Isolation and characterization of riboflavin-binding protein from pregnant-rat serum

A high-affinity riboflavin -binding protein was isolated and characterized for the first time from pregnant-rat sera by affinity chromatography on a lumiflavin-agarose column. The purified protein was homogeneous by the criteria of analytical polyacrylamide-gel disc electrophoresis, gel-filtration chromatography on Sephadex G-100 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It had a molecular weight of 90000+/-5000 and interacted with [14C]riboflavin with a 1:1 molar ratio with a dissociation constant (Kd) of 0.42 micron.

Characterization of protoporphyrin as the physiological regulator of delta-aminolevulinate dehydratase in Neurospora crassa

In Neurospora crassa, the activity of δ-aminolevulinate dehydratase, the second and rate-limiting enzyme of the heme-biosynthetic pathway, is low in normal cells compared to the activity detected in plants, animals and bacteria. The activity is almost undetectable when Neurospora crassa is grown under iron-deficient conditions. The enzyme activity increases strikingly on addition of iron to iron-deficient cultures. This increase can be blocked by the addition of protoporphyrin, the penultimate product of the heme-biosynthetic pathway, to the cultures. The question whether iron directly acts at the genetic level or acts merely by removing protoporphyrin, converting the latter into heme prosthetic groups of hemoproteins, has been investigated by studying the effect of inhibition of heme synthesis on the induction of δ-aminolevulinate dehydratase. It has been found that treatments with levulinic acid or cyanide which inhibit the formation of the porphyrin moiety, induce δ-aminolevulinate dehydratase, whereas treatments which inhibit at a step after protoporphyrin formation (iron-deficiency and cobalt treatment) repress the enzyme. The endogenous levels of protoporphyrin are strictly controlled: a decrease below the optimum level causing induction and an increase above the optimum level leading to repression of δ-aminolevulinate dehydratase. Levulinic acid and cyanide can induce the enzyme in iron-deficient cultures in the absence of added iron, indicating that the metal iron acts only by converting protoporphyrin to heme fixed in hemoproteins in Neurospora crassa. Therefore it is suggested that protoporphyrin is the physiological regulator of δ-aminolevulinate dehydratase in Neurospora crassa.

3-Amino-1,2,4-triazole is an inhibitor of protein synthesis on mitoribosomes in Neurospora crassa

The effects of the herbicide, 3-amino-1,2,4-triazole, an inhibitor of heme synthesis in rat liver, have been examined in the mold Neurospora crassa. The drug is a potent inhibitor of the growth of the mold and produces biochemical changes identical to those produced by chloramphenicol. 3-Amino-1,2,4-triazole, like chloramphenicol, is a direct and specific inhibitor of protein synthesis on mitoribosomes. A decrease in the levels of mitochondrial proteins which are completely or partly made on mitoribosomes and an accumulation in the levels of mitochondrial proteins of cytosolic origin have been observed. Both drugs depress porphyrin and heme levels, but there is actually an elevation in the levels of δ-aminolevulinate dehydratase, the rate-limiting enzyme of the heme-biosynthetic pathway in Neurospora crassa. In liver the enzyme is present in non-limiting amounts and the levels are depressed under conditions of 3-amino-1,2,4-triazole treatment. In Neurospora crassa the ‘derepression’ of δ-aminolevulinate dehydratase under conditions of 3-amino-1,2,4-triazole or chloramphenicol treatment is only partial because the drugs inhibit protein synthesis on mitoribosomes. It is concluded that an optimal rate of protein synthesis on mitoribosomes is necessary to maintain an adequate rate of heme synthesis.

Identification of active-site residues of sheep liver serine hydroxymethyltransferase

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5'-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.

Functional changes in rat liver mitochondria on administration of 2-methyl-4-dimethylaminoazobenzene

Administration of 2-methyl-4-dimethylaminobenzene in the diet (0.1%, w/w) for 85-90 days doubled the content of mitochondria in the livers of rats. The azodye was covalently bound to liver proteins, and about 15% of the amount found in liver was associated with the mitochondrial fraction. Mitochondria isolated from the livers of azodye-fed animals showed drastically lowered ability to oxidize NAD+-linked substrates. The inhibited electron-transfer step was the reduction of ubiquinone. The organelles showed a large increase in succinate oxidase activity. The activity of cytochrome oxidase and the content of cytochrome aa3 were substantially higher in these organelles. Azodye-fed animals showed depressed serum cholesterol concentrations. The content of ubiquinone in liver also registered a small increase.

Role of prolactin(prl) in regulation of the nocturnal surge of testosterone (t) in the bonnet monkey (macaca radiata)

Earlier studies from this lebordory have shown thet adult male bonnet monkeys exhibit nychthemrel rhythmicity la the secretion of serum 'T' the levele reehlng peek by 22OOhr. Of the gonedotropine cnelyeed only serum PRL showed a concommitent increme with T(Biol.of Reprod. 24,814, 1981). In the present study mMinietretion of l rgobromocryptin (EBC) either by i.v.route(2mg)or by naeel l pr~(100~)reeulted in blockade of nocturnal increase of both PRL end T(Controle T-18.6ng/ml: PRL 130=29ng/ml: EBC treated T-2.2&1.2ng/ml; PRL n.d.to 15nng/ml). Adminietretion of N oPRL could not reverse the effect of EBC. Although, increaeed serum PRL induced by injection of Chlorprommine did not result in increase in serum 'T' during the dey time, the nocturnel 'T' surge could not be obeeerved. EBC treeted monkeys, however, showed normal testosterone response to exogenous hCG. These IeSUlte a0 SwgeStive of high levels of PRL me&in6 reeponeiveneee of testes to tonic levels of serum IX. (Aided by grant8 from ICMR, Kew Delhi, WHO, Geneva eld FPF, India).

Indolic compounds in the leaves of Tecoma stans

Indole, tryptophan, tryptamine and skatole were isolated from the leaves of Tecoma stans. Anthranilic acid was also identified in its free form, in contrast to its glucoside, in Jasminum grandiflorum. The presence of both indole and anthranilic acid in the leaves of Tecoma stans indicates that they are the true substrate and product of indole oxygenase from the leaves of Tecoma stans.

Chemical Modification Studies On Ricinus-Communis (Castor Bean) Agglutinin

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.

Trehalose toxicity in cuscuta-reflexa - cell-wall synthesis is inhibited upon trehalose feeding

a,a-Trehalose induced a rapid blackening of the terminal 2.5-centimete region of excised Cuscuta relexa Roxb. vine. The incorporation of radioactivite from [I'C]glucose into alkali-insoluble fraction of shoot tip was markedly inhibited by 12 hours of trehalose feeding to an excised vine. This inhibition was confied to the apical segment of the vine in which cell elongation occurred. The rate of blackening of shoot tip explants was hastened by the addition of gibberellic acid A3, which promoted elongationgrowth of isolated Cuscuta shoot tips. The symptom of trehalose toxicity was duplicated by 2-deoxygucose, which has been shown to ba potent inhibitor of ceD wall synthesis in yeast. The observations suggest that trehalose interferes with the synthesis of ceDl wail polysaccharides, the chief component of which was presumed to be cellulose.

A comparative study of tbe regulation of cytochrome P-450 and glutathione transferase gene expression in rat liver

A cDNA clone for the Ya subunit of glutathione transferase from rat liver was constructed in E.coli. The clone hybridized to Ya and Yc subunit messenger RNAs. On the basis of experiments involving cell-free translation and hybridization to the cloned probe, it was shown that prototype inducers of cytochrome P-450 such as phenobarbitone and 3-methylcholanthrene as well as inhibitors such as CoCl2 and 3-amino-l,2,4-triazole enhanced the glutathione transferase (Ya+Yc) messenger RNA contents in rat liver. A comparative study with the induction of cytochrome P-450 (b+e) by phenobarbitone revealed that the drug manifested a striking increase in the nuclear pre-messenger RNAs for the cytochrome at 12 hr, but did not significantly affect the same in the case of glutathione transferase (Ya+Yc). 3-Amino-l, 2,4-tnazole and CoCl- blocked the phenobarbitone mediated increase in cytochrome P-450 (b+e) nuclear pre-messenger RNAs. These compounds did not significantly affect the glutathione transferase (Ya+Yc) nuclear pre-messenger RNA levels. The polysomal, poly (A)- containing messenger RNAs for cytochrome P-450 (b+e) increased by 12–15 fold after phenobarbitone administration, reached a maximum around 16hr and then decreased sharply. In comparison, the increase in the case of glutathione transferase (Ya+Yc) mesenger RNAs was sluggish and steady and a value of 3–4 fold was reached around 24 hr. Run-off transcription rates for cytochrome P-450 (b+e) increased by nearly 15 fold in 4 hr after phenobarbitone administration, whereas the increase for glutathione transferase (Ya+Yc) was only 2.0 fold. At 12 hr after the drug administration, the glutathione transferase (Ya+Yc) transcription rates were near normal. Administration of 3-amino-l,2,4-triazole and CoCl2 blocked the phenobarbitone-mediated increase in the transcription of cytochrome P-450 (b+e) messenger RNAs. These compounds at best had only marginal effects on the transcription of glutathione transferase (Ya+Yc) messenger RNAs. The half-life of cytochrome P-450 (b+e) messenger RNA was estimated to be 3–4 hr, whereas that for glutathione transferase (Ya+Yc) was found to be 8-9 hr. Administration of phenobarbitone enhanced the half-life of glutathione transferase (Ya+Yc) messenger RNA by nearly two fold. It is suggested that while transcription activation may play a primary role in the induction of cytochrome P-450 (b+e), the induction of glutathione transferase (Ya+Yc) may essentially involve stabilization of the messenger RNAs.

Factor VIII-hydrolyzing IgG in acquired and congenital hemophilia

Anti-factor VIII (FVIII) inhibitory IgG may arise as alloantibodies to therapeutic FVIII in patients with congenital hemophilia A, or as autoantibodies to endogenous FVIII in individuals with acquired hemophilia. We have described FVIII-hydrolyzing IgG both in hemophilia A patients with anti-FVIII IgG and in acquired hemophilia patients. Here, we compared the properties of proteolytic auto- and allo-antibodies. Rates of FVIII hydrolysis differed significantly between the two groups of antibodies. Proline-phenylalanine-arginine-methylcoumarinamide was a surrogate substrate for FVIII-hydrolyzing autoantibodies. Our data suggest that populations of proteolytic anti-FVIII IgG in acquired hemophilia patients are different from that of inhibitor-positive hemophilia A patients.

Identification of a novel nucleolin related protein (NRP) gene expressed during rat spermatogenesis

Nucleolin is a major nucleolar phosphoprotein involved in various steps of ribosome biogenesis in eukaryotic cells. As nucleolin plays a significant role in ribosomal RNA transcription we were interested in examining in detail the expression of nucleolin across different stages of spermatogenesis and correlate with the transcription status of ribosomal DNA in germ cells.

Adriamycin affects plasma membrane redox functions

Adriamycin (Doxorubicin) stimulates NADH oxidase activity in liver plasma membrane, but does not cause NADH oxidase activity to appear where it is not initially present, as in erythrocyte membrane. NADH dehydrogenase from rat liver and erythrocyte plasma membranes shows similar adriamycin effects with other electron acceptors. Both NADH ferricyanide reductase and vanadate-stimulated NADH oxidation are inhibited by adriamycin, as is a cyanide insensitive ascorbate oxidase activity, whereas NADH cytochrome c reductase is not affected. The effects may contribute to the growth inhibitory (control) and/or deleterious effects of adriamycin. It is clear that adriamycin effects on the plasma membrane dehydrogenase involve more than a simple catalysis of superoxide formation.