Proteins In Various Tissues Research Articles

Glucose Transporter Levels in Tissues of Spontaneously Diabetic Zucker fa/fa Rat (ZDF/drt) and Viable Yellow Mouse (Avy/a)

We used antibodies to the fat/muscle glucose transporter (GLUT4) and the liver glucose transporter (GLUT2) to measure levels of these proteins in various tissues of two rodent models of non-insulin-dependent (type II) diabetes mellitus: the obese spontaneously diabetic male Zucker fa/fa rat (ZDF/drt) and the male viable yellow Avy/a obese diabetic mouse. The ZDF/drt strain generally develops overt diabetes associated with decreased plasma insulin levels. Depending on the age of the animals, the ZDF/drt rats can be arbitrarily segregated into age-matched obese, mildly diabetic (blood glucose less than 11 mM) and obese, and severely diabetic (blood glucose greater than 20 mM) groups. Avy/a mice are comparably hyperglycemic but unlike the ZDF/drt rats are severely hyperinsulinemic. In both groups of diabetic animals, GLUT4 in adipose tissue, heart, and skeletal muscle was reduced 25-55%, and GLUT2 in liver was increased 30-40%, relative to lean, age-matched controls. However, when the mildly diabetic ZDF/drt rats were compared to the lean controls, the only significant difference was a 25% reduction of GLUT4 in heart. Within all of the ZDF/drt rats (excluding the lean controls), GLUT2 in liver and GLUT4 in adipose tissue, heart, and skeletal muscle correlated significantly with glycemia. These data suggest that, in these two models of type II diabetes, glucose transporter levels in muscle, adipose tissue, and liver are regulated in a tissue-selective manner in response to changes in insulin and glucose. Furthermore, at least in the ZDF/drt rat, alterations in GLUT2 and/or GLUT4 protein levels appear not to be associated with obesity per se but appear to be secondary to the severely diabetic state.(ABSTRACT TRUNCATED AT 250 WORDS)

Automated two-dimensional liquid chromatographic system for mapping proteins in highly complex mixtures

An automated two-dimensional liquid chromatographic system was developed for systematic protein separations which could serve for analytical mapping and preparative separations of proteins. The system applies the principles of the column-switching technique, and consists of two different columns connected in tandem through an electrical column switching valve, two pumping systems to operate each column independently and a system controller to perform sequential chromatography on the two columns. A protein mixture is applied to the first-dimensional anion-exchange column and is separated by stepwise elution with an increasing sodium chloride concentration. The eluent is introduced directly to the second-dimensional reversed-phase column, and is further separated by gradient elution with an increasing acetonitrile concentration. The two elution stages are synchronized by a computer program. By this system, very complex protein mixtures such as crude cerebellar extracts were resolved reproducibly into ca. 200 peaks within 12 h. The method can be used for the total analysis of proteins in various tissues and cells without complicated premanupulation of samples, and allows the simultaneous analysis of a protein isolated by chromatography. The isolated protein is most suitable for use in the strategy of protein and gene sequence analysis.

Effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail

1. 1. The effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail were investigated. 2. 2. The level of liver NAD was not affected by niacin deficiency whereas the level of pectoral muscle NAD was markedly reduced. 3. 3. In all dietary treatments the liver had the highest turnover rates of proteins, heart and brain had intermediate rates, and pectoral muscle had the lowest rates. 4. 4. Relative turnover rates of proteins in all tissues (particularly pectoral muscle) of the niacin deficient group were significantly higher than those of pair-fed control group, although there were no significant differences in turnover rate between pair-fed control and control groups. 5. 5. The high turnover rate of proteins in niacin deficiency was primarily attributed to enhanced degradation rate of proteins rather than enhanced synthesis rate of proteins. 6. 6. Optical density scanning (or densitometric) of water-soluble pectoral muscle proteins separated by isoelectric focusing revealed several additional minor protein bands between major protein bands in the niacin deficient group which were more pronounced in the acidic region of the gel. 7. 7. These results suggest that proteins with a low pI value in pectoral muscle of the niacin deficient animal are highly sensitive to protein degradation.

Immunohistochemical Localization of Nuclear 3,5,3'-Triiodothyronine Receptor Proteins in Rat Tissues Studied with Antiserum against C-ERB A/T3Receptor*

We have previously raised an anti-c-erb A peptide antibody (designated 4B II) which immunoprecipitated in vitro transcription/translation products of c-erb A alpha 1 and beta. 4B II could recognize nuclear T3 receptor (NT3R) without distinction between difference in species and tissues. Using 4B II, we studied immunohistochemical localization of NT3R proteins in various tissues of the rat. Cryostat sections (4-6 microns) of selected rat tissues were incubated with 4B II at 4C overnight, followed by fluorescein-isothyocianate-conjugated anti-rabbit immunoglobulin G for 60 min at 25 C. The cellular localization of fluorescence in all tissues examined was exclusively nuclear. Under the same conditions, control sections stained with antiserum which had previously absorbed with c-erb A peptide or inactive serum showed no specific staining. In the brain the large nuclei, supposed to be neuronal, were strongly stained in the cerebral cortex and the granular layer of the cerebellum. In the kidney, cells in the glomerulus, the distal, but not the proximal, tubules, and the collecting ducts exhibited nuclear staining. Nuclear fluorescence was observed homogeneously in the heart and liver, but the intensity was much weaker in the latter. Less intense fluorescence was seen in the testis and spleen, although specific immunostaining was clearly observed in the nuclei of spermatocytes, Leydig cells, and the heads of the sperms in the testis, and many lymphocytes in the spleen. Nuclei of follicular cells of the thyroid exhibited very strong fluorescence, suggesting existence of plenty of NT3R proteins. The anterior pituitary showed strong immunostaining in most nuclei, and clear nuclear fluorescence was also detected in the intermediate lobe of the pituitary. The present study showed that NT3R distributes selectively in certain types of cells in many tissues and that the content of NT3R proteins seems to correlate with the concentration of c-erb A mRNA alpha 1 and beta among many organs.

Cellular retinol-binding protein messenger RNA levels in normal and retinoid-deficient rats.

A study was conducted to determine the levels of cellular retinol-binding protein (CRBP) mRNA and protein in various tissues of the rat, to explore relationship between CRBP mRNA and protein levels in different tissues, and to examine the effects of changes in retinol nutritional status on the tissue distribution and levels of CRBP mRNA. Previous studies have shown that tissue CRBP protein levels are reduced in totally retinoid-deficient rats, but are otherwise minimally affected by changes in retinoid status. Three groups of male rats were compared: normal controls, retinoid-deficient, and retinol-repleted deficient rats. CRBP mRNA levels were measured by RNase protection assay and CRBP protein levels by radioimmunoassay in seven tissues. High levels of both CRBP mRNA and CRBP protein were found in the proximal epididymis, kidney, and liver; lower levels were seen in lung, testis, spleen, and small intestine. Tissue CRBP mRNA and protein levels were highly correlated (P less than 0.01) with each other. Retinoid deficiency did not alter the levels of CRBP mRNA found in the proximal epididymis, kidney, and liver. In contrast, CRBP mRNA levels in the lung, testis, spleen, and small intestine were reduced substantially in retinoid-deficient rats, to values that were only 23% to 50% of the corresponding values in the tissues of control rats. After oral repletion with retinol (4-18 h earlier), CRBP mRNA levels for these latter four tissues were found to have risen to control or near-control levels. The suggestion is raised that retinol repletion may have directly induced the expression of the CRBP gene in these particular tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the food mutagens MeIQx and PhIP on the expression of cytochrome P450IA proteins in various tissues of male and female rats

The promutagenic 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) found in cooked food are converted to their active forms mainly by cytochrome P450 forms IA1 and IA2. By induction of these isoenzymes the food mutagens could thus influence their own rate of activation. Male and female Wistar rats were given MeIQx, PhIP, beta-naphthoflavone (BNF) or saline i.p. at 50 mg/kg body wt on three consecutive days. On the fourth day the rats were killed and lungs, kidneys, liver and intestines taken. The microsomal fraction from each organ was prepared as well as 9000 g supernatant from the liver. The induction of cytochrome P450IA was measured at the protein level by enzymatic assays (ethoxyresorufin-O-deethylation, Ames' mutagenicity test) and immunoassays (Western blot) and at the pretranslational level by RNA hybridization (Northern blot). The binding affinities of MeIQx, PhIP and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-receptor were studied by evaluation of the competition with 3H-labelled TCDD for specific binding. Ethoxyresorufin-O-deethylase (EROD) activity was significantly increased in the liver (males 2.1-fold, females 3.3-fold), kidneys (males 2.1-fold, females 1.8-fold) and lungs (males 4.3-fold, females 3-fold) of the MeIQx-treated rats. Furthermore, the levels of cytochrome P450IA proteins were increased in these animals. It was not possible, however, to detect the corresponding mRNA. In the case of the PhIP-treated animals a significantly increased EROD activity (2.7-fold) and an increased cytochrome P450IA protein level were seen only in the male lungs. Only a very weak TCDD-receptor affinity was observed for PhIP, whereas MeIQx or IQ did not appear to compete significantly with [3H]TCDD for binding to the TCDD-receptor. It is concluded that MeIQx is a weak inducer of cytochrome P450IA in several organs of the rat, while PhIP induced these isoenzymes only in the male lungs. More work is needed to clarify the mechanism(s) whereby this induction occurs.

Isolation of the alpha subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized beta gamma subunits.

Immobilized beta gamma subunits of GTP-binding regulatory proteins (G proteins) were used to isolate alpha subunits from solubilized membranes of bovine tissues and to separate specific alpha subunits based on their differential affinities for beta gamma subunits. The beta gamma subunits were cross-linked to omega-aminobutyl agarose. Up to 7 nmol of alpha subunit could bind to each milliliter of beta gamma-agarose and be recovered by elution with AIF4-. This affinity resin effectively separated the alpha subunits of Gi1 and Gi2 from "contaminating" alpha subunits of Go, the most abundant G protein in bovine brain, by taking advantage of the apparent lower affinity of the alpha subunits of Go for beta gamma subunits. The beta gamma-agarose was also used to isolate mixtures of alpha subunits from cholate extracts of membranes from different bovine tissues. alpha subunits of 39-41 kDa (in various ratios) as well as the alpha subunits of Gs were purified. The yields from extracts exceeded 60% for all alpha subunits examined and apparently represented the relative content of alpha subunits in the tissues. This technique can rapidly isolate and identify, from a small amount of sample, the endogenous G proteins in various tissues and cells. So far, only polypeptides in the range of 39-52 kDa have been detected with this approach. If other GTP-binding proteins interact with these beta gamma subunits, the interaction is either of low affinity or mechanistically unique from the alpha subunits isolated in this study.

Transblot identification of biotin-containing proteins in rat liver

Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 p I) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 p I) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.

Folate coenzyme and antifolate transport proteins in normal and neoplastic cells

The transport systems for folate coenzymes and antifolate compounds into various types of normal and neoplastic cells display considerable diversity in their pharmacokinetics and also in terms of the apparent molecular weights of the proteins involved. Further, several uptake routes may exist in a given cell type. A variety of neoplastic tissues have been reported to rely upon a single major transport system that has a relatively high affinity for the reduced form of folate compounds and for antifolates such as methotrexate. Using a photoaffinity analogue of methotrexate, we have identified the involvement of a 48 kDa membrane protein and a 38 kDa cytosolic or peripheral membrane protein in the transport of this compound into murine L1210 leukemia cells. Such an uptake is absent in mutant L1210 cells that are defective in methotrexate transport. We propose a model for the uptake of reduced folate coenzymes in L1210 cells in which the compound is initially transported across the cell membrane by the 48 kDa protein and delivered on the cytoplasmic surface to the 38 kDa protein; the 38 kDa protein then carries the folate compound to a specific enzyme of folate metabolism. Antibodies to the membrane folate binding protein from human placenta cross-react with the 48 kDa protein in L1210 cell membranes indicating an immunological relationship between these two proteins. Comparison of the amino acid sequences of peptides of the placental receptor obtained by digestion with S. aureus V8 protease indicate the presence of two homologous forms of the folate binding protein in placenta; one of these forms appears to have an identical sequence to the soluble and membrane associated folate binding proteins in human epidermoid carcinoma (KB) cells, which in turn share the primary structure of the soluble and membrane associated folate binders in human milk in the regions that have been sequenced. These results indicate that the folate coenzyme transport proteins in various tissues may be structurally related and in several instances may even be identical. In the latter cases the observed differences in apparent molecular weights may be due to differences in glycosylation and/or proteolysis. In support of this view is our observation that the deglycosylated and/or partially proteolyzed placental receptor retains the ability to bind folate.

Changes in the expression of the carbohydrate epitope HNK-1 associated with mesoderm induction in the chick embryo

We report that a monoclonal antibody, HNK-1, identifies specific regions and cell types during primitive streak formation in the chick blastoderm. Immunohistochemical studies show that the cells of the forming hypoblast are HNK-1 positive from the earliest time at which they can be identified. Some cells of the margin of the blastoderm are also positive. The mesoderm cells of the primitive streak stain strongly with the antibody from the time of their initial appearance. In the epiblast, some cells are positive and some negative at pre-primitive-streak stages, but as the primitive streak develops a gradient of staining intensity is seen within the upper layer, increasing towards the primitive streak. At later stages of development, the notochord and the mesenchyme of the headfold are positive, while the rest of the mesoderm (lateral plate) no longer expresses HNK-1 immunoreactivity. This antibody therefore reveals changes associated with mesodermal induction: before induction, it recognizes the 'inducing' tissue (the hypoblast) and reveals a mosaic pattern in the responding tissue (the epiblast); after primitive streak formation, the mesoderm of the primitive streak that results from the inductive interactions expresses the epitope strongly. Affinity purification of HNK-1-related proteins in various tissues was carried out, followed by SDS-PAGE to identify them. The hypoblast, mesoderm and epiblast of gastrulating chick embryos have some HNK-1-related proteins in common, while others are unique to specific tissues. Attempts have been made to identify these proteins using Western blots and antibodies known to recognize HNK-1-related molecules, but none of the antibodies used identify the bands unique to any of the tissues studied. We conclude that these proteins may be novel members of the HNK-1/L2 family, and that they may have a role in cell interactions during early development.

Expression of ras oncogene p21 during human fetal development as determined by monoclonal antibodies RAP-5, Y13-259, and DWP

In this report we describe the expression of the ras proto-oncogene p21 protein in various tissues during normal fetal development. Conventional, formalin fixed and paraffin-embedded sections of normal organs were examined from fetuses ranging 9 to 42 weeks of gestation. Immunohistochemical localization of ras p21 was accomplished using the broadly reactive, mouse monoclonal antibodies RAP-5 and Y13-259. The monoclonal antibody DWP, which is specific for a mutated form of ras p21 having a valine/cysteine at amino acid position 12, was also used. Detectable expression of the p21 protein was seen at different time periods during fetal development depending on the tissue. The expression of ras p21 (as detected by RAP-5 and Y13-259) was noted in a wide range of cell types and tissues; intense immunostaining was noted in epithelial cells of the gastrointestinal tract, exocrine and endocrine pancreas, renal tubules and transitional urotheliem, as well as in other tissues. This immunostaining generally, but not invariably, corresponded with patterns previously reported in benign and/or malignant neoplasms of adult tissues. In most instances ras p21 expression, when present, occurred during periods of rapid growth in given organ systems. However, some actively proliferating fetal tissues such as thymus and spleen, failed to express detectable ras p21 suggesting that factors other than cell cycle may influence its expression. No reactivity with DWP was noted in any of the tissues, suggesting that the mutated forms detected by this monoclonal antibody are not expressed during normal human embryogenesis. These data show that there is regulated expression, and broad distribution of this gene product in normal developing human fetal tissue.

The synthesis in vivo of proteins in various tissues in chickens adapted to intermittent feeding.

1. Protein synthesis was estimated in vivo in breast (superficial pectoral) and tibia (gastrocnemius) muscles, liver, kidney, pancreas, crop, duodenum, jejunum and ileum, using L-[U-14C]lysine injection. The effect on incorporation of [14C]lysine 1 and 2 h after injection was examined in five chickens adapted or not adapted to intermittent feeding. 2. Incorporation of [14C]lysine into tissue decreased in magnitude in the following descending order: pancreas greater than jejunum, duodenum greater than ileum, crop, liver greater than kidney greater than tibia, breast muscle and blood plasma. 3. The incorporation of [14C]lysine into muscle protein was higher in chicks after 24 h of refeeding than after 24 h of food deprivation. These differences were higher in adapted than in non-adapted birds. On days of refeeding the rate of incorporation exceeded that found in chickens fed ad lib. 4. Bound 14C from lysine in the intestinal segments was less than in control birds after food deprivation and greater after refeeding in non-adapted chicks only. 5. A negative relation was observed between bound and free 14C in muscles and in other tissues. 6. Short- and long-term adaptations to feeding regimens are discussed.

Androgen-regulated epididymal secretory proteins associated with post-testicular sperm development.

A considerable body of information has now been assembled with regard to the major androgen-dependent secretory proteins of the rat epididymis. The proteins have been purified, specific antibodies have been developed against them, and their primary structure has been determined from cDNA clones. The antibody and cDNA probes have, in turn, been used to study the androgenic regulation of the synthesis and secretion of the proteins, the distribution of the proteins in various tissues and animal species, and the association of the proteins with the sperm surface during epididymal maturation. Despite this intensive effort, the precise physiological functions of the proteins still remain obscure, although circumstantial evidence indicates that glycoproteins D and E may be associated with surface receptors responsible for gamete recognition. Elucidation of the physiological roles of the proteins is clearly the area that warrants the greatest attention in future work.

Tissue distribution of rat S100 alpha and S100 beta and S100-binding proteins.

To understand the physiological role of the calcium-binding proteins S100 alpha and S100 beta, it is necessary to determine the distribution of these proteins and detect their intracellular targets in various tissues. The distribution of immunoreactive S100 alpha and S100 beta in various rat tissues was examined by radioimmunoassay. All tissues examined contained detectable S100, but the S100 beta/S100 alpha ratio in each tissue differed. Brain, adipose, and testes contained 18- to 40-fold more S100 beta than S100 alpha; skin and liver contained approximately equivalent amounts and kidney, spleen, and heart contained 8- to 75-fold more S100 alpha than S100 beta. Analysis of S100-binding proteins by gel overlay showed that each tissue possessed its own complement of binding proteins. The S100 beta-binding profile was indistinguishable from the S100 alpha-binding profile and both of these profiles were distinct from the calmodulin-binding profile. These observations suggest that the differential distribution and quantity of the individual S100 polypeptides and their binding proteins in various tissues may be important factors in determining S100 function.

Regulation of prostaglandin formation by glucocorticoids and their second messenger, lipocortins

Glucorcorticoids induce the synthesis of a family of phospholipase inhibitory proteins, lipocortins. This family of lipocortins includes inhibitory proteins on phospholipase A 2, phospholipase C and phosphatidylinositol phospholipase C. Hence, glucocorticoids reduce the formation of prostaglandins and leukotrienes by inhibiting cellular phospholipases, enzymes that degrade membrane phospholipids to release arachidonic acid, a precursor. The induction by glucocorticoids requires 1 h for the synthesis of mRNA and 5 h for the synthesis of proteins in various tissues and cells. However, glucocorticoids often exert their suppressive effects before the induction of lipocortins. This is now attributed to the nonenzymic formation of the adducts between glucocorticoids and lipocortins. These adducts are easily inserted into the membranes and more resistant to digestion of proteases, thus being more biologically potent with respect to suppression of the release of arachidonic acid, a precursor of prostaglandins and leukotrienes.

1202 TRANSCRIPTION OF THE INSULIN GENE IN THE BRAIN AND SUBMANDIBULAR GLAND OF THE RAT

Analysis of RNA from day old rat brain neuronal cell cultures and adult salivary gland tissue revealed a 2.4Kb polyA+ RNA species that hybridized to both cloned human and rat insulin genomic probes. Hybridization was not observed between glial cell RNA and either insulin DNA probe. The abundance of the 2.4Kb RNA species was 83 copies/neuronal cell and 14.5 copies/cell in the submandibular gland. Mature preproinsulin mRNA (PPImRNA) was not detected in either tissue. The 2.4Kb RNA was present in the adult pancreas at very low levels (<10 copies/cell vs. 1600 copies/cell for the PPImRNA). Immunofluorescent studies with purified anti-insulin antibodies detected a sub-population of neuronal cells (5%) and scattered ductal epithelium in the submandibular gland as sources of extrapancreatic insulin synthesis. Others have postulated the extrapancreatic synthesis of insulin based on the presence of low but detectable levels of protein in various tissues. Most controversial has been the discussion centered on the brain as a site of insulin synthesis. Our results suggest that two distinct tissues, the brain and the submandibular gland, possess unique cell types that contain both (i) a 2.4Kb RNA species that hybridizes with an insulin gene and (ii) positive anti-insulin immunofluorescence staining patterns. This may indicate that the insulin gene(s) is expressed in brain and salivary gland and that both tissues are potential de novo synthetic sites.

Lipid transfer proteins

Lipid transfer proteins were discovered in our labe ratory as a result of studies that began with the question: How are lipid components of plasma lipoproteins taken up by the liver cell? It soon became apparent that these cells contained a cytosolic factor ( I ) , which transferred PC from one membrane to another. Upon demonstrating that the factor was nondialyzable, heat-labile, and sensitive to trypsin, the factor was assumed to be a protein. In as much as the factor accelerated the transfer of PC between microsomes and mitochondria to approximately equal extents, the protein was named phospholipid exchange protein (2). Similar observations soon followed from other laboratories. In addition, the systematic efforts to purify the exchange protein uncovered the presence of a variety of proteins that could accelerate the transfer of different phospholipid classes and the less polar lipids as well. Moreover, under the proper conditions, the proteins were shown to mediate net transfer reactions as well as equimolar lipid exchanges. Hence, by agreement between the major groups working in this area, the designation of these proteins was changed to “lipid transfer proteins” for generic usage. The more specific designations, for example, would then be “PC-specific transfer protein”, abbreviated as PC-TP and “nonspecific transfer protein”, abbreviated ns-TP. Most of the transfer proteins discovered thus far have been identified as cytosolic proteins in various tissues of animals and plants. One cannot exclude the possibility, however, that the proteins were originally membraneassociated, but were released by the homogenization procedure. Such an occurrence has been observed for a lipid transfer protein, which appears to be associated largely with the microsomal fraction, but that can be released by the treatment of microsomes with low ionic strength buffers (3). Even during the homogenization of liver with 250 mM sucrose, 50 mM Tris, 1 mM EDTA, some of this transfer activity is found in cytosol, but was probably microsomal in origin. One other class of lipid transfer proteins is found in blood plasma of some animal species (4, 5), including human plasma (6-14). These proteins may be rather nonspecific (I 5) or may accelerate the transfer of phospholipid preferentially (1 6, 17). In subsequent sections I intend to review the distribution of lipid transfer proteins, how these proteins have been utilized to study lipid asymmetry in biological and in artificial membranes, and the effect of membrane lipid modification on enzyme activities. Some of these aspects of lipid transfer protein biochemistry have also been reviewed recently (1 8). Methodologies of assays, as well as other topics, have been discussed by Zilversmit and Hughes (1 9), Wirtz (20), Wetterau and Zilversmit (21), Akeroyd and Wirtz (22), Kader (25, 24), Bloj and Zilversmit (25, 26), Bergelson and Barsukov (27), and Rothman and Lenard (28). From these reviews it is evident that many varieties of lipid transfer proteins have been discovered in animal and plant cells and a more limited number in unicellular organisms.

Cellular retinol-binding protein. Quantitation and distribution.

The distribution of cellular retinol-binding protein was localized in three tissues involved in the transport of vitamin A using the peroxidase-antiperoxidase technique. In addition, a sensitive radioimmunoassay was developed to quantitate cellular retinol-binding protein in various tissues. In liver, the protein was found in the hepatocytes and in the perisinusoidal fat-storing cells. The columnar epithelial cells of the jejunum and the cells of the proximal tubuli also exhibit high concentrations of cellular retinol-binding protein. Estimations of the content of the protein in various tissues from normal and retinol-deficient rats showed that the retinol status did not affect the tissue levels or the subcellular distribution. The appearance of high amounts of this protein in cells involved in the dietary uptake, storage, mobilization, and resorption of vitamin A suggest that one function of the cellular retinol-binding protein is to act as a vehicle in the intracellular transport of retinol through these organs.

Ubiquitous binding of benzo[ a]pyrene metabolites to DNA and protein in tissues of the mouse and rabbit

The in vivo formation of benzo[ a]pyrene (BP) metabolite-DNA adducts in several tissues of mice and rabbits was examined. Included were tissues with widely divergent xenobiotic metabolizing capabilities such as liver and brain. The major adduct identified in each tissue was the (+)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-BP (BPDEI)-deoxyguanosine adduct. A 7β,8α-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro-BP (BPDEII)-deoxyguanosine adduct, a (−)-BPDEI-deoxyguanosine adduct and an unidentified adduct were also observed. These adducts were present in all of the tissues of the mice and in the lungs of the rabbits; only BPDEI and BPDEII were seen in the rest of the rabbit tissues. In all of the tissues studied, the DNA adduct levels were unexpectedly similar. For example, the BPDEI-DNA adduct levels in muscle and brain of mice were approx. 50% of those in lung and liver at each oral BP dose examined. After an i.v. dose of BP in rabbits, the BPDEI adduct levels in lung were three times those in brain or liver and twice those in muscle. The binding of BP metabolites to protein was also determined in these tissues. The tissue-to-tissue variation in protein binding levels of BP metabolites was greater than that for BPDEI-DNA adducts. There are several possible explanations for the in vivo binding of BP metabolites to DNA and protein of various tissues. First, oxidative metabolism of BP in each of the examined tissues might account for the observed binding. Second, reactive metabolites could be formed in tissues such as liver and lung and be transported to cells in tissues such as muscle and brain where they bind to DNA and protein. In any case, the tissue-to-tissue variations in protein and DNA binding of BP-derived radioactivity do not correlate with differences in cytochrome P-450 activity.

Immunochemical study of the proteins of various tissues in Crustacea (Decapoda): nature, role, origin

The main proteins of the haemolymph of Crustacea Decapoda have been identified and analysed: haemocyanin, plasma coagulogen, heteroagglutinins, vitellogenins, and molt-related proteins. All these complex components exhibit a high molecular weight and as oligomeric fractions are able to aggregate or dissociate in subunits according to the composition of medium and experimental procedures. Besides their important rôle in the defense mechanism, some proteins are involved in the edification of diverse tissues. They are detected within different compartments: soft integument, calcified carapace and hepatopancreas. They are either in transit or sequestered or synthetized within these tissues. In the crayfish Astacus leptodactylus, some components have been identified in different compartments: --in aqueous extracts from soft integument: the haemocyanin, coagulogen and both fraction F1 (lipoprotein with an approximate molecular weight of 45 kdal) and fraction F2 related to the molt. Both coagulogen and fraction F2 appear sometimes as melanized. These two latter fractions exhibit some glucose-mannose residues and they occur with a higher relative amount than in the blood. --in soluble extracts from calcified cuticle: among the numerous fractions showing a high molecular weight, the haemocyanin and coagulogen are detected. --in aqueous extracts from hepatopancreas: both haemocyanin and coagulogen appear with a little relative amount. Components termed as Fa and Fb are found with a high concentration. One minor fraction is also detected. --in aqueous extracts from eggs: the haemocyanin and fraction Fb are present. Other proteins showing only some antigenic identities with those of the haemolymph are also detected in all these tissues. The haemolymph proteins are not present within these compartments following a passive diffusion. Indeed, their relative amount varies according to the tissue investigated and is different from that found in the blood. Except the haemocyanin detected in all tissues with different aggregation states, the haemolymph proteins identified vary in the organs studied. A qualitative and quantitative selection occurs when the blood proteins enter the other compartments. Perhaps some other proteins are not detected following alterations underwent either in the epithelial barriers or during the tannage process or the chitino-proteic complex formation or due to experimental procedures. On the other hand, each tissue has its own proteins. The integument contains crustacyanins alpha, beta, gamma; the eggs are mainly constituted of lipovitellins and the hepatopancreas is rich in small molecular weight proteins and digestive enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)