Histone gene expression in early development of Xenopus laevis

Abstract. This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis , and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3.
Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.     

Expression of rat liver canalicular sulfate carrier in Xenopus laevis oocytes

Poly(A)+ RNA (mRNA)extracted from rat liver was injected into Xenopus laevis oocytes and the expression of sulfate transport was determined by measuring [35S] sulfate uptake. Compared to water-injected oocytes, which exhibited virtually no sulfate uptake, injection of rat liver mRNA resulted in a time- and dose-dependent increase in uptake of sulfate. Depending on the method used for the isolation of the mRNA, sulfate uptake was stimulated after injection (40 ng after 6 days) between 8- and 72-fold compared to water-injected oocytes. Sulfate uptake of oocytes injected with mRNA was found to be sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (IC50 less than 20 microM) and could also be inhibited by thiosulfate. Sulfate uptake of injected oocytes showed Michaelis-Menten kinetics (apparent Km, 0.31 mM) which is similar to the Km of the sulfate/bicarbonate antiporter of rat liver canalicular plasma membranes. After fractionation by a sucrose density gradient, the mRNA encoding for the expressed rat liver sulfate carrier was found in fractions containing messages of 3.5-4.0 kilobases in length.     

Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots

Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9 +/- 1.0 microm). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 microm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 microm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.     

Placental, renal, and ileal sulfate transporter gene expression in mouse gestation

Sulfate is important for mammalian growth and development. During pregnancy, maternal circulating sulfate levels increase by 2-fold, enhancing sulfate availability to the fetus. We used quantitative real-time PCR to determine sulfate transporter mRNA levels during mouse gestation in three tissues: kidney and ileum, to identify transporters involved in sulfate absorption and maintaining high maternal circulating sulfate level; and placenta, to build a model of directional sulfate transport from mother to fetus. In the kidney, Slc13a1 and Slc26a1 were the most abundant sulfate transporter mRNAs, which increased by ≈2-fold at E4.5 or E6.5, whereas lower levels of Slc26a2, Slc26a6, and Slc26a7 mRNA increased by ≈3- to 6-fold from E4.5. Ileal sulfate transporter mRNA levels were not increased in gestation, but slight decreases (by ≈30-40%) were found for Slc26a3 and Slc26a6. In placentae, Slc13a4 and Slc26a2 mRNAs were most abundant, with levels increasing from E10.5 and peaking (≈8-fold) from E14.5 to E18.5, whereas Slc26a1 increased by ≈3-fold at E18.5. The spatial expression of placental mRNAs was determined by in situ hybridization showing Slc13a4 and Slc26a6 in yolk sac, Slc26a1 in spongiotrophoblasts, and Slc13a4, Slc26a2, Slc26a3, and Slc26a7 in the labyrinthine layer. Within the labyrinth, cell-specific staining revealed Slc13a4 expression in syncytiotrophoblast-II (SynT-II) and Slc26a2 in SynT-I. Together, these data show kidney Slc13a1 and Slc26a1 and placental Slc13a4 and Slc26a2 to be the most abundant sulfate transporter mRNAs in mouse gestation, which likely play important physiological roles in maintaining high maternal serum sulfate levels during pregnancy and mediating sulfate supply to the fetus.     

Cell-free translation of adenovirus 2 E1a- and E1b-specific mRNAs and evidence that E1a-related polypeptides are produced from E1a-E1b overlapping mRNA

We have characterized the polypeptides translated in vitro by mRNAs of early region 1 (E1) of human adenovirus (Ad) type 2. Poly (A+) polyribosomal RNA was isolated from early Ad2-infected cells, the viral specific mRNAs were selected by hybridization to Ad2 E1a and E1b DNA, and the mRNAs were translated in vitro using [35S]methionine as a labeled precursor with a rabbit reticulocyte lysate. E1a-selected mRNA was translated to the 45-58-kDa cluster of polypeptides. We show here that E1b-selected mRNA can also be translated to the 45-58-kDa cluster of polypeptides in addition to the major 19-kDa polypeptide. The E1b 58-kDa polypeptide was produced only at a low level unless E1b mRNA is fractionated before translation to enrich for the 58-kDa mRNA. Translation of E1b region-selected mRNAs that have been fractionated by size shows that the 22 S mRNA fraction is translated to at least the 53-58-kDa E1a-related polypeptides as well as to E1b 58- and 19-kDa polypeptides. Our experiments suggest that the 22 S mRNA fraction includes E1a-E1b overlapping mRNA which was translated to E1a-related polypeptides as well as E1b 22 S mRNA. When compared by two-dimensional gel electrophoresis and by tryptic peptide mapping, the cluster of polypeptides translated from E1a-selected mRNA and the cluster translated from E1b-selected mRNA were distinguishable. A possible explanation for this is discussed, based upon splicing sites of the E1a-E1b overlapping mRNA which would result in an amino acid sequence with a COOH-terminal end slightly different from that of E1a polypeptides.     

Expression profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of primary and secondary sulfur-related pathways in Arabidopsis thaliana

The treatment of Arabidopsis thaliana with methyl jasmonate was used to investigate the reaction of 2467 selected genes of primary and secondary metabolism by macroarray hybridization. Hierarchical cluster analysis allowed distinctions to be made between diurnally and methyl jasmonate regulated genes in a time course from 30 min to 24 h. 97 and 64 genes were identified that were up- or down-regulated more than 2–fold by methyl jasmonate, respectively. These genes belong to 18 functional categories of which sulfur-related genes were by far strongest affected. Gene expression and metabolite patterns of sulfur metabolism were analysed in detail, since numerous defense compounds contain oxidized or reduced sulfur. Genes encoding key reactions of sulfate reduction as well as of cysteine, methionine and glutathione synthesis were rapidly up-regulated, but none of the known sulfur-deficiency induced sulfate transporter genes. In addition, increased expression of genes of sulfur-rich defense proteins and of enzymes involved in glucosinolate metabolism was observed. In contrast, profiling of primary and secondary sulfur metabolites revealed only an increase in the indole glucosinolate glucobrassicin upon methyl jasmonate treatment. The observed rapid mRNA changes were thus regulated by a signal independent of the known sulfur deficiency response. These results document for the first time how comprehensively the regulation of sulfur-related genes and plant defense are connected. This interaction is discussed as a new approach to differentiate between supply- and demand-driven regulation of the sulfate assimilation pathway.     

Interferon-gamma binds to heparan sulfate by a cluster of amino acids located in the C-terminal part of the molecule

Using three different approaches (domain mapping with monoclonal antibodies, limited enzymatic digestion and competition with synthetic peptides), we demonstrated that a cluster of basic amino acids on interferon-gamma is involved in its binding to heparan sulfate. This cluster (Lys125-Arg131) is localized in the C-terminal part of IFN-gamma. Once bound to heparin sulfate, IFN-gamma is protected against protease attack.     

Cysteine does not repress adenosine-5′-phosphosulfate reductase through its conversion to either sulfate or glutathione

Cysteine (Cys) represses the activity of several key regulatory enzymes in the plant sulfate assimilatory pathway. However, it is not clear whether this effect arises from Cys itself or through its conversion to either sulfate or glutathione (GSH). Therefore, we examined this phenomenon by analyzing the activity of adenosine-5′-phosphosulfate (APS) reductase. Both APS reductase (AR) activity and mRNA levels were decreased by treatingArabidopsis thaliana roots with 1 mM Cys. The intracellular sulfate concentration was not affected, whereas enzymatic activity and, to some extent, the mRNA level, declined. Cys treatment in sulfur-starved plants also diminished both parameters. However, this response to Cys was more efficient than when plants were treated with an equal amount of sulfate. When Cys was removed from both Cys-and sulfate-fed plants, AR activity was recovered; the same removal of sulfate was not so effective. Moreover, buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, did not influence the repression of AR by Cys. Finally the AR enzyme was inhibited by cysteinein vitro. These results indicate that Cys represses AR by inhibiting mRNA expression and by directly repressing enzymatic activity, rather than through its conversion to either sulfate or GSH.     

Regulation of expression of c-fos and c-myc in rat lymphoma Nb-2 cells

This study examined the effects of the mitogen, prolactin and the cell cycle inhibitors, cyclosporin A and neomycin sulfate, on expression of the proto-oncogenes c-fos and c-myc in the rat lymphoma Nb-2 cell line. Stimulation of quiescent cultures with prolactin resulted in a 2-3-fold increase in the constitutive levels of c-myc mRNA which peaked at 4 h and declined thereafter. c-Fos mRNA was not detected in quiescent or prolactin-stimulated cultures. Cyclosporin A or neomycin sulfate reversibly blocked the mitogenic effect of prolactin on Nb-2 cells, but had little effect on constitutive levels of c-myc. However, the release of Nb-2 cells from a cyclosporin A or a neomycin sulfate block resulted in a rapid transient induction of c-fos which peaked at 0.5-1 h and declined rapidly thereafter. These results indicate that the rapid transient expression of c-fos following release from cell cycle blockage was not sufficient to elicit cell division, but these cells were competent to respond to prolactin. Prolactin allows progression through the cell cycle and enhances c-myc mRNA levels.     

Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4–like heparan sulfate proteoglycan and a distinct chondroitin sulfate form

Proteoglycan accumulation by thioglycollate-elicited mouse peritoneal macrophages and a panel of murine monocyte-macrophage cell lines has been examined to determine whether these cells express plasma membrane-anchored heparan sulfate proteoglycans. Initially, cells were screened for heparan sulfate and chondroitin sulfate glycosaminoglycans after metabolic labeling with radiosulfate. Chondroitin sulfate is secreted to a variable extent by every cell type examined. In contrast, heparan sulfate is all but absent from immature pre-monocytes and is associated predominantly with the cell layer of mature macrophage-like cells. In the P388D1 cell line, the cell-associated chondroitin sulfate is largely present as a plasma membrane-anchored proteoglycan containing a 55 kD core protein moiety, which appears to be unique. In contrast, the cell-associated heparan sulfate is composed of a proteoglycan fraction and protein-free glycosaminoglycan chains, which accumulate intracellularly. A fraction of the heparan sulfate proteoglycan contains a lipophilic domain and can be released from cells following mild treatment with trypsin, suggesting that it is anchored in the plasma membrane. Isolation of this proteoglycan indicates that it is likely syndecan-4: it is expressed as a heparan sulfate proteoglycan at the cell surface, it is cleaved from the plasma membrane by low concentrations of trypsin, and it consists of a single 37 kD core protein moiety that co-migrates with syndecan-4 isolated from NMuMG mouse mammary epithelial cells. Northern analysis reveals that a panel of macrophage-like cell lines accumulate similar amounts of syndecan-4 mRNA, demonstrating that this proteoglycan is expressed by a variety of mature macrophage-like cells. Syndecan-1 mRNA is present only in a subset of these cells, suggesting that the expression of this heparan sulfate proteoglycan may be more highly regulated by these cells. © 1993 Wiley-Liss, Inc.     

The presence of an iron-sulfur cluster in adenosine 5'-phosphosulfate reductase separates organisms utilizing adenosine 5'-phosphosulfate and phosphoadenosine 5'-phosphosulfate for sulfate assimilation

It was generally accepted that plants, algae, and phototrophic bacteria use adenosine 5'-phosphosulfate (APS) for assimilatory sulfate reduction, whereas bacteria and fungi use phosphoadenosine 5'-phosphosulfate (PAPS). The corresponding enzymes, APS and PAPS reductase, share 25-30% identical amino acids. Phylogenetic analysis of APS and PAPS reductase amino acid sequences from different organisms, which were retrieved from the GenBank(TM), revealed two clusters. The first cluster comprised known PAPS reductases from enteric bacteria, cyanobacteria, and yeast. On the other hand, plant APS reductase sequences were clustered together with many bacterial ones, including those from Pseudomonas and Rhizobium. The gene for APS reductase cloned from the APS-reducing cyanobacterium Plectonema also clustered together with the plant sequences, confirming that the two classes of sequences represent PAPS and APS reductases, respectively. Compared with the PAPS reductase, all sequences of the APS reductase cluster contained two additional cysteine pairs homologous to the cysteine residues involved in binding an iron-sulfur cluster in plants. M?ssbauer analysis revealed that the recombinant APS reductase from Pseudomonas aeruginosa contains a [4Fe-4S] cluster with the same characteristics as the plant enzyme. We conclude, therefore, that the presence of an iron-sulfur cluster determines the APS specificity of the sulfate-reducing enzymes and thus separates the APS- and PAPS-dependent assimilatory sulfate reduction pathways.     

Mutants of Chlamydomonas with Aberrant Responses to Sulfur Deprivation

In the absence of sulfur, Chlamydomonas reinhardtii, a unicellular green alga, increases its rate of sulfate import and synthesizes several periplasmic proteins, including an arylsulfatase (Ars). These changes appear to help cells acclimate to a sulfur-deficient environment. The elevated rate of sulfate import results from an increase in the capacity and affinity of the transport system for sulfate. The synthesis of Ars, a periplasmic enzyme that cleaves sulfate from aromatic compounds, enables cells to use these molecules as a source of sulfur when free sulfate is not available. To characterize the ways in which C. reinhardtii perceives changes in the sulfur status of the environment and regulates its responses to these changes, we mutagenized cells and isolated strains exhibiting aberrant accumulation of Ars activity. These mutants were characterized for Ars activity, ars mRNA accumulation, periplasmic protein accumulation, and sulfate transport activity when grown in both sulfur-sufficient and sulfur-deficient conditions. All of the mutants exhibited pleiotropic effects with respect to several of these responses. Strains harboring double mutant combinations were constructed and characterized for Ars activity and ars mRNA accumulation. From the mutant phenotypes, we inferred that both positive and negative regulatory elements were involved in the acclimation process. Both the epistatic relationships among the mutations and the effects of the lesions on the responses of C. reinhardtii to sulfur limitation distinguished these mutants from similar mutants in Neurospora crassa.     

Iron overload of human colon adenocarcinoma cells studied by synchrotron-based X-ray techniques

Fast- and slow-proliferating human adenocarcinoma colorectal cells, HT-29 and HCA-7, respectively, overloaded with transferrin (Tf), Fe(III) citrate, Fe(III) chloride and Fe(II) sulfate were studied by synchrotron radiation total-reflection X-ray spectrometry (TXRF), TXRF-X-ray absorption near edge structure (TXRF-XANES), and micro-X-ray fluorescence imaging to obtain information on the intracellular storage of overloaded iron (Fe). The determined TfR1 mRNA expression for the investigated cells correlated with their proliferation rate. In all cases, the Fe XANES of cells overloaded with inorganic Fe was found to be similar to that of deliquescent Fe(III) sulfate characterized by a distorted octahedral geometry. A fitting model using a linear combination of the XANES of Tf and deliquescent Fe(III) sulfate allowed to explain the near edge structure recorded for HT-29 cells indicating that cellular overload with inorganic Fe results in a non-ferritin-like fast Fe storage. Hierarchical cluster analysis of XANES spectra recorded for Fe overloaded HT-29 and HCA-7 cells was able to distinguish between Fe treatments performed with different Fe species with a 95 % hit rate, indicating clear differences in the Fe storage system. Micro-X-ray fluorescence imaging of Fe overloaded HT-29 cells revealed that Fe is primarily located in the cytosol of the cells. By characterizing the cellular Fe uptake, Fe/S content ratios were calculated based on the X-ray fluorescence signals of the analytes. These Fe/S ratios were dramatically lower for HCA-7 treated with organic Fe(III) treatments suggesting dissimilarities from the Tf-like Fe uptake.     

Heparan sulfate-degrading enzymes induce modulation of smooth muscle phenotype.

Macrophages cocultured with rabbit aortic smooth muscle cells at a ratio of 1:3 degraded all the 35S-labeled heparan sulfate proteoglycan from the smooth muscle surface into free sulfate (Kav of 0.84 on Sepharose 6B). Concomitantly, the same macrophages induced a decrease in the volume fraction of myofilaments (Vvmyo) of the smooth muscle cells and a decrease in alpha-actin mRNA as a percentage of total actin mRNA. Both macrophage lysosomal lysate at neutral pH and heparinase degraded cell-free 35S-labeled matrix deposited by smooth muscle cells into fragments which eluted at a Kav of 0.63 and which were identified as heparan sulfate chains by their complete degradation in the presence of low pH nitrous acid. At acid pH the macrophage lysosomal lysate completely degraded the heparan sulfate to free sulfate (Kav 0.84). Both macrophage lysosomal lysate and commercial heparinase at neutral pH induced smooth muscle phenotypic change while other enzymes such as trypsin and chondroitin ABC lyase had no effect. It was therefore suggested that the active factor present in the macrophages is a lysosomal heparan sulfate-degrading endoglycosidase (heparinase). Only a small amount of heparan sulfate-degrading activity was released into the incubation medium by living macrophages, and there was no heparinase activity on their isolated plasma membranes, although proteolytic enzymes were evident in both instances. In pulse-chase studies, high Vvmyo smooth muscle cells were seen to constantly internalize and degrade 35S-labeled heparan sulfate proteoglycan from their own pericellular compartment, suggesting that this may be the mechanism by which smooth muscle phenotype is maintained under normal circumstances and that removal of heparan sulfate from the surface of smooth muscle cells and its degradation by macrophages temporarily interrupts this process, inducing smooth muscle phenotypic change.     

Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Cysteine synthesis from sulfide and O-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. Using Lemna minor, we analyzed the effects of omission of CO(2) from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5'-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO(2) led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO(2) on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO(2), APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO(2) also recovered both enzyme activities, with OAS again influenced only APR. (35)SO(4)(2-) feeding showed that treatment in air without CO(2) severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of (35)S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of (35)S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.     

Interferon-gamma C-terminal function: new working hypothesis. Heparan sulfate and heparin, new targets for IFN-gamma, protect, relax the cytokine and regulate its activity

Structure and function of the interferon-gamma C-terminal extremity has been widely studied. A basic amino acid cluster located in this domain is involved in the tridimentional structure of the protein and is essential for the biological activity. This specific group of amino acid is also involved in the binding of interferon-gamma to basement membrane or cell surface heparan sulfate. Once bound to heparan sulfate, interferon-gamma is protected from proteolytic cleavage and it is suggested that the protein folds in a new relaxed conformation, with increased stability.     

Coordinated expression of sulfate uptake and components of the sulfate assimilatory pathway in maize

A high-affinity-type sulfate transporter (Group 1: ZmST1;1, Accession No. AF355602) has been cloned from maize seedlings by RT-PCR. Tissue and cell specific localisation of this sulfate transporter has been determined along the developmental gradient of the root and in leaves of different ages. In S-sufficient conditions there was uniform low expression of ZmST1;1 in the root and very low expression in the leaves. Increased mRNA abundance and sulfate influx capacity indicated that S-starvation increased ZmST1;1 expression in roots, especially at the top of the root (just behind the seed, the area possessing most laterals and root hairs) compared to the root tip. Similarly a group 2, probable low affinity-type sulfate transporter, ZmST2;1, and also ATP-sulfurylase and APS-reductase but not OAS(thiol)lyase were induced by S-starvation and showed highest expression in the upper section of the root. S-starvation increased root/shoot ratio by 20 % and increased root lateral length and abundance in the region closest to the root tip. As the increase in root proliferation was not as great as the increase in mRNA pools, it was clear that there was a higher cellular abundance of the mRNAs for sulfate transporters, ATP-sulfurylase, and APS-reductase in response to sulfur starvation. In the leaves, the sulfate transporters, ATP-sulfurylase and APS-reductase were induced by S-starvation with the most mature leaf showing increased mRNA abundance first. In situ hybridization indicated that ZmST1;1 was expressed in epidermal and endodermal cell layers throughout the root whilst OAS(thiol)lyase was highly expressed in the root cortex.     

Sulfate-dependent iron oxidation by Thiobacillus ferrooxidans: characterization of a new EPR detectable electron transport component on the reducing side of rusticyanin

Iron(II) oxidation by pH 2.5 HCl-washed cells of Thiobacillus ferrooxidans is known to be sulfate dependent. Sulfate dependence of the autooxidation of a novel component in the electron transport pathway is demonstrated. This component exhibits an electron paramagnetic resonance (EPR) signal in the oxidized state at g = 2.005 distinguishable from the g = 2.08 signal attributed to rusticyanin. The novel component is proposed to be a three-iron-sulfur cluster based upon the g value, lineshape, and temperature dependence. Oxyanion specificity for the EPR signal has the same dependence on sulfate as does iron(II) oxidation. By using azide to inhibit electron transfer to oxygen, sulfate was shown to be involved in electron transfer from the g = 2.005 component to the copper of rusticyanin.