Comparison of the high-mobility-group chromosomal proteins in rainbow-trout (Salmo gairdnerii) liver and testis.

Chromatography and characterization of the proteins extracted by 5% (w/v) HClO4 from rainbow-trout (Salmo gairdnerii) liver and testis show that the two tissues present a characteristically different spectrum of high-mobility-group (HMG) proteins. A variant subfraction of HMG C is found in liver, but is not detectable in testis, where even the main fraction of HMG C is present in only very low quantity. A protein, F, which appears to be related to protein H6 has similarly been isolated only from liver and not from testis. Quantification of the HMG proteins in total 5%-HClO4 extracts of trout liver and testis nuclei shows that, in relation to DNA, levels of HMG T1 and T2, and D are more than 2-fold, and C, 20-fold higher in liver than in testis. However, these differences do not result merely from the sequential withdrawal of HMG proteins at the same time that histones are replaced by protamines in the developing spermatid, since in testis, at some stages of maturation, levels of H6 are almost 2-fold higher than in liver. The implications of these findings for the function of HMG proteins are discussed.     

High mobility group (HMG) proteins in the tammar wallaby Macropus eugenii: quantitative variations between tissues and testis-specific co-extracted proteins

1. Tammar wallaby (Macropus eugenii, Marsupialia) proteins with similar electrophoretic mobilities to calf non-histone chromosomal proteins HMG 1, 2, 14 and 17 are perchloric acid extracted from whole tissues (liver, kidney, spleen, brain and testis) and purified liver nuclei (using PCA or 0.35 M NaCl). 2. Tammar and calf HMG 1 have similar amino acid compositions. 3. Two testis-specific basic proteins co-extracting with HMG-like proteins from both tammar and red kangaroo (Megaleia rufa) are found in whole testis, purified testis nuclei, but not epididymis. 4. Tammar HMG 2 separates into two components on both acid urea and SDS gels. The larger, more basic protein, HMG 2b, is relatively abundant in proliferating tissues (testis, spleen).     

Possible endogenous substrate proteins for cytosolic protein-tyrosine kinase from porcine spleen.

1. Endogenous phosphate acceptor proteins by cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) were studied using subcellular fractions of porcine spleen and supernatant fraction of rat various tissues. 2. At least 13 phosphate acceptor proteins ranging from 94 to 26 kDa were observed in all but mitochondrial subcellular fractions. 3. Among the supernatant fraction of rat tissues, brain, testis and spleen contained many phosphate acceptor proteins. 4. The most heavily phosphorylated band of around 55 kDa which was commonly recognized among various tissues was confirmed as tubulin by the immunoreactivity with anti-tubulin antibody. 5. The results obtained in this paper indicate that CPTK-40 has the potential to catalyze the phosphorylation of numerous endogenous proteins including tubulin.     

DNA-protein interactions in the CCAAT box region of the murine lactate dehydrogenase C promoter

Electrophoretic Mobility (EMSA), using oligonucleotides containing CCAAT box sequences from the murine Idhc promoter show the presence of CCAAT binding proteins in nuclear extracts from liver and testis. In a liver extract, a single shifted band is seen. However, in the testis extract, two shifts are observed, one of which may be due to a testis specific isoform of CCAAT binding factor (CBF). Southwestern analysis with an oligonucleotide probe containing these sequences reveals the presence of a protein of approximately 120 kD in the testis extract. In the liver extract, a 70-kD protein binds the probe. An antibody against HeLa CBF causes a supershift in testis nuclear extract.     

In vitro ADP-ribosylation of nuclear proteins in mouse tissues. Identification of an unusual group of acceptors in testis nuclei

1. Acceptor proteins for poly(ADP-ribose) have been identified in nuclei from mouse testis, liver, kidney and spleen. Purified nuclei were incubated in vitro with [14C]NAD, extracted sequentially with 5% HClO4 and 0.25 N-HCl and labelled proteins analysed on acetic acid/urea polyacrylamide gels pH 2.9. 2. Results show that: (a) in vitro there are significant differences between tissues in the extent of poly(ADP-ribosylation) of nuclear proteins; (b) in testis nuclei two tissue specific proteins are poly(ADP-ribosylated) to higher specific activity than histones; (c) there are significant differences between in vivo and in vitro studies on poly(ADP-ribosylation) of nuclear proteins in testis nuclei.     

Comparison of the high mobility group proteins and their chromatin distribution in trout testis and liver

The trout testis contains two major high mobility group (HMG) proteins HMG-T and H6 which, although related to the four mammalian HMGs, exhibit distinct variation as evidenced by differences in electrophoretic mobility and amino acid sequence. Previous work using various endonucleases as probes has shown that HMG-T and H6 are located at specific sites in the testis chromatin. The differentiation of testis cells during spermatogenesis is characterized by a unique transition from a histone-packaged genome to one bound by a class of small molecular weight, highly basic proteins, the protamines. Questions arise as to whether any of the HMG variability may be unique to the process of spermatogenesis and whether the histone-protamine transition occurring in most testis cells affects the HMG protein distribution and/or the specificity of the probe. In an attempt to answer these questions, the distribution of the HMG proteins in the chromatin of trout liver, a tissue lacking protamine, has been studied and comparisons made with testis. Liver HMGs exhibit the same electrophoretic characteristics as the testis HMGs indicating that the variability when compared to mammalian HMGs is primarily phylogenetic in origin rather than tissue-specific. Furthermore, micrococcal nuclease digestion of liver nuclei and its effect on the subsequent HMG protein distribution during chromatin fractionation yields a pattern very similar to that for testis, suggesting that the interaction of the HMGs with the remaining testis nucleohistone is not significantly altered by the ongoing transition to nucleoprotamine. Finally, the HMGs represent an unusually high proportion of the total testis non-histone protein population; the implications of this are discussed.     

A mammalian sperm lectin related to rat hepatocyte lectin-2/3

In rat liver the asialoglycoprotein receptor is composed of three polypeptides, RHL-1, RHL-2 and RHL-3 [6]. In rat testis and spermatozoa a galactosyl receptor (RTG-r) which is immunologically related to RHL-2/3 has been described [7]. We now report that in addition to its presence in the rat, an antigenic species of 54 kDa related to RHL-2/3 is present on rabbit, human, pig and mouse spermatozoa. Purified rabbit testis galactosyl receptor (RbTG-r) consists of two major proteins of 54 and 49 kDa, while purified rabbit liver galactose lectin consists of two major proteins of 43 and 40 kDa. In an ELISA the purified rabbit testis galactosyl receptor was shown to bind biotinylated heat solubilized rabbit zonae, while the purified liver galactose lectin did not. We conclude that one of the mammalian sperm's zona binding proteins is a galactose lectin of 54 kDa related to rat liver RHL-2/3.     

1,25-Dihydroxyvitamin D effects in the kidney: induction of calmodulin binding proteins.

A marked induction of 125I-calmodulin binding proteins (185kDa and 115kDa) occurred in the rat kidney in response to treatment with 1,25-dihydroxyvitamin D3 (100 ng/day s.c. x 7d). These 125I-calmodulin binding proteins, measured by the gel overlay procedure, exhibited calcium dependence and were abolished in the presence of excess unlabelled calmodulin. The response was tissue specific: there was no change in 125I-calmodulin binding in rat testis, heart, and brain and only a modest elevation of binding to one calmodulin binding protein in the intestinal mucosa. These results are particularly important in suggesting that the calmodulin signal transduction mechanism may, via changes in its acceptor proteins, participate in mediating some biological effects of 1,25-dihydroxyvitamin D3.     

Identity of ligandin in rat testis and liver.

1. One of the main problems in the field of multifunctional proteins such as ligandin is the possibility that multiple forms and isoproteins may exist. Because liver ligandin [GSH (reduced glutathione) S-transferase B] consists of equal amounts of Ya (22 000 Da) and Yc (25 000 Da) subunits, and testis ligandin, prepared by the standard technique of anion-exchange and molecular-exclusion chromatography, contains more Yc subunit than Ya, it has been claimed that testis and liver ligandin are different entities. 2. We purified testis ligandin by immunoaffinity chromatography and have obtained a product identical with liver ligandin (Yc = Ya). This suggests that the differences previously described may be due to contamination of testis ligandin by a closely related species. In fact sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis of testis GSH S-transferases separated by CM-cellulose chromatography showed that GSH S-transferase AA, present in large amounts, migrated in the same region as Yc subunit. 3. Testis ligandin prepared by the standard technique was similar to that reported [Bhargava, Ohmi, Listowsky & Arias (1980) J. Biol. Chem. 255, 724-727] and contained more Yc subunit than Ya. CM-cellulose chromatography of this 'pure' preparation revealed significant amounts of GSH S-transferase AA migrating as Yc subunit, in addition to ligandin consisting of equal amounts of Ya and Yc subunits. 4. Our studies show that testis ligandin is identical with liver ligandin. Previously described differences are due to a contaminant identified as GSH S-transferase AA.     

Differential expression of transforming growth factor-beta in the interstitial tissue of testis during aging

Transforming growth factor-betas (TGF-betas) have significant effects on testis development. The pattern of TGF-beta expression in aging testis has not been established to date. We examined age-related changes in the expression of TGF-beta and its receptors in the testis using Western blot analysis. TGF-beta1 expression increased continuously in aging rat testis, whereas no age-associated changes were observed for TGF-beta3. Strong expression of TGF-beta2, as well as type I and II receptors was observed in 12-month-old testis, but following this time, expression decreased dramatically. Interestingly, TGF-beta2 and -beta3 displayed strong and similar expression patterns in liver, regardless of age, suggesting that the down-regulation of TGF-beta2 is testis-specific. We observed significant induction of p53 and p21WAF1 in 18-month-old testis that appeared to correspond with aging. Moreover, caloric restriction (CR) prevented age-related decrease in TGF-beta2 expression. Using immunohistochemistry, we showed that all TGF-beta1, -beta2, and -beta3 proteins are expressed primarily in interstitial cells, which are located in the space between adjoining seminiferous tubules. Our data collectively indicate that aging in the testis is regulated by differential expression of TGF-beta proteins, and decreased levels of TGF-beta2 contribute to the aging process.     

Selenoprotein P receptor from rat

Radioreceptor assay technology was used to show the presence in the rat of a receptor that binds selenoprotein P, a selenocysteine-containing rat plasma protein. 75Se-labeled selenoprotein P bound to testis, kidney, and liver membranes. The binding was specific in that increasing amounts of partially-fractionated rat plasma specifically displaced the binding of 75Se-labeled selenoprotein P to testis membrane in a competitive manner. 75Se-labeled selenoprotein P binding was saturable in the presence of increasing amounts of testis membranes. The binding of 75Se-labeled selenoprotein P was optimal at about pH 4.2. Several proteins and blood fractions had little or no significant effect on binding of 75Se-labeled selenoprotein P to testis membranes. All plasma sources tested specifically displaced 75Se-labeled selenoprotein P from testis membrane, indicating that selenoprotein P-related proteins may be widespread in nature. The study indicated that selenoprotein P has a receptor and is involved in selenium transport.     

Cadmium metabolism in cdm/cdm mice

The uptake and metabolism of cadmium by cadmium-susceptible (+/+) and cadmium-resistant (cdm/cdm) strains of mice have been compared. These strains did not differ with respect to the quantitative uptake of cadmium into liver, kidney, or testis. After intraperitoneal administration of a nontoxic dose, more than 80% of the cadmium in liver and testis of both strains is bound to low molecular weight proteins. The chromatographic behavior of these cadmium-binding proteins is not affected by cdm genotype.This research was supported in part by NIH Grant GM 24872 and the Michigan Memorial Phoenix Project.     

Electrophoretic analysis of liver and testis histones of the frog Rana pipiens

Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3.     

Structural study of the porcine NA+/H+ exchanger NHE1 gene and its 5′-flanking region

The poly(ADP-ribosyl)ation system, associated with different nuclear fractions of rat testis, has been analyzed for both pADPR and pADPR acceptor proteins. The DNase I sensitive and resistant chromatin contain 35% and 40%, respectively, of the total pADPR synthesized in intact nuclei incubated with [³²P]NAD. Moreover, the residual 25% were estimated to be associated with the nuclear matrix.Three different classes of pADPR are present in the nuclei. The longest and branched ADPribose polymers modify proteins present in the DNase I resistant (2 M NaCl extractable) chromatin and in the nuclear matrix, whereas polymers of > 20 residues interact with the components of the DNase I sensitive chromatin and oligomers of 6 ADPribose residues are bound specifically to the acid-soluble chromosomal proteins, present in isolated nuclear matrix. The main pADPR acceptor protein in all the nuclear fractions is represented by the PARP itself (auto-modification reaction). The hetero-modification reaction occurs mostly on histone H1 and core histones, that have been found associated to DNase I sensitive and resistant chromatin, respectively. Moreover, an oligo(ADP-ribosyl)ation occurs on core histones tightly-bound to the matrix associated regions (MARs) of chromatin loops.     

Transfer of fluorescent fatty acids from liver and heart fatty acid-binding proteins to model membranes

Fatty acid binding proteins (FABP) are a family of 14-15 kDa proteins found in high abundance in many mammalian cell types. The physiological functions of the FABP remain unknown. It is also not known whether each FABP has a unique function, or whether all FABP function in a similar manner in their respective tissues. In this report the rate of transfer of anthroyloxy-labeled free fatty acid (ffa) from FABP to phospholipid bilayers is monitored using a fluorescence resonance energy transfer assay. A comparison is made between heart muscle FABP and liver FABP, and the results show that the rate of ffa transfer from the heart protein is an order of magnitude greater than the rate of transfer from the liver protein. Ffa transfer rates from both liver and heart FABP are independent of acceptor concentration and composition, suggesting that, at least in the case of model membrane acceptor vesicles, the mechanism of transfer is via aqueous diffusion rather than via collision of FABP with membranes. Since the rate of ffa transfer is likely to be important to cellular ffa traffic, these studies suggest that heart FABP may function differently within the myocyte than does liver FABP within the hepatocyte.     

Immunochemical distribution and immunohistochemical localization of 20β-hydroxysteroid dehydrogenase in neonatal pig tissues

Immunochemical distribution of 20β-hydroxysteroid dehydrogenase (HSD) in neonatal pig tissues was investigated by Western blot analysis of the proteins reacting with anti-20β-HSD antibody. 20β-HSD was present in all organs investigated: brain, lung, thymus, submandibular gland, heart, liver, kidney, spleen, adrenal gland, testis, epididymis, prostate, vas deferens and seminal vesicle. In particular, high concentrations of 20β-HSD were detected in the testis, followed by the kidney and liver, by the [¹²⁵I]-protein A binding method. Immunohistochemical localization of the enzyme was achieved in paraffin sections of the testis, kidney, liver, epididymis, and vas deferens by the streptoavidin-biotin complex method. In the testis, very strong immunostaining was found only in interstitial Leydig cells, whereas the cells in seminiferous tubules, such as Sertoli cells and spermatogenic cells, were entirely negative. In the kidney, strong immunostaining was detected in epithelial cells of Henle's loop. The immunoreactive proteins were also localized in the hepatic lobules of the liver, tall columnar cells of the ductus epididymidis of the epididymis, and mucosal epithelium cells and muscularis of the vas deferens. These observations indicate that tissue distribution of 20β-HSD is similar to that of carbonyl reductase in the human and rat. However, the specific and abundant expression of 20β-HSD in testicular Leydig cells of the neonatal pig, which are concerned with the synthesis of androgens, suggests that 20β-HSD has a very important physiological role in testicular function during the neonatal stage.     

Distribution of high mobility group proteins in different tissues of rats during aging

The distribution of high mobility group (HMG) proteins has been studied in the liver, brain, kidney, lung, spleen, testis, thymus, and heart of young (19 weeks) and old (118 weeks) rats. These proteins were extracted with perchloric acid, fractionated by CM-Sephadex column chromatography, and analysed by acetic acid-urea polyacrylamide slab gel electrophoresis. As compared with that in young rats, the level of total HMG proteins in the old increased in liver and lung, decreased in thymus, heart, brain, and kidney, and remained unchanged in spleen and testis. In particular, the levels of HMG 1 and 2 were maximum in the thymus of young rats and dropped drastically in the old. However, the amount of HMG 17 was high in the spleen of both young and old rats, though it was comparatively higher in the former. Such age-dependent variation in the level of HMG proteins of different tissues denotes indirectly differences in the functional state of chromatin, and in growth and activity of cells, during aging.     

Evidence that translational control mechanisms operate to optimize antifreeze protein production in the winter flounder

In fall and winter, the liver of the winter flounder produces large amounts of alanine-rich (60 mol %) antifreeze proteins for export to the circulation. We have examined the tRNA in the liver to see if the seasonal production of antifreeze protein is accompanied by changes in tRNAAla isoacceptors. Total tRNA from the liver of winter fish showed an approximate 40% increase in alanine acceptor capacity over tRNA from summer fish. In contrast, the acceptor capacities for other amino acids showed no seasonal difference. When labeled alanyl-tRNAs were separated by reverse phase chromatography-5 chromatography, a large proportion of the increase in alanine acceptor capacity was in one of three main peaks. Measurements of the optimum temperatures for various flounder amino-acyl-tRNA synthetases suggest that alanyl-tRNA synthetase functions best between 0 and 5 degrees C, which is the sea water temperature when antifreeze protein synthesis occurs, while prolyl- and valyl-tRNA synthetases are most active between 20 and 30 degrees C. These differences in temperature optima and the seasonal variation in tRNAAla levels and isoaccepting species may both serve to optimize antifreeze protein production by increasing the translational efficiency of its mRNA.