gα_o, on the other hand, only present as oligomers that were difficult to disaggregate into monomers. The effect of palmitoylation on oligomerization of Gα_o was further investigated by several other biochemical and biophysical methods including gel filtration chromatography, analytical ultracentrifugation and atomic force microscopy analysis. The results consistently demonstrated that palmitoylation facilitated oligomerization of the Gα_o protein. Autoradiography indicated that [¹⁴C]-palmitoylated Gα_o would in no case disaggregate into monomers after treatment with GTPγS. [³⁵S]-GTPγS binding activity assay showed that palmitoylated Gα_o was saturated at only 7.8 nmol/mg compared to 21.8 nmol/mg for non-palmitoylated Gα_o. Fluorescent quenching studies using BODIPY FL-GTPγS as a probe showed that the conformation of GTP-binding domain of Gα_o tended to become more compact after palmitoylation. These results implied that palmitoylation may regulate the GDP/GTP exchange of Gα_o by influencing the oligomerization state of Gα_o and thereby modulate the on-off switch of the G protein in G protein-coupled signal transduction.     

A molecular mechanics and semiempirical molecular orbital study on the conformation of polynorbornene chains

The conformational analysis of polynorbornene (PNB) chains was investigated with the AM1, MM2, AMBER and OPLS methods taking into consideration the possibility of binding of norbornene monomers to each other at various positions, i.e. exo–exo, exo–endo, endo–endo. The chain that is formed by connecting exo–endo positions of the monomers has lower torsional barrier energy than those formed with bonds at other positions and has more flexibility. It is determined that the thredisyndiotactic chain formed by exo–endo addition adopts a helix structure and has a coil shape. The disyndiotactic chain formed by connecting norbornene monomers in mixed type has a linear structure. It is found that the repeat unit conformations of thredisyndiotactic and disyndiotactic chains of PNB are TGTG^– and (TGTG^–)₂, respectively.     

Fractal dimension analysis and mathematical morphology of structural changes in actin filaments imaged by electron microscopy

In this work, we examined structural changes of actin filaments interacting with myosin visualized by quick freeze deep-etch replica electron microscopy (EM) by using a new method of image processing/analysis based on mathematical morphology.In order to quantify the degree of structural changes, two characteristic patterns were extracted from the EM images. One is the winding pattern of the filament shape (WP) reflecting flexibility of the filament, and the other is the surface pattern of the filament (SP) reflecting intra-molecular domain-mobility of actin monomers constituting the filament. EM images were processed by morphological filtering followed by box-counting to calculate the fractal dimensions for WP (D_WP) and SP (D_SP). The result indicates that D_WP was larger than D_SP irrespective of the state of the filament (myosin-free or bound) and that both parameters for myosin-bound filaments were significantly larger than those for myosin-free filaments. Overall, this work provides the first quantitative insight into how conformational disorder of actin monomers is correlated with the myosin-induced increase in flexibility of actin filaments along their length as suggested by earlier studies with different techniques. Our method is yet to be improved in details, but promising as a powerful tool for studying the structural change of protein molecules and their assemblies, which can potentially be applied to a wide range of biological and biomedical images.     

Kinetics of formation of fibrin oligomers. I. Theory

The course of formation of fibrin oligomers is treated theoretically for the condition that self-assembly of fibrin monomers is rapid compared with the loss of A peptides by the enzymatic action of thrombin. The rate constant for removal of the second A peptide is taken to be larger than that for the first by an arbitrary factor q; the association of activated A sites with their complementary a sites is assumed to be random and independent of oligomer size. Two types of oligomers are considered: noncovalently bonded protofibrils formed by the staggered overlap of thrombin-activated monomers and covalently bonded linear oligomers formed by factor XIIIa-mediated end-to-end ligation of adjacent monomers within protofibrils. Oligomers of the first type, if ligated, are dissociated to oligomers of the second type by solubilization in SDS–urea. Theoretical curves are presented for x _w and x ′_w (weight-average degree of polymerization of staggered overlap and linear ligated oligomers, respectively) and for the weight fractions of monomer, dimer, and decamer of both ligated and unligated species as functions of y, the fraction of A peptide removed; and also for w_x and w′_x, the weight fractions of x-mer of the respective oligomer types, as a function of x at y = 0.5. With increasing q, the maximum w_x or w′_x that a low oligomer will reach during the reaction decreases and the size distribution is broadened toward larger oligomers. Comparison with experiment is made in a companion paper.     

Genetical control and linkage relationships of Isozyme markers in sugar beet (B. vulgaris L.). 2. NADP- and NAD-specific malate dehydrogenases, 6-P-gluconate dehydrogenase,shikimate dehydrogenase,diaphorase and aconitase

Summary The NADP-specific malate dehydrogenase isozymes were controlled by multiple gene systems. Three genes coding for dimeric enzymes segregated in a dependent fashion (NADP-Mdh ₁, NADP-Mdh ₂, NADP-Mdh ₃). A fourth gene (NADP-Mdh ₄), also coded for dimers, but was not polymorphic in B. vulgaris. A fifth gene (NADP-Me ₁) coded for enzymes active as monomers. Two genes were found to control the main zone of NAD-specific malate dehydrogenase: one coded for dimers (Mdh ₁), while a second (Mdh ₂) was not polymorphic in the assessions studied. 6-P-Gluconate dehydrogenase was not polymorphic in B. vulgaris; the two types detected on SGE₁ electrophoresis were due to developmental expression of the different systems. No genetical segregations could be detected in progeny of crosses of the distinct phenotypes. A shikimate dehydrogenase gene (Skdh ₁) that coded for monomers was identified. The diaphorase system was rather complex, but one gene (Dia ₁) coding for monomeric enzymes could be identified. Aconitase was found to be controlled by two independent genes (Aco ₁, Aco ₂), both polymorphic and coding for proteins active as monomers. Tight linkage was found between the genes NADP-Mdh ₁, NADP-Mdh ₂ and NADP-Mdh ₃. Linkage was also found between a pollen fertility restorer (Z) and the Mdh ₁ gene. The identification of linkage with Aco ₁ needs further investigation. R segregated independently from Mdh ₁, Aco ₁ and Dia ₁. Independent segregations were scored for isozyme genes Pgm ₂, Icd ₁, Ak ₁, Gpi ₁, Aco ₁ and Dia ₁.Abbreviations Tris-HCl Tris (hydroxymethyl) aminomethane-HCl - NADP nicotinamide adenine dinucleotide phosphate - NBT nitro-blue tetrazolium chloride monohydrate - PMS phenazine methosulphate