Periodic NADH oxidase activity associated with an endoplasmic reticulum fraction from pig liver. Response to micromolar concentrations of retinol

An endoplasmic reticulum fraction from pig liver enriched in transitional endoplasmic reticulum vesicles capable of forming 50-60 nm buds in the presence of ATP and retinol was assayed for retinol-responsive oxidation of NADH and cleavage of a dithiodipyridine (DTDP) protein disulfide-thiol interchange substrate. Maxima for the two activities alternated giving rise to a 24 min period. The NADH oxidase activity was inhibited by micromolar and submicromolar concentrations of retinol. Retinol at 0.1 mM stimulated the activity. The inhibition was confined to two activity maxima separated in time by about 5 min. In contrast, with the DTDP substrate, the activity was stimulated by retinol and the stimulations were in the part of the oscillatory pattern where retinol inhibition of NADH oxidation was observed. The findings support an earlier proposed mechanism whereby retinol exerted opposing effects on NADH oxidation and protein disulfide reductions.     

Retinol activates tyrosine hydroxylase acutely by increasing the phosphorylation of serine40 and then serine31 in bovine adrenal chromaffin cells

Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of the catecholamines. It has been reported that retinol (vitamin A) modulates tyrosine hydroxylase activity by increasing its expression through the activation of the nuclear retinoid receptors. In this study, we observed that retinol also leads to an acute activation of tyrosine hydroxylase in bovine adrenal chromaffin cells and this was shown to occur via two distinct non-genomic mechanisms. In the first mechanism, retinol induced an influx in extracellular calcium, activation of protein kinase C and serine40 phosphorylation, leading to tyrosine hydroxylase activation within 15 min. This effect then declined over time. The retinol-induced rise in intracellular calcium then led to a second slower mechanism; this involved an increase in reactive oxygen species, activation of extracellular signal-regulated kinase 1/2 and serine31 phosphorylation and the maintenance of tyrosine hydroxylase activation for up to 2 h. No effects were observed with retinoic acid. These results show that retinol activates tyrosine hydroxylase via two sequential non-genomic mechanisms, which have not previously been characterized. These mechanisms are likely to operate in vivo to facilitate the stress response, especially when vitamin supplements are taken or when retinol is used as a therapeutic agent.     

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity in the somatic cells of the seminiferous tubules. II. Effect of retinol

The effect of retinol on cyclic AMP dependent protein kinase activity of Sertoli cells and peritubular cells isolated from prepubertal rats has been investigated. Treatments longer than six hours induced a significant inhibition of type I protein kinase activity of Sertoli cells without appreciable variation of type II protein kinase. Short time treatments with the vitamin did not affect the Sertoli cell protein kinase activity. The vitamin A addition did not induce any appreciable variation of peritubular cell protein kinase activity.     

Retinoids as ligands and coactivators of protein kinase C alpha.

Whereas retinoic acids control nuclear events, a second class of retinol metabolites, that is, the hydroxylated forms exemplified by 14-hydroxy-retro-retinol (HRR), operate primarily in the cytoplasm. They function as regulatory cofactors for cell survival/cell death decisions. In accordance with these biological aspects, we demonstrate that these retinoids bound protein kinase C (PKC) alpha with nanomolar affinity and markedly enhance the activation of PKC alpha and the entire downstream MAP kinase pathway by reactive oxygen species. HRR was 10 times more efficient than retinol, and the optimum doses are 10-7 and 10-6 M, respectively. PKC alpha activation was reversed rapidly by imposition of reducing conditions. The retinoid binding site was mapped to the first cysteine-rich region in the regulatory domain, C1A, yet was distinct from the binding sites of diacylglycerol and phorbol esters. The C1B domain bound retinoids poorly. The emerging theme is that retinoids serve as redox regulators of protein kinase C.     

Xanthine dehydrogenase processes retinol to retinoic acid in human mammary epithelial cells

Retinoic acid is considered to be the active metabolite of retinol, able to control differentiation and proliferation of epithelia. Retinoic acid biosynthesis has been widely described with the implication of multiple enzymatic activities. However, our understanding of the cell biological function and regulation of this process is limited. In a recent study we evidenced that milk xanthine oxidase (E.C. 1.17.3.2.) is capable to oxidize all-trans-retinol bound to CRBP (holo-CRBP) to all-trans-retinaldehyde and then to all-trans-retinoic acid. To get further knowledge regarding this process we have evaluated the biosynthetic pathway of retinoic acid in a human mammary epithelial cell line (HMEC) in which xanthine dehydrogenase (E.C. 1.17.1.4.), the native form of xanthine oxidase, is expressed. Here we report the demonstration of a novel retinol oxidation pathway that in the HMEC cytoplasm directly conduces to retinoic acid. After isolation and immunoassay of the cytosolic protein showing retinol oxidizing activity we identified it with the well-known enzyme xanthine dehydrogenase. The NAD⁺ dependent retinol oxidation catalyzed by xanthine dehydrogenase is strictly dependent on cellular retinol binding proteins and is inhibited by oxypurinol. In this work, a new insight into the biological role of xanthine dehydrogenase is given.     

Inhibition of Starfish Oocyte Maturation by Tumor-Promoting Phorbol Esters*

Effect of tumor promoters including phorbol esters and teleocidin on 1-methyladenine (1-MeAde)-induced oocyte maturation was studied in the starfish. When isolated immature oocytes were treated with 1-MeAde and 12-O-tetradecanoylphorbol-13-acetate (TPA), 1-MeAde-induced maturation was completely inhibited at more than 2.5 μg/ml. However, if TPA was added after the hormone-dependent period (the minimum period wherein 1-MeAde is required), such maturation-inhibiting effect was no longer observed. Pretreatment with TPA for 5 min showed that its inhibitory action is irreversible. However, when TPA-injected oocytes were treated with 1-MeAde, all oocytes underwent germinal vesicle breakdown (GVBD). GVBD was induced in TPA-treated oocytes upon injection of the cytoplasm of maturing oocytes containing maturation-promoting factor (MPF). These facts show that TPA acts on the oocyte surface to inhibit the production of MPF. Retinoids including retinal, retinol and retinoic acid reversed the inhibitory effect of TPA on 1-MeAde-induced maturation. Experiments with various phorbol esters showed a good correlation between their maturation-inhibiting activity and their known tumor-promoting activity. Further, telecoidin, which is structurally unrelated to phorbol esters, inhibited 1-MeAde action. Since both tumor-promoting phorbol esters and teleocidin are known to activate Ca²⁺ -activated, phospholipid-dependent protein kinase (protein kinase C) and their activation effect is inhibited by retinoids, it appears that the activation of protein kinase C by tumor promoters is involved in blocking of 1-MeAde action.     

A molecular basis for retinol stimulation of vesicle budding in vivo and in vitro

Retinol stimulates the formation of transition vesicles in situ and in all free systems based on rat liver. The stimulation is on vesicle formation from transitional endoplasmic reticulum and not on vesicle fusion with donor membranes. Vesicle budding in the cell free system requires a nucleoside triphosphate and is sensitive to inhibition by thiol reagents. In this report we develop and test a model whereby a retinol-modulated NADH:protein disulfide reductase (NADH oxidase) with protein disulfide-thiol interchange activity is implicated in the vesicle budding mechanism. The protein has the ability to restore activity to scrambled, inactive RNase A and is stimulated or inhibited by retinol depending on the redox environment. Under reducing conditions and in the presence of a chemical reductant such as GSH, the partial reaction stimulated by retinol appears to be the oxidation of membrane disulfides. This is the first report of an enzymatic mechanism to explain specific retinol effects both in vivo and in vitro on membrane trafficking not given by retinoic acid.     

Vitamin A acid-induced activation of Ca2+-activated, phospholipid-dependent protein kinase from rabbit retina

Ca2+-activated, phospholipid-dependent protein kinase from rabbit retina was partially purified. Vitamin A acid (retinoic acid) stimulated this protein kinase in the presence of Ca2+, while other metabolites of vitamin A such as retinol or retinal were less effective. The order of the extent of phosphorylation of the various substrate proteins by this protein kinase was identical in the presence of vitamin A acid or phosphatidylserine. The major spots of the 32P labeled peptide from histone H1 phosphorylated in the presence of vitamin A acid by this protein kinase did not differ from those obtained from histone H1 phosphorylated in the presence of phosphatidylserine. Retinol caused a further enhancement of the enzymatic activity, whereas the addition of retinal inhibited the activation by vitamin A acid. Thus, vitamin A and its metabolites may play an important role in the regulation of Ca2+-activated, phospholipid-dependent protein kinase activity in the retina.     

Stimulation by Bt2cAMP of epidermal mucous metaplasia in retinol-pretreated chick embryonic cultured skin, and its inhibition by herbimycin A, an inhibitor for protein-tyrosine kinase.

Epidermal mucous metaplasia of cultured 13-day-old chick embryonic tarsometatarsal skin can be induced by culture in medium containing retinol (20 microM) for only 8-24 h and then in a chemically defined medium without vitamins or serum for 6 days. In the induction of mucous metaplasia, retinol primarily affects the dermal cells and a signal(s) induced in the dermis by excess retinol alters epidermal differentiation toward secretory epithelium. In this work we found that Bt2cAMP (2 mM) stimulated mucous metaplasia severalfold when added to retinol-pretreated skin but inhibited epidermal mucous metaplasia when added together with retinol. Forskolin (100 microM), an activator of adenylate cyclase, also stimulated mucous metaplasia when added to retinol-pretreated skin. On the other hand, transduction in the epidermal cells of a signal(s) induced in dermal cells by excess retinol was inhibited by herbimycin A (500 ng/ml), an inhibitor of protein-tyrosine kinases, and TPA (0.1 microM), an activator of protein kinase C. Hence these findings indicated that cAMP stimulated signal-induced mucous metaplasia, and that transduction of the signal(s) in the epidermal cells required protein-tyrosine kinase and was inhibited by protein kinase C.     

Studies on phospholipase activities in Neurospora crassa conidia

Cytosol retinyl ester lipoprotein complex from rat liver was capable of transferring its unesterified retinol component to serum aporetinol-binding protein. In the presence of serum albumin and aporetinol-binding protein, 68% of retinyl ester was hydrolyzed and up to 30% of unesterified retinol was transferred from cytosol retinyl ester lipoprotein complex to serum aporetinol-binding protein in 24 h at 30 °C. The reconstituted retinol-retinol-binding protein complex showed biochemical and biophysical properties similar to native retinol-retinol-binding protein. Both native and reconstituted retinol-retinol-binding proteins had identical uv, CD, and fluorescence spectra as well as binding affinity to prealbumin. Treatment of cytosol retinyl ester lipoprotein with sulfhydryl reagent, with 1 n NaCl, or with diisopropyl fluorophosphate (0.14 mm) abolished the hydrolysis of retinyl ester; however, the activity of retinol transfer from cytosol retinyl ester lipoprotein complex to serum retinol-binding protein was still unaffected. The activity of retinol transfer was proportional to the amount of retinol content in the complex and the amount of aporetinol-binding protein. These experiments suggest that the cytosol retinyl ester lipoprotein complex serves three major functions: (i) as a storage form of retinyl ester and retinol; (ii) as an enzyme for hydrolyzing its own retinyl ester ligand; and (iii) as a medium for transfer of unesterified retinol to serum retinol-binding protein.     

Rapid effects of phorbol esters on isolated rat adipocytes. Relationship to the action of protein kinase C

Treatment of isolated rat adipocytes with tumor-promoting phorbol esters, caused a fivefold stimulation of glucose oxidation, determined as 14CO2 production from [1-14C]glucose and a fivefold increase in the rate of lipid synthesis from [14C]glucose. Treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate increased the rate of 86Rb+ uptake into the cells. Also phospholipase C was able to stimulate the rate of glucose oxidation; phospholipase C and 12-O-tetradecanoylphorbol 13-acetate stimulated glucose oxidation in a non-synergistic fashion, indicating a common mechanism for their action. Active phorbol esters and, in part, also phospholipase C, caused a translocation of protein kinase C activity from the soluble to the particulate fraction of the adipocytes. This process was rapid, being complete 30 s after the addition of phorbol ester, and resulted in the appearance of the kinase mainly in the mitochondrial and plasma membrane fractions. A comparison between the binding characteristics of adipocyte protein kinase C and the metabolic effects of the phorbol esters on the adipocytes revealed that the dose-response relationship did not correlate with binding of the phorbol esters, but, rather, a correlation was observed between the dose of phorbol esters required for translocation of protein kinase C and the intracellular effects. The results indicate that the intracellular translocation of protein kinase C might be a trigger for the effects of phorbol esters on the adipocyte and that binding of the esters to protein kinase C is not a sufficient event to cause this effect. Furthermore, it is suggested that activation of protein kinase C might be partly the action of hormones, such as insulin, on the fat cells.     

Peripheral effect of alpha-melanocyte-stimulating hormone on fatty acid oxidation in skeletal muscle

To study the peripheral effects of melanocortin on fuel homeostasis in skeletal muscle, we assessed palmitate oxidation and AMP kinase activity in alpha-melanocyte-stimulating hormone (alpha-MSH)-treated muscle cells. After alpha-MSH treatment, carnitine palmitoyltransferase-1 and fatty acid oxidation (FAO) increased in a dose-dependent manner. A strong melanocortin agonist, NDP-MSH, also stimulated FAO in primary culture muscle cells and C2C12 cells. However, [Glu6]alpha-MSH-ND, which has ample MC4R and MC3R agonistic activity, stimulated FAO only at high concentrations (10(-5) M). JKC-363, a selective MC4R antagonist, did not suppress alpha-MSH-induced FAO. Meanwhile, SHU9119, which has both antagonistic activity on MC3R and MC4R and agonistic activity on both MC1R and MC5R, increased the effect of alpha-MSH on FAO in both C2C12 and primary muscle cells. Small interference RNA against MC5R suppressed the alpha-MSH-induced FAO effectively. cAMP analogues mimicked the effect of alpha-MSH on FAO, and the effects of both alpha-MSH and cAMP analogue-mediated FAO were antagonized by a protein kinase A inhibitor (H89) and a cAMP antagonist ((Rp)-cAMP). Acetyl-CoA carboxylase activity was suppressed by alpha-MSH and cAMP analogues by phosphorylation through AMP-activated protein kinase activation in C2C12 cells. Taken together, these results suggest that alpha-MSH increases FAO in skeletal muscle, in which MC5R may play a major role. Furthermore, these results suggest that alpha-MSH-induced FAO involves cAMP-protein kinase A-mediated AMP-activated protein kinase activation.     

Xanthine dehydrogenase processes retinol to retinoic acid in human mammary epithelial cells

Retinoic acid is considered to be the active metabolite of retinol, able to control differentiation and proliferation of epithelia. Retinoic acid biosynthesis has been widely described with the implication of multiple enzymatic activities. However, our understanding of the cell biological function and regulation of this process is limited. In a recent study we evidenced that milk xanthine oxidase (E.C. 1.17.3.2.) is capable to oxidize all-trans-retinol bound to CRBP (holo-CRBP) to all-trans-retinaldehyde and then to all-trans-retinoic acid. To get further knowledge regarding this process we have evaluated the biosynthetic pathway of retinoic acid in a human mammary epithelial cell line (HMEC) in which xanthine dehydrogenase (E.C. 1.17.1.4.), the native form of xanthine oxidase, is expressed. Here we report the demonstration of a novel retinol oxidation pathway that in the HMEC cytoplasm directly conduces to retinoic acid. After isolation and immunoassay of the cytosolic protein showing retinol oxidizing activity we identified it with the well-known enzyme xanthine dehydrogenase. The NAD+ dependent retinol oxidation catalyzed by xanthine dehydrogenase is strictly dependent on cellular retinol binding proteins and is inhibited by oxypurinol. In this work, a new insight into the biological role of xanthine dehydrogenase is given.     

Retinoids antagonize the induction of ornithine decarboxylase activity by phorbol esters and phospholipase C in rat tracheal epithelial cells

In this study we examined the action of phorbol esters, several phospholipases and retinoids on the induction of ornithine decarboxylase (ODC) activity in rat tracheal epithelial cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induces ODC activity in these cells in a dose-and time-dependent manner. This induction is inhibited by cycloheximide indicating a requirement for protein synthesis. Tracheal epithelial 2C5 cells contain two binding sites for phorbol esters, one with a high affinity KD,1 = 4.58 nM and one with a low affinity KD,2 = 344.8 nM. The ability of several phorbol esters to induce ODC correlates well with the described efficacy with which they bind to the receptor and is in agreement with the concept that phorbol ester receptors are involved in the induction of ODC. There is strong evidence that the phorbol ester receptor is the protein kinase C for which diacylglycerol is the physiological ligand. Treatment of cells with phospholipase C generates diacylglycerol and induces ODC activity in a dose- and time-dependent manner. Treatment with phospholipase A2 or D has no effect on ODC activity. These results support the concept that activation of protein kinase C is related to the induction of ODC activity. The induction of ODC by TPA as well as by phospholipase C is inhibited by retinoids. Specific cytosolic binding proteins for retinoids might be involved in at least some of the responses to these compounds. To examine whether the binding proteins are involved in the inhibition of ODC we determined the presence of these binding proteins and the structure-activity relationship of retinoids. Both retinol and retinoic acid-binding proteins can be detected in 2C5 cells, their levels are 1.06 and 3.36 pmoles/mg protein, respectively. The ability of several retinoids to inhibit ODC induction correlates well with their binding activity and support a role for these binding proteins in the action of retinoids on ODC induction.     

The assay of the activity of protein kinase C with the synthetic oligopeptide substrate designed for histone kinase II

The activity of histone kinase II was determined on the basis of its ability to phosphorylate the nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously as a specific substrate for this enzyme. Histone kinase II was purified from calf thymus extract by DEAE-cellulose chromatography followed by hydroxylapatite chromatography and high-performance liquid chromatography on a Protein Analysis column (I-125). The Mr value of histone kinase II estimated by the latter method was 50,000-55,000, but several observations indicated that histone kinase II was a product of a proteolytic process. Since the substrate specificity determinants for histone kinase II known from our previous investigations are very similar to those for protein kinase C, it was presumable that histone kinase II was the proteolytic fragment of protein kinase C. Therefore, the nonapeptide was tested as a substrate for protein kinase C prepared from rabbit brain extract by DEAE-cellulose chromatography. The activity of histone kinase II was also detected in brain extract. Histone kinase II was eluted from the DEAE-cellulose in the known position of the proteolytic fragment of protein kinase C. The nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide proved to be a better substrate than H1 histone for the detection of the activity of protein kinase C because it was not phosphorylated by the cAMP-dependent protein kinase and the Vmax of protein kinase C was about one order of magnitude higher with the peptide than with H1 histone. The apparent Km of protein kinase C for the peptide was identical with that of histone kinase II (0.2 mM).     

Induction of apoptosis by protein kinase C delta is independent of its kinase activity

Protein kinase C, a multigene family of phospholipid-dependent and diacylglycerol-activated Ser/Thr protein kinases, is a key component in many signal transduction pathways. The kinase activity was thought to be essential for a plethora of biological processes attributed to these enzymes. Here we show that at least one protein kinase C function, the induction of apoptosis by protein kinase C delta, is independent of the kinase activity. Stimulation of green fluorescent protein-protein kinase C delta fusion protein with phorbol ester or diacylglycerol led to its redistribution within seconds after the stimulus. Membrane blebbing, an early hallmark of apoptosis, was visible as early as 20 min after stimulation, and nuclear condensation was visible after 3-5 h. Apoptosis could be inhibited by expression of Bcl-2 but not by specific protein kinase C inhibitors. In addition, a kinase-negative mutant of protein kinase C delta also induced apoptosis to the same extent as the wild type enzyme. Apoptosis was confined to the protein kinase C delta-overexpressing cells. Stimulation of overexpressed protein kinase C epsilon did not result in increased apoptosis. Our results indicate that distinct protein kinase C isozymes induce apoptosis in vascular smooth muscle cells. More importantly, they show that some protein kinase C effector functions are independent of the catalytic activity.     

Identification of an endogenous protein kinase C activity and its intrinsic 15-kilodalton substrate in purified canine cardiac sarcolemmal vesicles

The cardiac sarcolemmal 15-kDa protein, previously shown to be the principal sarcolemmal substrate phosphorylated in intact heart in response to beta-adrenergic stimulation (Presti, C. F., Jones, L. R., and Lindemann J. P. (1985) J. Biol. Chem. 260, 3860-3867), was demonstrated to be the major substrate phosphorylated in purified canine cardiac sarcolemmal vesicles by an intrinsic protein kinase C activity. The intrinsic protein kinase C, detected by its ability to phosphorylate H1 histones, was most concentrated in cardiac sarcolemmal vesicles and absent from sarcoplasmic reticulum membranes. Unmasking techniques localized the intrinsic protein kinase activity and its principal endogenous substrate, the 15-kDa protein, to the cytoplasmic surfaces of sarcolemmal vesicles; phospholamban contaminating the sarcolemmal preparation was not significantly phosphorylated. The intrinsic protein kinase C required micromolar Ca2+ for activity, but not calmodulin. Half-maximal phosphorylation of the 15-kDa protein occurred at 10 microM Ca2+; optimal phosphorylation of the 15-kDa protein by protein kinase C and Ca2+ was additive to that produced by cAMP-dependent protein kinase. Exogenous phospholipids were not required to activate endogenous protein kinase C. However, heat-treated sarcolemmal vesicles, in which intrinsic protein kinase activities were inactivated, were sufficient to maximally activate soluble protein kinase C prepared from rat brain, suggesting that all the necessary phospholipid cofactors were already present in sarcolemmal vesicles. Of the many proteins present in sarcolemmal vesicles, only the 15-kDa protein was phosphorylated significantly in heat-inactivated sarcolemmal vesicles by soluble protein kinase C, confirming that the 15-kDa protein was a preferential substrate for this enzyme. Consistent with a protein kinase C activity in sarcolemmal vesicles, the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulated 15-kDa protein phosphorylation severalfold, producing approximately 70% of the maximal phosphorylation even in the absence of significant ionized Ca2+. The results are compatible with an intrinsic protein kinase C activity in sarcolemmal vesicles whose major substrate is the 15-kDa protein.     

Activation of adipocyte adenylate cyclase by protein kinase C

Adenylate cyclase activity in purified rat adipocyte membranes is stimulated by the calcium- and phospholipid-dependent enzyme protein kinase C. Over the concentration range of 100-1000 milliunits/ml, both highly purified (approximately 3000 units/mg of protein) protein kinase C from rat brain and partially purified (14 units/mg of protein) protein kinase C from guinea pig pancreas stimulate cyclase activity. The actions of both protein kinase C preparations on adenylate cyclase activity are dependent on added calcium, which is effective at concentrations less than 10 microM. Exogenous phospholipids are not required for stimulation of adenylate cyclase by protein kinase C; but, under typical cyclase assay conditions, the adipocyte membranes satisfy the lipid requirement for protein kinase C phosphorylation of histone. The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate enhances the kinase action on cyclase, and the phorbol ester is effective at concentrations equimolar with the kinase (less than 10 nM). With the brain protein kinase C, 12-O-tetradecanoylphorbol-13-acetate effects are especially evident at limiting calcium concentrations. Inhibitors of protein kinase C activity, such as chlorpromazine, palmitoylcarnitine, and polymyxin B, inhibit selectively that adenylate cyclase activity which is stimulated by protein kinase C plus calcium. It is concluded that protein kinase C acts directly on the adipocyte adenylate cyclase system.     

RDH13L,an Enzyme Responsible for the Aldehyde-Alcohol Redox Coupling Reaction (AL-OL Coupling Reaction) to Supply 11-cis Retinal in the Carp Cone Retinoid Cycle

Cone photoreceptors require effective pigment regeneration mechanisms to maintain their sensitivity in the light. Our previous studies in carp cones suggested the presence of an unconventional and very effective mechanism to produce 11-cis retinal, the necessary component in pigment regeneration. In this reaction (aldehyde-alcohol redox coupling reaction, AL-OL coupling reaction), formation of 11-cis retinal, i.e. oxidation of 11-cis retinol is coupled to reduction of an aldehyde at a 1:1 molar ratio without exogenous NADP(H) which is usually required in this kind of reaction. Here, we identified carp retinol dehydrogenase 13-like (RDH13L) as an enzyme catalyzing the AL-OL coupling reaction. RDH13L was partially purified from purified carp cones, identified as a candidate protein, and its AL-OL coupling activity was confirmed using recombinant RDH13L. We further examined the substrate specificity, subcellular localization, and expression level of RDH13L. Based on these results, we concluded that RDH13L contributes to a significant part, but not all, of the AL-OL coupling activity in carp cones. RDH13L contained tightly bound NADP⁺ which presumably functions as a cofactor in the reaction. Mouse RDH14, a mouse homolog of carp RDH13L, also showed the AL-OL coupling activity. Interestingly, although carp cone membranes, carp RDH13L and mouse RDH14 all showed the coupling activity at 15–37 °C, they also showed a conventional NADP⁺-dependent 11-cis retinol oxidation activity above 25 °C without addition of aldehydes. This dual mechanism of 11-cis retinal synthesis attained by carp RDH13L and mouse RDH14 probably contribute to effective pigment regeneration in cones that function in the light.     

Early effects of TPA on protein kinase activity in murine thymocytes. Reduction of protein kinase C activity in the cytosol and increase of Ca2+ and phospholipid-independent kinase activity in the particulate fractions

Brief treatment of intact thymocytes with TPA and other tumor promoters causes a reduction in protein kinase C activity from the cytosol and an increase in kinase activity in the particulate fraction. In contrast to the activity in the cytosol, which is absolutely dependent on the addition of Ca2+, phosphatidylserine and diolein, the activity in the particulate fraction is independent of these agents. Analysis of target specificity of the particulate kinase activity using exogenous and endogenous substrates suggests that the increased phosphorylation in the particulate fraction is catalysed by protein kinase C with altered catalytic properties. Although interleukin-1 and TPA are both co-mitogens for murine thymocytes, interleukin-1 does not share with TPA its property to alter protein kinase activity in the cytosolic and particulate fractions.