Cation distributions across the larval and pupal midgut of the lepidopteran, Hyalophora cecropia, in vivo.

1. The levels of potassium, sodium, magnesium and calcium in leaves, midgut contents, midgut tissue, and blood were analysed in seven developmental stages between feeding, fourth-instar larvae and new pupae of the Cecropia silkworm. 2. Three dramatic changes in cation levels were found: the K level in the contents drops from 284 /+- 51 mEquiv./1 tissue water in the fifth-instar larva to 51 +/- 6 mEquiv./1 in the new pupa; the Mg level in the midgut tissue increases from 28 +/- 3 mEquiv./1 at the time of gut evacuation to 1093 +/- 104 mEquiv./1 in the new pupa; and the Ca level in the contents drops temporarily from 56 +/- 12 mEquiv./1 in the feeding fourth instar larva to 17 +/- 5 mEquiv./1 in the new fifth instar larva. The Na level was nerve higher than 2.8 +/- 0.5 mEquiv./1. 3. The relative levels of the four cations were different for each tissue studied, but each tissue maintained the same relative levels during the developmental stages studied. The sequences are: leaf, Ca greater than K GREATEr than Mg greater than Na; midgut contents, K greater than Ca greater than Mg greater than Na; midgut tissue, K GREATEr than Mg greater than Ca greater than Na; and blood, Mg greater than K greater than Ca greater than Na. 4. There were three large concentration gradients across the midgut; the K level in the midgut contents is approximately 10 times the level in blood; the Mg level in contents is one-half to one-sixth the level in blood; and the Ca level in contents is 3-4 times the level in blood. The K gradient and the Ca gradient are opposed and the Mg gradient is favoured by the electrical gradient across the larval midgut, the contents being 100 mV positive with respect to the blood. The K gradient and the electrical gradient are not present across the pupal midgut while the Mg gradient and the Ca gradient persist. 5. The K gradient is presumably maintained by the midgut K pump, the Mg gradient is aided by the midgut Mg pump, and the Ca gradient suggests that the midgut may possess a Ca pump.     

Low speed preparation of microsomes: a comparative study

Microsomal fractions were isolated from hepatic tissue, housefly abdomen and southern armyworm midgut, either by centrifugation of post-mitochondrial supernatant for 1 h at 105 000×g or by calcium sedimentation and low speed centrifugation. A comparison of the two methods of isolation were made on the basis of: NADPH oxidase activity, O-demethylation of p-nitroanisole, spectral parameters of cytochrome P-450, and integrity of ultrastructural constituents. The method of isolation did not effect these parameters appreciably for microsomes isolated from hepatic tissue. Calcium sedimentation of insect microsomes resulted in a reduction of enzyme activity and an apparent decrease in cytochrome P-450. Ultrastructural integrity did not appear to be influenced by the method of sedimentation.     

Characterization of an insect ferritin subunit synthesized in a cell-free system

Ferritin, an iron-sequestering and -binding protein, is localized to the vacuolar system in Calpodes ethlius larvae. The amount of iron-loaded ferritin in intact larval midgut can be increased by pretreatment with iron. When poly(A)+ RNA from control or iron-treated larvae was translated in vitro, a 24 kilodalton (kDa) protein was a major translation product. If the cell-free system was supplemented with dog pancreatic microsomes, the 24-kDa protein was not detectable: the major translation product was 28-30 kDa. The 24-kDa and 28- to 30-kDa proteins were identified as ferritin subunits by immunoprecipitation with anti-Manduca ferritin antibodies. Proteinase K digestion of the translation products showed that the 28- to 30-kDa subunit was targeted into the lumen of, and protected by, the microsomes. The change in molecular mass of the ferritin monomer was attributed to glycosylation of the 24-kDa subunit within the lumen of the microsomes. This was demonstrated by (i) the ability of the 28- to 30-kDa subunit, but not the 24-kDa subunit, to bind concanavalin A on Western blots and (ii) inhibition of the change in molecular mass from 24 to 28-30 kDa if tunicamycin is added to the microsomes. The results indicate that the Calpodes ferritin subunit was synthesized, targeted to microsomes, and glycosylated within their lumen in a rabbit reticulocyte cell-free system primed with midgut poly(A)+ RNA extracted from control or iron-treated larvae.     

Peptidergic regulation of the Limulus midgut

1. The morphology and innervation of the midgut (intestine) in the horseshoe crab, Limulus polyphemus was investigated. The organization of this tissue was examined with routine histology. Radioimmunoassay, immunohistochemistry and high performance liquid chromatography were employed to detect, localize and identify peptidergic innervation of the midgut. The actions of synthetic and native proctolin-like and FMRFamide-like peptides were compared on the isolated midgut preparation. 2. Levels of proctolin and FMRFamide were determined in extracts of Limulus midgut tissue using radioimmunoassay. High levels of proctolin-like immunoreactivity (69.5 +/- 11.3 ng/g) were detected, while levels of FMRFamide-like immunoreactivity (0.8 +/- 0.2 ng/g) were less. Proctolin levels were equally distributed, while the levels of FMRFamide-like immunoreactivity exhibited an anterior bias. 3. Proctolin- and FMRFamide-like immunoreactivities in the Limulus midgut were localized with immunohistochemistry. Proctolin- and FMRFamide-immunoreactive elements were detected in intestinal nerve branches and individual fibers running along the surface of the midgut in whole-mount preparations. In sectioned tissue, staining for these peptides was observed throughout the midgut, typically associated with muscle bands and fibers. Only a few immunoreactive cell bodies were observed. 4. Proctolin, and several FMRFamide-like peptides produced distinct and opposing actions on the isolated Limulus midgut preparation. Proctolin elicited contracture and rhythmic contractions of this tissue, while FMRFamide and N-terminally extended analogs of FLRFamide relaxed gut tension. FMRFamide-like peptides partially reversed the excitatory actions of proctolin. 5. Proctolin- and FMRFamide-like peptides in Limulus midgut extracts were partially characterized with high performance liquid chromatography. One peak of proctolin-like activity was detected on a linear gradient of 18 to 31.5% acetonitrile. The native proctolin-like peptide produced excitatory actions on the isolated midgut preparation which were indistinguishable from those produced by synthetic proctolin. Several peaks of FMRFamide-like bioactivity (Busycon radula protractor muscle assay) were detected with a linear gradient of 5 to 30% acetonitrile. Fractions from two distinct peaks produced FMRFamide-like inhibitory effects on the isolated Limulus midgut preparation. These findings suggest a role for proctolin-like and FMRFamide-like peptides as regulators of intestinal motility in Limulus.     

Preparation and characterization of total, rough and smooth microsomes from the lung of control and methylcholanthrene-treated rats.

Optimal conditions for the preparation of relatively pure microsomes and microsomal subfractions from rat lung have been determined. The most importnat of these conditions is homogenization of a 20% (w/v) suspension of lung tissue in 0.44 M sucrose/1% (w/v) bovine serum albumin with four up-and-down strokes at 440 rev./min in a Potter-Elvehjem homogenizer. The 10000 X g supernatant prepared from this homogenate can be centrifuged at 105000 X g to obtain total microsomes or subfractionated into rough and smooth microsomes on a Cs+-containing discontinuous sucrose gradient. The total, rough and smooth microsomes have been characterized in terms of their chemical composition, enzymatic activity, and morphology. These preparations should prove useful in studies of various enzymes in lung (e.g. benzpyrene monooxygenase, epoxide hydrase, enzymes of phospholipid and ascorbic acid synthesis) and in subfractionations designed to reveal heterogeneites in the lateral plane of the lung endoplasmic reticulum.     

Aryl hydrocarbon hydroxylase in larvae of the southern armyworm (Spodoptera eridania)

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity in midgut microsomes of southern armyworm (Spodoptera eridania) larvae was induced 11-fold and 5.6-fold respectively following three days of feeding on diets containing pentamethylbenzene or naphthalene (both 0.2% w/v). β-Naphthoflavone and Aroclor 1254 were less effective inducers of AHH activity, phenobarbital was only slightly active and 3-methylcholanthrene caused a decrease in enzyme activity. AHH activity in microsomes from untreated and induced larvae was susceptible to inhibition by α-naphthoflavone, 1-phenylimidazole and piperonyl butoxide. Equilibrium dialysis studies with 1-(4′-³H-phenyl)imidazole showed that control and induced armyworm midgut microsomes contained a class of cytochrome(s) P-450 with a uniformly high affinity for phenylimidazole. It is concluded that AHH activity in the armyworm is catalyzed by a class of cytochrome(s) P-450 with characteristics intermediate between mammalian cytochrome(s) P-450 and P-448.     

Preparation and characterization of total,rough and smooth microsomes from the lung of control and methylcholanthrene-treated rats

Optimal conditions for the preparation of relatively pure microsomes and microsomal subfractions from rat lung have been determined. The most important of these conditions is homogenization of a 20% (w/v) suspension of lung tissue in 0.44 M sucrose/1% (w/v) bovine serum albumin with four up-and-down strokes at 440 rev./min in a Potter-Elvehjem homogenizer. The 10 000 × g supernatant prepared from this homogenate can be centrifuged at 105 000 × g to obtain total microsomes or subfractionated into rough and smooth microsomes on a Cs⁺-containing discontinuous sucrose gradient. The total, rough and smooth microsomes have been characterized in terms of their chemical composition, enzymatic activity, and morphology. These preparations should prove useful in studies of various enzymes in lung (e.g. benzpyrene monooxygenase, epoxide hydrase, enzymes of phospholipid and ascorbic acid synthesis) and in subfractionations designed to reveal heterogeneites in the lateral plane of the lung endoplasmic reticulum.     

家蚕消化管内桑叶的银染法研究

通过银染法对家蚕整体染色,结果表明:家蚕消化管内的桑叶由叶表皮、叶肉和叶脉组成。叶表皮包括上表皮和下表皮。上表皮细胞可分为三种:钟乳体细胞、绿色表皮细胞和黄色表皮细胞;下表皮内含有气孔;叶肉组织内含有晶体,其中海绵组织内的最多。家蚕消化管由前向后可分为前肠、中肠和后肠,由外向内依次为肌层、底膜、上皮细胞层、内膜。中肠最为发达,其发达的上皮细胞向内表面突起形成许多大的指突形皱褶;上皮细胞层内有圆筒形细胞、杯形细胞两种细胞,两者在形状、功能以及嗜银性等方面有所差异。家蚕消化管对桑叶不同组织的消化吸收效率有差异,上表皮吸收效率最高,下表皮和栅栏组织次之,最低的是海绵组织。采用动物细胞染色方法对植物细胞进行染色,并与常规植物学染色方法进行了比较;依据细胞嗜银性的不同,可将桑叶的上表皮细胞分为两种亚型。     

Isolation of separate apical,lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.     

Stem cells from midguts of Lepidopteran larvae: clues to the regulation of stem cell fate

Previously, we showed that isolated stem cells from midguts of Heliothis virescens can be induced to multiply in response to a multiplication protein (MP) isolated from pupal fat body, or to differentiate to larval types of mature midgut cells in response to either of 4 differentiation factors (MDFs) isolated from larval midgut cell-conditioned medium or pupal hemolymph. In this work, we show that the responses to MDF-2 and MP in H. virescens stem cells decayed at different time intervals, implying that the receptors or response cascades for stem cell differentiation and multiplication may be different. However, the processes appeared to be linked, since conditioned medium and MDF-2 prevented the action of MP on stem cells; MP by itself appeared to repress stem cell differentiation. Epidermal growth factor, retinoic acid, and platelet-derived growth factor induced isolated midgut stem cells of H. virescens and Lymantria dispar to multiply and to differentiate to mature midgut cells characteristic of prepupal, pupal, and adult lepidopteran midgut epithelium, and to squamous-like cells and scales not characteristic of midgut tissue instead of the larval types of mature midgut epithelium induced by the MDFs. Midgut stem cells appear to be multipotent and their various differentiated fates can be influenced by several growth factors.     

The peritrophic membrane of Spodoptera frugiperda: secretion of peritrophins and role in immobilization and recycling digestive enzymes

A peritrophin from the Spodoptera frugiperda peritrophic membrane (PM) and microvillar proteins from S. frugiperda anterior midgut cells were isolated and used to raise antibodies in a rabbit. These antibodies, as well as a Tenebrio molitor amylase antibody that cross-reacts with S. frugiperda amylases, and wheat-germ aglutinin were used in immunolocalization experiments performed with the aid of confocal fluorescence and immunogold techniques. The results showed that the peritrophin was secreted by anterior midgut columnar cells in vesicles pinched-off the microvilli (microapocrine secretion). The resulting double membrane vesicles become single membrane vesicles by membrane fusion, releasing peritrophin and part of the amylase and trypsin. The remaining membranes still containing microvillar proteins and membrane-bound amylase and trypsin are incorporated into a jelly-like material associated with PM. Calcofluor-treated larvae lacking a PM were shown to lose the decreasing gradient of trypsin and chymotrypsin observed along the midgut of control larvae. This gradient is thought to be formed by a countercurrent flux of fluid (in the space between PM and midgut cells) that powers enzyme recycling.     

Monitoring stem cell proliferation and differentiation in primary midgut cell cultures from Heliothis virescens larvae using flow cytometry

In the midgut of Heliothis virescens larvae, proliferation and differentiation of stem cell populations allow for midgut growth and regeneration. Basic epithelial regenerative function can be assessed in vitro by purifying these two cell type populations, yet efficient high throughput methods to monitor midgut stem cell proliferation and differentiation are not available. We describe a flow cytometry method to differentiate stem from mature midgut cells and use it to monitor proliferation, differentiation and death in primary midgut stem cell cultures from H. virescens larvae. Our method is based on differential light scattering and vital stain fluorescence properties to distinguish between stem and mature midgut cells. Using this method, we monitored proliferation and differentiation of H. virescens midgut cells cultured in the presence of fetal bovine serum (FBS) or AlbuMAX II. Supplementation with FBS resulted in increased stem cell differentiation after 5 days of culture, while AlbuMAX II-supplemented medium promoted stem cell proliferation. These data demonstrate utility of our flow cytometry method for studying stem cell-based epithelial regeneration, and indicate that AlbuMAX II-supplemented medium may be used to maintain pluripotency in primary midgut stem cell cultures.     

Characterization of the Mg2+-activated ATPase activity in smooth-muscle membranes. NADH oxidase and adenylate kinase interfere with the NADH-coupled enzyme assay.

The apparent Mg2+-activated ATPase activity measured by the continuous NADH-coupled enzyme assay was studied in a number of microsomal preparations obtained from smooth muscle of the myometrium from pregnant or 17 beta-oestradiol-pretreated rats, the bovine aorta, the guinea-pig taenia coli, the rabbit ear artery and pig antrum. It was shown that this ATPase assay is prone to the effects of a number of artefacts that are tissue-dependent. The apparent Mg2+-ATPase activity in microsomes (microsomal fractions) from myometrium, aorta and taenia coli declines non-linearly during the assay. Its initial high rate gradually diminishes over 15-60 min, depending on the type of smooth muscle, to a constant value. This decline depends on the presence of ATP and can be partially prevented by concanavalin A. The non-linearity is limited in microsomes from rabbit ear artery. In microsomes from antrum the apparent Mg2+-ATPase activity actually increases with time, albeit gradually. Storage on ice of the microsomes of the aorta, and especially of myometrium of pregnant rats and of taenia coli, is accompanied over a few hours after their preparation by a gradual suppression of the component of the Mg2+-ATPase activity that is inhibited by ATP. The Mg2+-ATPase activity in microsomes from antrum remains constant. NADH oxidase activity accounts for 10% of the Mg2+-ATPase activity in microsomes from stomach smooth muscle. The apparent initial non-linearity of the Mg2+-ATPase activity in that tissue is due to a time-dependent decrease of a rotenone-sensitive NADH oxidase activity. The adenylate kinase activity, as deduced from the effect of the adenylate kinase inhibitor P1,P5-di(adenosine-5') pentaphosphate, could account for 45.0, 35.0 and 31.0% respectively of the Mg2+-ATPase activity in microsomes from stomach, myometrium and aorta. No adenylate kinase activity could be detected in microsomes from ear artery and taenia coli. When microsomes from stomach smooth muscle were separated on a sucrose gradient, the contribution of adenylate kinase and NADH oxidase to the Mg2+-ATPase activity was most pronounced in the higher-density fractions. Part of the NADH oxidase activity and of the Mg2+-ATPase activity, and most of the adenylate kinase activity, are not sedimented at 224000 gmax. for 30 min and may therefore be present as soluble enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

SUBFRACTIONATION OF SMOOTH MICROSOMES FROM RAT LIVER

Total smooth microsomes from rat liver isolated on a Cs⁺-containing sucrose gradient were concentrated and subsequently fractionated by zone centrifugation on a stabilizing sucrose gradient. The prerequisite for fractionation is to prepare total smooth microsomes in a nonaggregated condition, as well as to utilize a procedure which counteracts enzyme inactivation. The median equilibrium density of the various smooth microsomal vesicles ranges from 1.10 to 1.18. The phospholipid/protein ratio is identical in all subfractions, but cholesterol, on a PLP basis, is enriched in the subfractions with the highest sedimentation velocity. The enzyme distribution pattern reveals a pronounced heterogeneity. A number of NADH- and NADPH-oxidizing enzymes are concentrated in the upper part of the gradient and exhibit a certain degree of separation from G6Pase. Mg⁺⁺-ATPase and AMPase are enriched in the lower part of the gradient. No specific enrichment of newly synthesized NADPH-cytochrome c reductase activity occurs in any of the subfractions after phenobarbital treatment. These data demonstrate that smooth microsomes, by adequate fractionation procedure, can be separated into subfractious of heterogeneous composition.     

Quantitative determination of the lipid peroxidation product 4-hydroxynonenal by high-performance liquid chromatography

4-Hydroxynonenal is a product formed in tissue and tissue fractions from polyunsaturated membrane lipids through a free radical-induced lipid peroxidation process. The biological properties of this aldehyde have been studied in many respects. This article describes for the first time a sensitive and reproducible method for quantitative analysis of 4-hydroxynonenal in biological samples as well as in lipid-containing foodstuffs. The method involves extraction of the aldehyde by dichloromethane from cells or microsomes trapped on an Extrelut column. Oils and foodstuffs are extracted with excess water. After additional sample cleanup by solid-phase extraction on a disposable octadecyl silica gel (ODS) extraction column, the sample is analyzed by high-performance liquid chromatography using an ODS column and methanol/water 65/35 (v/v) or acetonitrile/water 40/60 (v/v) as eluant; the detection wavelength is 220 nm. The method developed has a high precision with coefficients of variation of 1.4% (microsomes) to 3.5% (olive oil). The recovery depends on the sample type and lies between 45% (control microsomes) and 96% (solution of hydroxynonenal in water). The method has been used for the determination of 4-hydroxynonenal in microsomes, platelets, and various foodstuffs.     

Feeding state influences the content of FMRFamide- and tachykinin-related peptides in endocrine-like cells of the midgut of Locusta migratoria

The midgut of 5th instar male African migratory locust, Locusta migratoria, was found to contain endocrine-like cells that stained positively for FMRFamide-like immunoreactivity. These cells have cell bodies which are tear-drop in shape with processes extending from the cell body. FMRFamide-like immunoreactivity has been described in similar cells in adult midgut tissue [16]. The midgut tissue content of FMRFamide-like immunoreactivity is differentially distributed throughout various regions of the midgut (gastric cecae, anterior and posterior midgut) in 5th instar and varied ages of adult. FMRFamide-like immunoreactivity in midgut tissues decreases significantly by 24 h of starvation, whereas locustatachykinin I-like immunoreactivity does not decrease until 48 h of starvation indicating that there are differential timing effects of these two peptide families on midgut content. HPLC analysis, combined with RIA, of different regions of the midgut tissue from both fed and starved locusts revealed that the relative proportions of the members of the two peptide families vary depending upon the feeding state. These results indicate that the contents of these endocrine-like cells appears to be differentially influenced by the feeding state of the locust.     

Juvenile hormone metabolism in the plasma,integument, midgut,fat body,and brain during the last instar of the tobacco hornworm,Manduca sexta (L.)

Juvenile hormone (JH) III esterase and JH III epoxide hydrolase activity was found in the integument, midgut, fat body, and brain during last instar development of the tobacco hornworm, Manduca sexta. JH esterase activity was primarily located in the cytosol in these tissues while the majority of the JH epoxide hydrolase activity was found in the microsomes. A prewandering (on day 3) and postwandering (on day 8) peak in plasma JH III esterase activity occurs in the last instar of gate I M. sexta. The JH esterase activity profile in integument, midgut, fat body, and brain followed a similar pattern to that of the plasma. The only exception to this was the absence of the postwandering, prepupal (on day 8) JH esterase peak in the fat body. The topical application of the juvenoid, (RS)-methoprene, failed to induce fat body JH esterase activity but increased activity in the plasma, integument, midgut, and brain in M. sexta prepupae. These results indicate that the source of plasma JH esterase activity is not always the fat body as previously hypothesized. The developmental profile of tissue JH epoxide hydrolase activity was also similar to that of JH esterase suggesting that both enzymes may be regulated partly by the same factors and that JH epoxide hydrolase may also have an important, previously unrecognized functional role in JH regulation and insect metamorphosis. Multiple isoelectric forms of tissue-specific JH esterases and JH epoxide hydrolases were found in integument, midgut, fat body, and brain. The JH esterases in these tissues had isoelectric points more acidic than that for plasma. Tissue α-naphthyl acetate esterase, developmental profiles, and inhibitor sensitivity to 3-(octylthio)-1,1,1-trifluoropropan-2-one differed significantly from that for JH esterase, suggesting that they represent different enzymes. ©1992 Wiley-Liss, Inc.     

家蚕中肠组织抗核型多角体病毒病的相关蛋白分析

家蚕中肠上皮是病毒经口侵入遇到的第一个组织。昆虫幼虫抵御杆状病毒的感染,可通过选择性的使感染的中肠上皮细胞发生调亡并在释放病毒粒子进入血淋巴之前使感染的细胞从中肠脱落。为研究家蚕抗核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)病的机制,通过对BmNPV高度抗性和高度敏感性的家蚕品系杂交和回交构建了近等基因系。本文对家蚕高抗,敏感及近等基因系5龄起蚕中肠组织的蛋白质表达谱进行了二维电泳 (two-dimensional gel electrophoresis,2-DE) 分析,并利用基质辅助激光解吸电离飞行时间 (matrix-assisted laser desorption/ionization-time of flight, MALDI-TOF) 质谱对差异蛋白进行鉴定。结果发现了5个差异表达的蛋白。推测这些蛋白可能与家蚕中肠对BmNPV的抗性或感性有关。     

Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants.

Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.