The interaction of riboflavin with a protein isolated from hen's egg white: a spectrofluorimetric study.

The interaction of riboflavin with a protein isolated from egg white has been studied spectrofluorimetrically at different pH values. In 0.1 M phosphate buffer pH 7.0; 1:1 complex formation occurs with the association constant Ka = 7.7-10(7) M-1. In the presence of 0.033% sodium dodecyl sulphate, the complex dissociated with a rate constant of 4-10(-2) sec-1 at 29 degrees C. The binding was sensitive to pH and to the antibodies produced against the protein. On lowering the pH from 7 to 4 the binding affinity decreased approximately 100-fold and below pH 4, the binding could not be detected at all. These data, together with those obtained by measuring the fluorescence intensities of riboflavin in presence of N-bromosuccinimide oxidized- and disulphide reduced apoprotein, suggest that carboxyl functions, 1-2 tryptophan residues and 2-3 disulphide bridges are essential for binding. The emission spectra of the protein under different conditions upon excitation at 280 and 295 nm were analyzed to calculate the quantum yield (Q) and the efficiency of energy transfer (e) from tyrosine to tryptophan residues. From these data it was concluded that the energy transfer did not occur with equal efficiency under all conditions and that the tryptophan residues responsible for the riboflavin binding are more accessible to N-bromosuccinimide oxidation than others.     

STRUCTURAL ALTERATIONS OF AMINO ACIDS AT THE LEVEL OF AMINOACYL-tRNAS: IDENTIFICATION OF THE TRANSFORMATION PRODUCTS OF DICARBOXYLIC AMINO ACIDS

Abstract— Glutamyl-, glutaminyl-, aspartyl- and asparaginyl-tRNAs were separated into different isoacceptor species by reverse phase column chromatography. RNase hydrolysates of any of the isoacceptor [¹⁴C]aminoacyl-tRNAs for a given amino acid gave radioactivity profiles, on paper electrophoresis, very similar to unfractionated tRNA. This suggested a lack of tRNA specificity for the transformation reaction involving the aminoacyl moieties of asparaginyl and glutaminyl-tRNAs. GnP₂ and AnP₂ detected in the products of deaminoacylation of glutaminyl and asparaginyl-tRNA showed a number of properties in common with GnE₃ and AnE₃ present in the RNase hydrolysates of the same tRNAs. Thus, GnP₂ and GnE₃ chromatographed in the same position in the phenol: water solvent and both yielded glutamate on acid hydrolysis and a mixture of glutamine and isoglutamine on alkaline hydrolysis. Similarly, AnP₂ and AnE₃ had the same R_F value in phenol :water chromatography and gave aspartate or a mixture of asparagine and isoasparagine when hydrolyzed with acid or alkali. On the basis of these results and other evidence, GnP₂ and GnE₃ were assigned the structure, α-aminoglutarimide; AnP₂ and AnE₃ were identified as α-aminosuccinimide. These cyclic compounds are presumed to be formed by nucleophilic attack of the amide nitrogen of asparagine or glutamine on the carbon of the aminoacyl ester carbonyl group. The cyclization-deesterification appeared to be facilitated by RNase hydrolysis of aminoacyl-tRNA indicating that the aminoacyl-tRNA is probably more resistant to this reaction than aminoacyladenosine. Neither the imides nor the amino acid isoamides were detected in the reaction mixture in which aminoacylation of tRNA was performed, suggesting that a mechanism may exist for inhibition of the cyclization reaction under conditions of active aminoacylation.     

Inhibition of phenylalanine hydroxylase activity by alpha-methyl tyrosine, a potent inhibitor of tyrosine hydroxylase.

Inhibitors of phenylalanine hydroxylase and tyrosine hydroxylase were used in the assay of phenylalanine hydroxylase in liver and kidney of rats and mice. Parachlorophenylalanine (PCPA), methyl tyrosine methyl ester and dimethyl tyrosine methyl ester showed 5–15% inhibition while α-methyl tyrosine seemed to inhibit phenylalanine hydroxylase to the extent of 95–98% at concentrations of 5 × 10 ⁻⁵M –1 × 10 ⁻⁴M. After a phenylketonuric diet (0.12% PCPA + 3% excess phenylalanine), the liver showed 60% phenylalanine hydroxylase activity and kidney 82% that present in pair-fed normals. Hepatic activity was normal after 8 days refeeding normal diet whereas kidney showed 63% of normal activity. The PCPA-fed animals showed 34% in liver and 38% in kidney as compared to normals; in both cases normal activity was noticed after refeeding. The phenylalanine-fed animals showed activity similar to that seen in phenylketonuric animals. The temporary inducement of phenylketonuria in these animals may be due to a slight change in conformation of the phenylalanine hydroxylase molecule; once the normal diet is resumed, the enzyme reverts back to its active form. This paper also suggests that α-methyl tyrosine when fed in conjunction with the phenylketonuric diet may suppress phenylalanine hydroxylase activity completely in the experimental animals thus yielding normal tyrosine levels as seen in human phenylketonurics.     

Studies on metabolism of vitamin A. Absorption of retinal (vitamin A aldehyde) in rats

1. Young rats with low reserves of vitamin A were dosed with retinal in groundnut oil, and the stomach, the contents, mucosa and muscles of small intestine, the blood and the liver were analysed at periods up to 24hr. after dosing. 2. Up to 6hr. after the dose, retinal was present in high concentrations in the contents, mucosa and muscles of the intestine. Small but significant amounts were present in blood and liver at all times. 3. The intestinal mucosa and muscles always contained small amounts of retinol and its esters. 4. A study of the distribution of the three forms of the vitamin within the mucosal cell showed that most of the mucosal retinal enters the cell unchanged. 5. When protein-depleted rats were similarly given retinal, the rate of reduction of the aldehyde, and the consequent deposition in the liver of retinol and its esters, progressively decreased with reduced protein intake.     

X-ray diffraction analysis of dehydrated myelin

Improved X-ray diffraction data from dry nerve myelin are presented. In addition to the spacings of approx. 150 Å, 60 Å, 44 Å and 34.6 Å, which have been previously reported, we identify a 14 Å series. The data suggests that the hydrocarbon chains in the single bilayer ($\text{≈ 60}\text{A}\text{?}$) is ordered, whereas in the double bilayer ($\text{≈ 150}\text{A}\text{?}$) and in the fluid phase ($\text{≈ 44}\text{A}\text{?}$) it is disordered. It is shown that cholesterol ($\text{≈34.6}\text{A}\text{?}$) exists as a bilayer, and the 14 Å series is probably another cholesterol phase.     

Preliminary crystallization studies of calmodulin-dependent protein phosphatase (calcineurin) from bovine brain

Molecular and Cellular Biochemistry - Calcineurin is a serine/threonine protein phosphatase which catalyzes the hydrolysis of both phosphoseryl/phosphothreonyl and phosphotyrosyl proteins as well...     

Thidiazuron-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogaea): Endogenous growth regulator levels and significance of cotyledons

The regulatory role of thidiazuron (TDZ) and explant factors in imparting somatic embryogenic potential was assessed in relation to endogenous growth regulator levels in peanut ( Arachis hypogaea L. cv. Tango). TDZ induced somatic embryogenesis over a range of concentrations (0.5 to 10 μ M ). Culture of seedlings for just 2 days on TDZ-supplemented medium was sufficient to induce somatic embryogenesis. Seedling age and retention or removal of cotyledons during culture influenced morphogenic potential significantly. The younger the seedlings, the better was the embryogenic response to TDZ treatment. The embryogenic potential of seedlings was limited for explants with no cotyledons and they did not respond to increasing levels of TDZ. In contrast, retention of at least one or both the cotyledons resulted in increased response to TDZ. Endogenous levels of cytokinins, auxins and abscisic acid were influenced by TDZ treatment, while TDZ itself was detected only in the cotyledons. The cytokinin N⁶-(2-iso-pentenyl)adenine (2iP) fluctuated on a 10-day cycle in the cotyledons; TDZ treatment suppressed this change and caused an overall decrease in the pool of available 2iP in the tissue. It appeared that TDZ induced somatic embryogenesis in peanut by influencing endogenous levels of both auxin and cytokinins.     

Studies on molecular evolution and structural features of double-headed inhibitors of α-amylase and trypsin in plants

Plant seeds usually have high concentrations of proteinase and amylase inhibitors. These inhibitors exhibit a wide range of specificity, stability and oligomeric structure. In this communication, we report analysis of sequences that show statistically significant similarity to the double-headed α-amylase/trypsin inhibitor of ragi (Eleusine coracana). Our aim is to understand their evolutionary and structural features. The 14 sequences of this family that are available in the SWISSPROT database form three evolutionarily distinct branches. The branches relate to enzyme specificities and also probably to the oligomeric state of the proteins and not to the botanical class of the plant from which the enzymes are derived. This suggests that the enzyme specificities of the inhibitors evolved before the divergence of commercially cultivated cereals. The inhibitor sequences have three regions that display periodicity in hydrophobicity. It is likely that this feature reflects extended secondary structure in these segments. One of the most variable regions of the polypeptide corresponds to a loop, which is most probably exposed in the native structure of the inhibitors and is responsible for the inhibitory property.     

Induction of high-frequency somatic embryogenesis in geranium (Pelargonium x hortorum Bailey cv Ringo Rose) cotyledonary cultures

Summary The cv Ringo Rose of hybrid seed geranium (Pelargonium x hortorum Bailey), previously shown to be recalcitrant in culture, produced somatic embryos when cotyledonary explants were cultured on regeneration medium containing thidiazuron (TDZ), forchlorfenuron (CPPU), or a combination of indole-3-acetic acid and N⁶ benzylaminopurine (IAA+BAP). Amendment of the basal medium with TDZ (0.5 M) was the most effective treatment. Addition of amino acids to the medium promoted the growth of somatic embryos. Retention of the proximal region of the cotyledon was crucial for regeneration, but the removal of the distal 1/3 to 1/2 cotyledon had no significant effect on somatic embryogenesis. Cotyledonary explants formed somatic embryos in higher frequency and much earlier than hypocotyl explants cultured on the same medium. The somatic embryos induced on cotyledonary explants were germinated on basal medium. More than 70% of the somatic embryos were converted into plants and transferred to soilAbbreviations BAP N⁶-benzylaminopurine - CPPU N-(2-chloro-4-pyridyl)-N'-phenylurea (forchlorfenuron) - IAA indole-3-acetic acid - TDZ N-phenyl-N'-1,2,3,-thiadiazol-5ylurea (thidiazuron)     

Particle pathways of Niagara river water in Lake Ontario affecting bottom sediment contamination

The results of integrated multidisciplinary studies of the transport and pathways of toxic contaminants entering Lake Ontario through the Niagara River are summarized. The factors controlling the transport and distribution of contaminants are: (i) Niagara River mixing characteristics; (ii) prevailing local wind conditions and large scale lake-wide circulation features, and (iii) suspended sediment load and the partitioning of the contaminants onto the suspended solids. Physical limnological studies included a variety of direct measurements to delineate the nearfield and farfield mixing characteristics of the river plume in terms of the prevailing winds and large scale lake circulation. In the nearfield region of the plume, around the river mouth, contaminant transport was affected by the initial momentum and buoyancy of the river water mass and a sharp thermal front that is generally present through the stratified season. After the initial momentum is dissipated, the weakly buoyant river plume responds to the prevailing winds and lake-wide circulation. Adsorbed contaminants rapidly settle once the initial momentum of the river plume is dissipated but the dissolved contaminants are transported into the farfield regions in the lake and after becoming entrained in the strong south shore eastward flowing boundary current are carried toward the St. Lawrence River outflow. Only about ten percent of this eastward flowing transport exits via the St. Lawrence River, the remainder is recirculated back into the main lake. A contaminant transport model of the Niagara River Plume incorporating a hydrodynamic model, observed plume mixing characteristics and chemical partitioning was used to simulate the compartmental distribution (adsorbed vs dissolved) of selected toxic chemicals distribution observed in the nearfield region of the plume.