Phototransformation of protochlorophyllideF657 in etiochloroplasts isolated from pine cotyledons; dark reformation of this pigment-complex from a pool of ALA-protochlorophyllideF635 in the presence of NADPH

Etiochloroplasts isolated from dark-grown pine cotyledons fed with -aminolevulinic acid (ALA) contain, in addition to the chlorophyll forms, two protochlorophyllide complexes which emit fluorescence around 635 nm and 657 nm respectively (ALA-PChlide_F635 and PChlide_F657). By a combination of light flashes and periods of darkness, it is possible to phototransform PChlide_F657 and thereafter, if NADPH is added to the system, to re-form this pigment-complex from the pool of ALA-PChlide_F635 during the dark periods. The process of phototransformation followed by the re-formation of PChlide_F657 in the presence of NADPH can be obtained in vitro five to six times consecutively. The role of NADPH in the formation of the PChlide_F657 complex is discussed.

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The effects of low pH on the properties of protochlorophyllide oxidoreductase and the organization of prolamellar bodies of maize (Zea mays).

Prolamellar bodies (PLB) contain two photochemically active forms of the enzyme protochlorophyllide oxidoreductase POR-PChlide640 and POR-PChlide650 (the spectral forms of POR-Chlide complexes with absorption maxima at the indicated wavelengths). Resuspension of maize PLB in media with a pH below 6.8 leads to a rapid conversion of POR-PChlide650 to POR-PChlide640 and a dramatic re-organization of the PLB membrane system. In the absence of excess NADPH, the absorption maximum of the POR complex undergoes a further shift to about 635 nm. This latter shift is reversible on the re-addition of NADPH with a half-saturation value of about 0.25 mm NADPH for POR-PChlide640 reformation. The disappearance of POR-PChlide650 and the reorganization of the PLB, however, are irreversible. Restoration of low-pH treated PLB to pH 7.5 leads to a further breakdown down of the PLB membrane and no reformation of POR-PChlide650. Related spectral changes are seen in PLB aged at room temperature at pH 7.5 in NADPH-free assay medium. The reformation of POR-PChlide650 in this system is readily reversible on re-addition of NADPH with a half-saturation value about 1.0 microm. Comparison of the two sets of changes suggest a close link between the stability of the POR-PChlide650, membrane organization and NADPH binding. The low-pH driven spectral changes seen in maize PLB are shown to be accelerated by adenosine AMP, ADP and ATP. The significance of this is discussed in terms of current suggestions of the possible involvement of phosphorylation (or adenylation) in changes in the aggregational state of the POR complex.     

Photoconvertible protochlorophyll(ide) in vivo: A single species or two species in dynamic equilibrium?

The decreasing absorbances in vivo of protochlorophyll(ide) at 635 and 650 nm bear the same relationships to one another during photoconversion to chlorophyll(ide) a in the leaves of dark-grown barley seedlings, regardless of whether the actinic light is absorbed primarily at 630, 640 or 671 nm. Accordingly, the absorption bands at 635–637 and 650 nm of photoconvertible protochlorophyll(ide) are attributed to a single species of membrane-bound protochlorophyll(ide) molecule or, alternatively, to two species which are in dynamic equilibrium.     

Relationship between Photoconvertible and Nonphotoconvertible Protochlorophyllides

Two forms of protochlorophyllide are found in dark-grown bean (Phaseolus vulgaris, var. Black Velentine) leaves, one (protochlorophyllide₆₅₀) which is directly photoconvertible to chlorophyllide and another (protochlorophyllide₆₃₂) which is not. Dark-grown leaves placed in solutions of δ-aminolevulinic acid accumulate protochlorophyllide₆₃₂. Protochlorophyllide₆₅₀ and protochlorophyllide₆₃₂ can be partially separated on sucrose density gradients. A nitrogen atmosphere blocks chlorophyll synthesis in light or the regeneration of protochlorophyllide₆₅₀ in the dark, even in the presence of excess δ-aminolevulinic acid, except when a stockpile of protochlorophyllide₆₃₂ is present in the leaf. Under the latter conditions chlorophyll synthesis or protochlorophyllide₆₅₀ regeneration is accompanied by a decrease in protochlorophyllide₆₃₂. These experiments suggest that protochlorophyllide₆₃₂ may be converted to protochlorophyllide₆₅₀.     

Rapid regeneration of protochlorophyllide(650)

The rate of regeneration of protochlorophyllide₆₅₀ was examined spectrophotometrically after a saturating light flash using 8- to 9-day-old dark-grown bean leaves. The regeneration occurred to the extent of 15% with a half rise time of about 20 seconds. Feeding δ-aminolevulinic acid to the excised leaves in the dark increased protochlorophyllides₆₃₅ but not the absorption at 650 nanometers, suggesting that the holochrome was normally saturated with protochlorophyllide and that the holochrome protein was not controlled by the level of protochlorophyllide. After a light flash, the excess protochlorophyllide, formed from exogenous δ-aminolevulinic acid, readily combined to regenerate the 650 nanometer absorbing species; the regeneration occurred to the extent of 60 to 80% with a half rise time of about 50 seconds. Regeneration was blocked at 0°, suggesting that there was some enzymic process required for regeneration, possibly the formation of a reductant component of the protochlorophyllides₆₅₀ holochrome.     

Measurement and Identification of NADPH:Protochlorophyllide Oxidoreductase Solubilized with Triton X-100 from Etioplast Membranes of Squash Cotyledons

Etioplast membranes were solubilized with 1 mM Triton X-100in the presence of excess NADPH and protochlorophyllide to isolateNADPH:protochlorophyllide oxidoreductase. The activity of thisreductase was assayed as the formation of chlorophyllide bya single flash and was equivalent to the amount of photoactiveprotochlorophyllide-NADPH-enzyme complex present before illumination.The rate of regeneration of the phtoactive complex was estimatedfrom the time course of chlorophyllide formation under a longflash. The highest rate was 651 nmol chlorophyllide formed min^–1mg^–1 protein. Photoconversion of protochlorophyllide to chlorophyllide andregeneration of the photoactive protochlorophyllide-NADPH-enzymecomplex were not much affected in a pH range from 6 to 8, atleast for several minutes. The apparent dissociation constantsof the photoactive complex were 0.039 µM for protochlorophyllideand 0.44 µM for NADPH. Triton-solubilized etioplast membraneswere fractionated by glycerol density gradient centrifugationto isolate the NADPH:protochlorophyllide oxidoreductase. Mostof the 36,000-dalton protein, the major protein of the prolamellarbody was recovered in the fraction enriched by NADPH:protochlorophyllideoxidoreductase and protochlorophyllide. Protochlorophyll andcarotenoids were present in different fractions. This is evidencethat the 36,000-dalton protein has the activity of NADPH:protochlorophyllideoxidoreductase and specifically binds protochlorophyllide. Themost highly purified fraction of the enzyme showed an activityof 7.8 nmol chiorophyllide formed flash^–1 mg^–1 proteinand bound 11.1 nmol protochlorophyllide mg^–1 of protein. (Received April 28, 1982; Accepted June 29, 1982)     

ADP/ATP and protein phosphorylation dependence of phototransformable protochlorophyllide in isolated etioplast membranes

The effects of modulated ADP/ATP and NADPH/NADP⁺ ratios, and of protein kinase inhibitors, on the in vitro reformation of phototransformable protochlorophyllide, i.e. the aggregated ternary complexes between NADPH, protochlorophyllide, and NADPH-protochlorophyllide oxidoreductase (POR, EC 1.3.1.33), in etioplast membranes isolated from dark-grown wheat (Triticum aestivum) were investigated. Low temperature fluorescence emission spectra (–196 °C) were used to determine the state of the pigments. The presence of spectral intermediates of protochlorophyllide and the reformation of phototransformable protochlorophyllide were reduced at high ATP, but favoured by high ADP. Increased ADP level partly prevented the chlorophyllide blue-shift. The protein kinase inhibitor K252a prevented reformation of phototransformable protochlorophyllide without showing any effect on the chlorophyllide blue-shift. Addition of NADPH did not overcome the inhibition. The results indicate that protein phosphorylation plays a role in the conversion of the non-phototransformable protochlorophyllide to POR-associated phototransformable protochlorophyllide. The possible presence of a plastid ADP-dependent kinase, the activity of which favours the formation of PLBs, is discussed. Reversible protein phosphorylation is suggested as a regulatory mechanism in the prolamellar body formation and its light-dependent dispersal by affecting the membrane association of POR. By the presence of a high concentration of phototransformable protochlorophyllide, prolamellar bodies can act as light sensors for plastid development. The modulation of plastid protein kinase and protein phosphatase activities by the NADPH/NADP⁺ ratio is suggested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photochemical reaction of chlorophyll biosynthesis at 4.2 K

Phototransformation of protochlorophyllide in etiolated leaves was shown to take place at 4.2 K. Upon illumination of the sample a photoactive form PChlide₆₅₀ was transformed into a non-fluorescent intermediate which was then transformed into fluorescent forms of chlorophyllide (Chlide) upon increase of temperature in the dark. Reported transformations are similar to those observed under illumination at 77 K.     

Shoot development and plant regeneration from protocorm-like bodies of <Emphasis Type="Italic">Zygopetalum mackayi</Emphasis>

Stem nodal segments of a sympodial orchid, Zygopetalum mackayi, were used as explants to induce protocorm-like body (PLB) formation on a hormone-free 1/2 Murashige and Skoog (1962) modified medium (1/2MS-0) or 1/2MS supplemented with 0.045–4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ] in light. After 1 mo of culture, pale to dark green, compact and irregular nodulars of PLBs formed from the explants. For PLB induction, TDZ had no significant effect on the percentage of PLB formation but promoted mean numbers of PLBs per responding explant at 0.045–4.54 μM. For plant conversion, PLBs were transferred onto the same basal medium devoid of TDZ. After 2–3 mo of culture, these PLBs successfully formed shoots and then roots with normal morphology. For PLB proliferation, TDZ has no significant effects on the fresh weight of PLB aggregates, but there is significantly retarded shoot development at 0.45–4.54 μM after 1 mo of culture. When transferring these PLB aggregates onto hormone-free medium for plant conversion, PLBs derived from TDZ-containing medium showed a decrease of shoot length (0.86–2.08 cm in shoot length) compared to those derived from 1/2MS-0 (2.74 cm in shoot length) after 1 mo of culture. Gibberellin A₃ [GA₃] at 0.29–8.66 μM significantly retarded PLB proliferation, but at 0.03 and 0.29 μM resulted in longer shoot length than the control treatment. Histological studies reveal that shoot development originated from the outer region of PLB aggregates. The young shoots initially connected to each other at their basal tissues with the parental PLBs. Plants were successfully obtained from PLBs and then gradually became more loosely connected with each other as well as with the parental aggregates. Several dozen plants were acclimatized in the greenhouse and showed normal morphology.     

Effect of Periodic Heat Shock on the Inner Membrane System of Etioplasts

Etiolated barley (Hordeum vulgare L.) seedlings were treated with heat shock (HS). The heat treatment was conducted daily for 1 h at 40°C over 6 days and led to shortening of leaves and coleoptiles, an increase in the etioplast volume and prothylakoid length, and to a decrease in the size of paracrystalline prolamellar bodies (PLB). As a result of HS treatment, stimulation of carotenoid and protochlorophyllide (Pchlide) synthesis as well as an increase in the relative content of the Pchlide short-wavelength form (Pchlide₆₃₀) were observed in the leaf tissue of seven-day-old seedlings 12 h after the last HS treatment. HS had no effect on the overall amount of Pchlide-oxidoreductase (POR) in leaves and PLB membranes and did not suppress the Pchlide photoreduction in vivo. PLB membranes, isolated from the HS-treated seedlings, possessed a higher Pchlide and carotenoid content as calculated on total protein basis. These membranes showed more intense protein fluorescence than PLB from untreated plants, whereas hydrophobicity of the microenvironment of the fluorescent amino-acid residues remained unchanged. Studies using pyrene (lipophilic fluorescent probe emitted in Pchlide and carotenoid absorption bands) showed that HS increases the fluidity of membrane lipids in PLB membranes and that the pigments accumulated in these membranes are located in the region of lipid–protein contact site. The results are discussed in relation to the adaptive role of protein–protein and pigment–protein–lipid interactions in etioplast membranes under stress.     

Efficient production of protocorm-like bodies and plant regeneration from flower stalk explants of the sympodial orchid <Emphasis Type="Italic">Epidendrum radicans</Emphasis>

Summary A simple and efficient micropropagation method was established for direct protocorm-like body (PLB) formation and plant regeneration from flower stalk internodes of a sympodial orchid, Epidendrum radicans. Small transparent tissues formed on surfaces and cut ends of flower stalk internodes on a modified half-strength Murashige and Skoog basal medium with or without thidiazuron (TDZ) after 1–2 wk of culture. In the light, the transparent tissues enlarged and turned into organized calluses on most of the explants. However, PLBs formed only on a medium supplemened with 0.45 μM TDZ within 2 mo. of culture. Sucrose, NH₄NO₃, and KNO₃ were used in media to test their effects on PLB proliferation and shooting. The best response on number of PLBs per tube was 23.6 at 40 gl⁻¹ sucrose, 825 mgl⁻¹ NH₄NO₃, and 950 mgl⁻¹ KNO₃, and the highest number of PLBs with shoots was found at 10 gl⁻¹ sucrose, 825 mgl⁻¹ NH₄NO₃, and 950 mgl⁻¹ KNO₃. Homogenized PLB tissues produced by blending were used to test the effects of four cytokinins [TDZ, N⁶-benzyladenine (BA), zeatin-riboside, and kinetin] on PLB proliferation and shoot formation. The best responses on number of PLBs per tube, proliferation rate, and number of PLBs with shoots per tube were obtained at 4.44 μM BA, 0.28 μM zeatin-riboside, and 1.39 μM kinetin, respectively. Normal plantlets converted from PLBs on the same TDZ-containing medium after 1 mo. of culture. The optimized procedure required about 12–13 wk from the initiation of PLBs to plantlet formation. The regenerated plants grew well with an almost 100% survival rate when acclimatized in a greenhouse.     

Separation and Characterization of Prolamellar Bodies and Prothylakoids from Squash Etioplasts

Intact etioplasts of squash cotyledons, which had been preparedby Percoll density gradient centrifugation, were ruptured hypotonicallyin the presence of deoxyribonuclease I then fractionated intoprolamellar bodies and prothylakoids by differential and Percolldensity gradient centrifugations. This procedure provided ahighly purified prolamellar body fraction that was composedmainly of a 36,000-dalton protein. This protein was identifiedas NADPH:protochIorophyllideoxidoreductase [Ikeuchi and Murakami(1982) Plant & Cell Physiol. 23: 1089]. The fraction alsohad a high content of protochlorophyllide that absorbed at 648nm and its NADPH:protochlorophyllide oxidoreductase had highactivity. When the fraction was illuminated, a chlorophyllidethat absorbed at 684–685 nm formed. In contrast, the prothylakoid fraction, which showed high activityfor the Ca²⁺-dependent ATPase of coupling factor 1, containedonly a small amount of the 36,000-dalton protein and showedvery low NADPH:protochlorophyllide oxidoreductase activity.The protochlorophyllide content of this fraction also was low,and the ratio of protochlorophyll to protochlorophyll(ide) high.The absorption peak in the prothylakoids was at 633–635nm, and after a brief illumination a chlorophyllide that absorbedat 672–673 nm formed. These results indicate that thephotoactive protochlorophyllide-NADPHreductase complex in etioplastsis concentrated in the prolamellar body and that the physicalstate of protochlorophyll(ide) in the prolamellar body differsfrom that of the prothylakoid. (Received April 28, 1982; Accepted November 15, 1982)     

Structural organisation of prolamellar bodies (PLB) isolated from Zea mays. Parallel TEM, SAXS and absorption spectra measurements on samples subjected to freeze-thaw, reduced pH and high-salt perturbation

Well-organised PLB gives rise to a X-ray diffraction pattern overlaid by a scattering pattern arising from individual tubules within less well-organised regions of the lattice. TEM and SAXS measurements were used to characterise the structural changes in PLB subjected to perturbation by freeze-thaw, exposure to pH 6.5, or resuspension in high-salt media. Comparison of SAXS patterns measured, before and after structural perturbation allows the separation of the contributions from ordered and disordered PLB. The diffraction pattern is shown to be based on a diamond cubic (Fd3m) lattice of unit cell a = 78 nm. Freeze-thaw and high-salt disruption lead to the breakdown of ordered PLB into disordered tubules of similar dimensions to those making up the original PLB lattice. Their scattering patterns suggest that they are approximately 26 nm in diameter with a central lumen about 16 nm in diameter. The tubules formed at pH 6.5 are appreciably narrower, probably reflecting changes in the pattern of ionisation of charged groups at the membrane surface. Absorption spectra of PLB in media containing different concentrations of salts indicated that the structural and spectral changes are related. NADPH, have a significant role in the protection of POR-PChlide₆₅₀ but to have only a relatively small effect on the preservation of PLB organisation indicating that the retention of POR-PChlide₆₅₀ in isolated PLB preparations is a poor guide to their structural integrity.     

Far-red induced changes of the protochlorophyllide components in wheat leaves

Biosynthesis of chlorophyll is partly controlled by the phytochrome system. In order to study the effects of an activated phytochrome system on the protochlorophyllide (PChlide) biosynthesis without accompanying phototransformation to chlorophyll, wheat seedlings (Triticum aestivum L. cv. Starke II Weibull) were irradiated with long wavelength far-red light of low intensity. Absorption spectra were measured in vivo after different times in the far-red light or in darkness. The relationship between the different PChlide forms, the absorbance ratio _{650nm636 nm} changed with age in darkness, and the change was more pronounced when the leaves were grown in far-red light. Absorption spectra of dark-grown leaves always showed a maximum in the red region at 650 nm. For leaves grown in far-red light the absorption at 636 nm was high, with a maximum at the 5 day stage where it exceeded the absorption at 650 nm. At the same time there was a maximum in the total amount of PChlide accumulated in the leaves, about 30% more than in leaves grown in darkness. But the amount of the directly phototransformable PChlide, mainly PChlide_650–657, was not increased. The amount of PChlide_628–632, or more probably the amount of (PChlide_628–632, + PChlide _636–657) was thus higher in young wheat leaves grown in far-red light than in those grown in darkness. After the 5 day stage the absorption at 636 nm relative to 650 nm decreased with age, and at the 8 day stage the spectra were almost the same in both types of leaves. Low temperature fluorescence spectra of the leaves also showed a change in the ratio between the different PChlide forms. The height of the fluorescence peak at 632 nm relative to the peak at 657 nm was higher in leaves grown in far-red light than in dark-grown leaves. – After exposure of the leaves to a light flash, the half time for the Shibata shift was measured. It increased with age both for leaves grown in darkness and in far-red light; but in older leaves grown in far-red light (7–8 days) the half time was slightly longer than in dark-grown leaves. – The chlorophyll accumulation in white light as well as the leaf unrolling were faster for leaves pre-irradiated with far-red light. The total length of the seedlings was equal or somewhat shorter in far-red light, but the length of the coleoptile was markedly reduced from 8.1 ± 0.1 cm for dark-grown seedlings to 5.2 ± 0.1 cm for seedlings grown in far-red light.     

Photoconvertible protochlorophyll(ide)635650 in vivo: A single species or two species in dynamic equilibrium?

The decreasing absorbances in vivo of protochlorophyll(ide) at 635 and 650 nm bear the same relationships to one another during photoconversion to chlorophyll(ide) a in the leaves of dark-grown barley seedlings, regardless of whether the actinic light is absorbed primarily at 630, 640 or 671 nm. Accordingly, the absorption bands at 635–637 and 650 nm of photoconvertible protochlorophyll(ide) are attributed to a single species of membrane-bound protochlorophyll(ide) molecule or, alternatively, to two species which are in dynamic equilibrium.     

Isolation and characterization of photoactive complexes of NADPH:protochlorophyllide oxidoreductase from wheat

A photoactive substrate-enzyme complex of the NADPH:protochlorophyllide oxidoreductase (POR; EC 1. 3. 1. 33) was purified from etiolated Triticum aestivum L. by gel chromatography after solubilization of prolamellar bodies by dodecyl-maltoside. Irradiation by a 1-ms flash induced the phototransformation of protocholorophyllide a (Pchlide) with −196 °C absorbance and emission maxima at 640 and 643 nm, respectively. The apparent molecular weight of this complex was 112 ± 24 kDa, which indicates aggregation of enzyme subunits. By lowering the detergent concentration in the elution buffer, a 1080 ± 250-kDa particle was obtained which displayed the spectral properties of the predominant form of photoactive Pchlide in vivo (−196 °C absorbance and fluorescence maxima at 650 and 653 nm). In this complex, POR was the dominant polypeptide. Gel chromatography in the same conditions of an irradiated sample of solubilized prolamellar bodies indicated rapid disaggregation of the complex after Pchlide phototransformation. High performance liquid chromatographic analysis of the POR complexes obtained using two detergent concentrations indicates a possible association of zeaxanthin and violaxanthin with the photoactive complex. Received: 25 February 1998 / Accepted: 8 June 1998     

Etioplast differentiation in arabidopsis: both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant.

The etioplast plastid type of dark-grown angiosperms is defined by the accumulation of the chlorophyll (Chl) precursor protochlorophyllide (Pchlide) and the presence of the paracrystalline prolamellar body (PLB) membrane. Both features correlate with the presence of NADPH:Pchlide oxidoreductase (POR), a light-dependent enzyme that reduces photoactive Pchlide-F655 to chlorophyllide and plays a key role in chloroplast differentiation during greening. Two differentially expressed and regulated POR enzymes, PORA and PORB, have recently been discovered in angiosperms. To investigate the hypothesis that etioplast differentiation requires PORA, we have constitutively overexpressed PORA and PORB in the Arabidopsis wild type and in the constitutive photomorphogenic cop1-18 (previously det340) mutant, which is deficient in the PLB and Pchlide-F655. In both genetic backgrounds, POR overexpression increased PLB size, the ratio of Pchlide-F655 to nonphotoactive Pchl[ide]-F632, and the amount of Pchlide-F655. Dramatically, restoration of either PORA or PORB to the cop1 mutant led to the formation of etioplasts containing an extensive PLB and large amounts of photoactive Pchlide-F655.     

Purification and properties of the membrane-bound NADH oxidase of a facultatively anaerobic alkaliphile

A membrane-bound NADH oxidase of an anaerobic alkaliphile, M-12 (a strain of Amphibacillus sp.), was solubilized with decanoyl N-methylglucamide and purified by chromatography on DEAE-Sepharose and hydroxyapatite. The purified enzyme appears to consist of a single polypeptide component with an apparent molecular mass of 56 kDa. The enzyme catalyzed the oxidation of NADH with the formation of H₂O₂ and exhibited a specific activity of 46 μmol NADH min^–1 (mg protein)^–1. NADPH did not serve as a substrate for the enzyme. The K _m for NADH was estimated to be 0.05 mM. The enzyme exhibited a pH dependence for activity, with a pH optimum at approximately 9.5. The enzyme required a high concentration of salt and exhibited maximum activity in the presence of 600 mM NaCl. Received: 3 August 1998 / Accepted: 23 December 1998     

Feeding with aminolevulinic acid increased chlorophyll content in Norway spruce (<Emphasis Type="Italic">Picea abies</Emphasis>) in the dark

In contrast to angiosperms, which accumulate protochlorophyllide after application of aminolevulinic acid in the dark, feeding with aminolevulinic acid (0.01–20 mM) via the roots in the 18-d-old seedlings of Norway spruce (Picea abies) stimulated not only protochlorophyllide but also chlorophyll accumulation.     

Proline Rich Polypeptide (PRP-1) Increases the Superoxide-Producing and Ferrihemoglobin Reducing Activities of Cytochrome B<Subscript>558</Subscript> Isoforms from Human Lymphosarcoma Tissue Cells

The two cytochromes (cyt) b₅₅₈ of acidic nature, one—95–100 kDa and another one, 60–70 kDa were isolated for the first time from the human’s lymphosarcoma tissue cells using gel filtration and ion exchange chromatography. These hemoproteins possess NADPH dependent O₂ ⁻-producing and ferrihemoglobin-reducing activities. The incubation of neuropeptide PRP-1 (5 μg) with cytochrome b₅₅₈, caused elevation of these activities. The gel filtration results indicated possible binding of PRP-1 to these cytochromes b₅₅₈. PRP-1 activated both NADPH dependent O₂ ⁻-producing and ferriHb-reducing activities of the cyt b¹ ₅₅₈ and cyt b² ₅₅₈, obtained from human lymphosarcoma tissue cells. One can assume that PRP-1 associated with cyt b₅₅₈ on the surface of the tumor cells by increasing both NADPH dependent O₂ ⁻-producing and ferriHb-reducing activities of cyt b₅₅₈, increases the oxidation- reduction status. Changing the oxidation–reduction status and oxygen homeostasis of the tumor cells by PRP-1 can serve as one of the possible explanation of antitumorigenic effect of this cytokine.