Computational constraints that may have favoured the lamination of sensory cortex

At the transition from early reptilian ancestors to primordial mammals, the areas of sensory cortex that process topographic modalities acquire the laminar structure of isocortex. A prominent step in lamination is granulation, whereby the formerly unique principal layer of pyramidal cells is split by the insertion of a new layer of excitatory, but intrinsic, granule cells, layer IV. I consider the hypothesis that granulation, and the differentiation between supra- and infra-granular pyramidal layers, may be advantageous to support fine topography in their sensory maps. Fine topography implies a generic distinction between where information, explicitly mapped on the cortical sheet, and what information, represented in a distributed fashion as a distinct firing pattern across neurons. These patterns can be stored on recurrent collaterals in the cortex, and such memory can help substantially in the analysis of current sensory input. The simulation of a simplified network model demonstrates that a non-laminated patch of cortex must compromise between transmitting where information or retrieving what information. The simulation of a modified model including differentiation of a granular layer shows a modest but significant quantitative advantage, expressed as a less severe trade-off between what and where. The further connectivity differentiation between infra-granular and supra-granular pyramidal layers is shown to match the mix of what and where information optimal for their respective target structures.     

Application of percolation theory principles to the analysis of interaction of adenylate cyclase complex proteins in cell membranes

Summary Lateral protein movement in cell membranes takes place in a medium with obstacles. These obstacles are: (a) aggregates of major integral proteins immobilized by submembraneous structures and cytoskeleton, and (b) membrane lipids in the gel phase. Hormonal activation of the adenylate cyclase complex is associated with lateral mobility of the constituent proteins. Modification of the interaction of these proteins due to variation of the fluid lipid fraction in reticulocyte membranes has been studied. A decrease in the percentage of fluid lipids in membranes resulted in the inhibition (up to the full cessation) of the interaction of -adrenoreceptors with regulatory N_s-proteins. The interaction of N_s-proteins with catalytic proteins stopped as well. On the other hand, an increase in the fluid lipid fraction led to a more intensive interaction. These facts do not arise from the functional damage of interacting proteins. Conseqently, hormonal activation of the adenylate cyclase complex depends on the fraction of fluid lipids in the membrane. The data obtained are in conformity with the percolation theory which makes it possible to characterize long-distance protein movement in a medium (fluid lipids) containing obstacles. Thus, interacting proteins prove to diffuse within distances greatly exceeding protein sizes. As a consequence, the intrinsic activity of a -agonist, isoproterenol, varies from 1 to 0 depending on the fluid lipid fraction. Our findings also suggest that in vitro there are no -receptors precoupled with N_s-proteins in rat reticulocyte membranes in the absence of guanine nucleotides.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Na,K-ATPase: Isoform structure,function, and expression

An interesting feature of the Na,K-ATPase is the multiplicity of and isoforms. Three isoforms exist for the subunit, 1, 2, and 3, as well for the subunit, 1, 2, and 3. The functional significance of these isoforms is unknown, but they are expressed in a tissue- and developmental-specific manner. For example, all three isoforms of the subunit are present in the brain, while only 1 is present in kidney and lung, and 2 represents the major isoform in skeletal muscle. Therefore, it is possible that each of these isoforms confers different properties on the Na,K-ATPase which allows effective coupling to the physiological process for which it provides energy in the form of an ion gradient. It is also possible that the multiple isoforms are the result of gene triplication and that each isoform exhibits similar enzymatic properties. In this case, the expression of the triplicated genes would be individually regulated to provide the appropriate amount of Na,K-ATPase to the particular tissue and at specific times of development. While differences are observed in such parameters as Na⁺ affinity and sensitivity to cardiac glycosides, it is not known if these properties play a functional role within the cell.Site-directed mutagenesis has identified amino acid residues in the first extracellular region of the subunit as major determinants in the differential sensitivity to cardiac glycosides. Similar studies have failed to identify residues in the second extracellular region involved in cardiac glycoside inhibition. Further analysis of the enzymatic properties of the enzyme, understanding the regulated expression of the genes, and structure-function studies utilizing site-directed mutagenesis should provide new insights into the enzymatic and physiological roles of the various subunit isoforms of the Na,K-ATPase.     

Uptake of a monoclonal antibody against CEA (Tumak 431/31) in a human colon tumor (Co-112) xenografted in the nude mouse

Summary A monoclonal antibody (Tumak) against carcinoembryonic antigen (CEA) was injected into nude mice bearing a human colon carcinoma (Co-112). The tumor uptake was found to be dependent on the size of the tumors: relative uptake (percentage of the injected dose/gram tumor (% i. d./g) decreased for tumors with weights up to 1 g, although the absolute uptake (% i. d./tumor) still increased over the same weight range. In the constant region (1 g) mean relative tumor uptake was 4% i. d./g.The same tumor size dependence was found for the relative Tumak uptake in the other mouse organs studied (e.g., blood, liver, spleen and muscle). Consequently tumor/organ ratios were found to be independent of tumor size.Tumor uptake was also studied for various doses of Tumak (0.07–120 g) in tumors of 1 g. Evidence was found for a threshold dose of 0.1 g under which no serious tumor uptake appeared. From 1 to 120 g no further dependence of Tumak distribution on applied dose was found: the relative uptake of all organs remained the same but the absolute uptake increased with dose.     

Physico-chemical and immunological properties of allophycocyanins

Allophycocyanins were purified from diverse cyanobacteria and one rhodophytan alga (Cyanidium caldarium). The native proteins are trimeric molecules with the structure ()₃. Representative native allophycocyanins and their and subunits were characterized with respect to molecular weight, amino acid composition, isoelectric point, absorption and fluorescence spectra and immunological properties. All of the allophycocyanins studied were strikingly similar with respect to each of these properties.Renatured and subunits of allophycocyanin were distinct immunologically from each other, and both cross-reacted with the antiserum to the native protein.Trimeric allophycocyanin was readily reconstituted from the purified and subunits. Formation of hybrid allophycocyanins was demonstrated by direct isolation and characterization of the hybrid proteins and by immunological techniques.The results support the view that allophycocyanins are a highly conserved group of proteins.Abbreviation Used SDS sodium dodecyl sulfate     

Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute     

Zum Ortstreue-Verhalten des Trauerschn?ppers(Ficedula hypoleuca) im westlichen Randbereich seines mitteleurop?ischen Verbreitungsgebietes

Zusammenfassung 1974–1981 wurden in einem Lärchen-Versuchsforst (320 ha; 600 künstliche Nisthöhlen) in Niedersachsen (52.27 N/7.15 E) 529 Brutvögel des Trauerschnäppers beringt. Geburtsortstreue — auf das Versuchsgebiet bezogen — konnte bei 78 Individuen (=5,2 % der ausgeflogenen Nestlinge) festgestellt werden. Die geburtsortstreuen wurden in der Regel schon im ersten Jahr als Brutvogel nachgewiesen, während die Hälfte der geburtsortstreuen bei der ersten Erfassung ein Alter von mindestens 2 Jahren besaß. In der mittleren Entfernung zwischen Geburts- und erstbekannter Bruthöhle ergab sich zwischen den Geschlechtern kein gesicherter Unterschied ( : 883 m, : 798 m). Geschwister verhielten sich bezüglich Ansiedlungsalter und Ansiedlungs-entfernung nicht einheitlich. Von den als Brutvögel beringten Trauerschnäppern konnten 38,2 % der und 24,0 % der in einem späteren Jahr wiedergefangen werden. Bei den lagen die in aufeinanderfolgenden Jahren besetzten Nisthöhlen näher beieinander als bei den (Mittelwerte: 371 m bzw. 534 m). In der untersuchten Population war in allen Jahren bei den der Prozentsatz brutortstreuer Individuen höher und der Anteil an Zuwanderern geringer als bei den (Mittelwerte: 48 % gegenüber 30 % bzw. 45 % gegenüber 64 %). Diese Befunde wurden unter anderem im Hinblick auf die Lage des Untersuchungsgebietes am Rande des Trauerschnäpper-Areals diskutiert. Die Ortstreue war geringer ausgebildet als in mehreren anderen Populationen.

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On place-fidelity behaviour in the Pied Flycatcher(Ficedula hypoleuca) in the western border area of its Central European range

Summary The evaluation is based on findings from 1974 to 1981, in a 320 ha. experimental larch forest (52.27N, 7.15E), with 600 nestboxes, in western Lower Saxony, from a total of 529 breeding Pied Flycatchers. Fidelity to birthplace — recovery in the study area — was recorded for 78 individuals (=5.2 % of the fledged nestlings). faithful to birthplace were traced, as a rule, in the first year, while half of the who showed this fidelity were at least 2 years old when first recorded. There was no significant difference between sexes in the mean distance between the hole in which they were hatched and the first known breeding hole ( : 883 m, : 798 m). There was no uniformity among siblings in relation to age of settlement and distance of settlement. 38.2 % of and 24.0 % of Pied Flycatchers ringed as breeding birds were recaptured in a later year. Nest boxes occupied in successive years were closer together in the case of than (mean: 371 m and 534 m respectively). The percentage of individuals showing fidelity to birthplace was higher, in all years, and the number of immigrants smaller, in the than in (mean: 48 % against 30 % and 45 % against 64 % respectively). These findings are discussed in relation to the border situation of the study area. The rate of recoveries was lower than in some other populations.

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Gefördert mit Hilfe von Forschungsmitteln des Landes Niedersachsen.     

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic β-cells and HIT cells

Summary ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic -cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 m and as high as 500 m, with maximal activation between 10 and 100 m ADP in mouse -cell membrane patches. At concentrations greater than 500 m, ADP inhibited K(ATP) channels while 10 mm virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mm ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of -cell membrane potential, increasing ADP in the 10 and 100 m range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize -cells.The results suggest that (i) K(ATP)-channel activity is not well predicted by the ATP/ADP ratio, and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10–100 m range as suggested by biochemical studies.We would like to thank Mr. Louis Stamps for expert technical assistance and Dr. Wil Fujimoto and Ms. Jeanette Teague for generously providing HIT cells obtained from Dr. Robert Santerre at Eli Lilly. We would also like to thank Dr. Michel Vivaudou for providing the program ALEX. Support was provided by the NIH and the Department of Veterans Affairs.     

Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells

The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA bovine serum albumin - CHO chinese hamster ovary - DHFR dihydrofolate reductase - FCS foetal calf serum - IFN- human interferon-gamma - q _IFN specific interferon production rate - specific growth rate - 2N doubly-gycosylated - 1N singly-glycosylated - ON non-glycosylated     

Vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibres in the penile cavernous tissue of green monkeys (Cercopithecus aethiops)

Summary The cavernous body of green monkeys contains many unmyelinated and few myelinated axons. The unmyelinated axons form terminals in the adventitia of the arteries, between trabecular muscle cells, in the interstitium, and close to endothelium cells of the sinuses. All terminals displayed predominantly small clear vesicles and very few large granular vesicles; small granular vesicles were not seen. However, in rabbit penises, terminals with many large granular vesicles are prominent. Immunohistochemistry (PAP technique) showed a dense network of VIP- and NPY-reactive fibres around the arteries and around trabecular muscles. The density of nerve fibres was particularly high around the subendothelial cushions of the helicine arteries. Double staining for NPY and VIP revealed that both peptides were colocalized. Immunocytochemistry (preembedding PAP technique) showed VIP- and NPY-reactivity in terminals with small clear vesicles; the reaction product was bound to the cytoplasmic face of different membrane types. Although the intracellular localization of the reaction product is probably due to artefactual displacement during preparation, the uniformity of the terminals questions the view that large and small granular vesicles in all species characterize peptidergic and noradrenergic terminals, respectively. The essential findings can be summarized as (1) a high degree of uniformity of nerve terminals, (2) colocalization of VIP and NPY, (3) heavy innervation of the subendothelial cushions of the helicine arteries, and (4) possible innervation of endothelial cells.     

Experimentally elevated testosterone increases status signalling in male Greylag geese (<Emphasis Type="Italic">Anser anser</Emphasis>)

Testosterone modulates male vertebrates sexual and social behaviour. We experimentally investigated the testosterone-sensitive behaviours in male greylag geese (Anser anser) by implanting silastic tubes containing crystalline testosterone during the mating season (February; 5 implanted and 5 control males) and in the early winter (November; 7 and 7). Focal animals were part of a semi-tame, unrestrained flock with fully intact social relationships. Excreted testosterone and corticosterone immunoreactive metabolites (TM, BM) were determined by enzyme immunoassay. Individual faecal samples and behavioural protocols were collected daily over a period of 5 weeks, including 1 control week before implantation. In February, no significant behavioural effects of the supplemental testosterone were observed, which may be due to the naturally occurring high systemic androgen levels in spring. In November, however, implanted males had higher TM excretion rates and performed status signalling behaviour (beak up) more frequently than control males. No differences between implanted and control males were found with respect to BM, agonistic interactions or vigilance behaviour. Furthermore, during the second week after implantation, TM positively correlated with the frequency of beak up of implanted males, whilst their female partners were attacked with lower latency by other members of the flock than the females of control males. Hence, status signalling in greylag ganders seems to be testosterone-sensitive year-long and inappropriate status signalling of males may draw attacks towards their females.     

Über die Kalkausscheidungen beiMonophyllaea horsfieldii (Gesneriaceae)

The lower surface of the leaf (macrocotyledon) ofM. horsfieldii is heavily calcified. SEM investigations reveal that the cristalline depositions of CaCO₃ (mainly needles, but also clump-like structures) are excreted by the head cells of two-cellular trichomhydathodes. First, a cap-like structure is formed. As the excretion continues, the cap takes on the shape of a hat with wide brim. Thus, the dense layer of CaCO₃ depositions is composed of hat-like structures whose brims at least partially touch. There is no evidence for the excretion of CaCO₃ by cells other than trichomhydathodes.

     

The isolation of barley-aleurone protoplasts

Summary A procedure is described for the isolation of large numbers of viable aleurone protoplasts. After treatment with Onozuka cellulase protoplasts were obtained which were surrounded by a thin, Onozuka-resistant wall. These cells were termed spheroplasts. Treatment of spheroplasts with Glusulase digested away the residual wall, yielding naked protoplasts. Measurements of respiration using a Clarke-type oxygen electrode indicated that aleurone cells isolated by this procedure were viable.Work supported by National Science Foundation grant GB-27468.     

Ultrastructure of motor nerve terminals on different types of muscle fibers in the atlantic hagfish (Myxine glutinosa,L.)

Summary White and intermediate parietal muscle fibers of Myxine are innervated focally at one end. Most synaptic vesicles are empty. These terminals also contain 1–2% large 800–1.100 Å dense-core vesicles. Red fibers of parietal and craniovelar muscle are innervated in a distributed fashion, and the presynaptic profiles contain a higher number of large dense-core vesicles (averaging 9% and 15%, respectively; up to 37%). For all terminals the synaptic gap is 450–600 Å wide, and postsynaptic folds are absent.Empty synaptic vesicles exist as round or elongated profiles. The proportion of elongated profiles increases by formation from round ones when increasing the molarity of the buffer in the aldehyde fixative. Furthermore, the proportion of elongated vesicle profiles in terminals on Myxine white fibers at different buffer molarities, is identical with that in mammalian motor terminals at similar molarities. On this basis the significance and mode of formation of elongated vesicle profiles is discussed. The conclusion is made that the susceptibility of flattening depends on the osmotic pressure of the vesicle contents once the aldehyde has influenced the vesicle membrane.The different vesicle populations in terminals on different types of muscle fibers are significant. Terminals on red fibers probably contain serotonin (5-HT) either as sole transmitter or in addition to acetylcholine.The author is indebted to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supply of hagfishes, and to Mrs. Jorunn Line Vaaland for expert technical assistance.     

A meta-analysis of frontalis EMG levels with biofeedback and alternative procedures

The lack of comparative reviews of the efficacy of EMG frontalis biofeedback versus alternative procedures for reduction of muscle tension prompted the present meta-analytic treatment of literature previously concluded to be equivocal. Twenty studies comparing EMG frontalis biofeedback with other tension-reduction procedures produced a total of 68 separate effect sizes suitable for meta-analysis. Differences between clinical and normal samples were nonsignificant, and data analyses revealed that EMG frontalis biofeedback was significantly superior to control (p<.05) but that alternative forms of muscle relaxation, while effective, did not reach statistical significance.     

Phosphorylation-dephosphorylation is involved in Ca2+-controlled cytoplasmic streaming of characean cells

Summary The mechanism of Ca²⁺ regulation of the cytoplasmic streaming in characean cells was studied in relation to protein phosphorylation and dephosphorylation. A tonoplast-free cell model was developed which was sensitive to Ca²⁺. Protein phosphatase-1 and its inhibitor-1 were applied into the tonoplast-free cells. A synthetic inhibitor of protein phosphatase, -naphthylphosphate, was applied either to tonoplast-free cells from inside or to the outside of plasmalemma-permeabilized cells which are known to be very sensitive to Ca²⁺. ATP--S applied to permeabilized cells strongly inhibited the recovery of the streaming which had been stopped by 10 M Ca²⁺. Both inhibitor-l and -naphthylphosphate inhibited the streaming even in the absence of Ca²⁺. On the other hand, protein phosphatase-l recovered the streaming even in the presence of Ca²⁺.The results indicate that characean streaming is regulated by the phosphorylation state of a regulatory and/or motile protein component. Streaming is activated when the component is dephosphorylated and inactivated when the component is phosphorylated. Ca²⁺ is assumed to stimulate both phosphorylation and dephosphorylation of the component. Involvement of Ca²⁺/calmodulin in the streaming recovery was discussed in terms of the stimulation of dephosphorylation.Abbreviations ATP -S, Adenosine-5-O-(3-thiotriphosphate) - -NP -naphthylphosphate - EGTA ethylenglycol-bis-(-aminoethylether)N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Pancreatic glucagon cells contain endorphin-like immunoreactivity

Summary On the basis of the histochemical activity of succinic dehydrogenase, only two fibre-types are distinguished in pigeon pectoralis major muscle. These are narrow Red and broad White. The histochemical activity of myofibrillar ATPase was studied in these two distinct fibre-types. Both fibre-types showed high activity for the ATPase. Red fibres of pigeon pectoralis were not alkali-labile, at incubation pH 9.4, as were the Type I fibres of both avian and mammalian muscles. Again unlike Type I fibres, the Red fibres of pigeon pectoralis lacked the characteristic activation of acid-preincubated ATPase reaction. Pigeon pectoralis Red fibres are known to posses some characteristics of fast-twitch fibres (e.g. high fat, considerable phosphorylase, fibrillenstruktur myofibrillar arrangement, focal en plaque pattern of nerve endings). It is emphasized, therefore, that the pigeon pectoralis Red fibres are not equivalent to Type I or slow-twitch, muscle fibres, but they are possibly fast-twitch fatigue resistent or Type II Red muscle fibres.     

A novel 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis: characterization,purification and kinetic properties

The enzyme, desacetoxyvindoline 4-hydroxylase, was purified to apparent homogeneity from Catharanthus roseus by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, green 19-agarose, hydroxylapatite, -kg sepharose and Mono Q. The 4-hydroxylase was characterized by its strict specificity for position 4 of desacetoxyvindoline suggesting it to catalyze the second to last step in vindoline biosynthesis. The molecular mass of the native and denatured 4-hydroxylase was 45 kDa and 44.7 kDa, respectively, suggesting that the native enzyme is a monomer. Two-dimensional isoelectric focusing under denaturing conditions resolved the purified 4-hydroxylase into three charge isoforms of pIs 4.6, 4.7 and 4.8. The purified 4-hydroxylase exhibited no requirement for divalent cations, but inactive enzyme was reactivated in a time-dependent manner by incubation with ferrous ions. The enzyme was not inhibited by EDTA or SH-group reagents at concentrations up to 10 mM. The mechanism of action of desacetoxyvindoline 4-hydroxylase was investigated. The results of substrate interaction kinetics and product inhibition studies suggest an Ordered Ter Ter mechanism where -kg is the first substrate to bind followed by the binding of O₂ and desacetoxyvindoline. Their K _m values for -kg, O₂ and desacetoxyvindoline are 45 M, 45 M and 0.03 M, respectively. The first product to be released was deacetylvindoline followed by CO₂ and succinate, respectively.Abbreviations -kg -ketoglutarate or 2-oxoglutarate - NMT N-methyltransferase - SAM S-adenosyl-l-methionine - TLC thin layer chromatography - VBL vinblastine - VCR vincristine