Delta-aminolevulinate dehydratase, a zinc dependent enzyme

Erythrocyte and liver tissue δ-aminolevulinate dehydratase activity was determined in rats fed a semipurified diet under controlled nutritional intake of zinc and copper. A significant decrease in enzymatic activity was observed in animals fed low zinc diet, while dietary copper had no effect. $\text{In vitro}$ addition of zinc to the erythrocyte preparations obtained from rats on low zinc diet produced a slight increase in enzymatic activity. It appears that, even though zinc may be the metal ion activator of δ-aminolevulinate dehydratase, the requirement of this metal is at the site of synthesis of this enzyme.     

Amino acid sequence of the N alpha-terminal 201 residues of human erythrocyte membrane band 3

We have determined the amino acid sequence of the N alpha-terminal portion of band 3, the anion transport protein of the human erythrocyte membrane. The material analyzed was a 201-residue, 23,053-Da fragment cleaved from the cytoplasmic end of band 3 by S-cyanylation. The sequence had these notable features. 1) The N alpha-terminal region was extraordinarily acidic, second only to a segment of similar size from the sigma factor of Escherichia coli RNA polymerase. The first 33 residues contained 6 aspartic acid and 12 glutamic acid residues, no basic residue, and a blocked N alpha-amino group. 2) The first 11 residues of the protein had a striking resemblance to the following 11 residues. 3) In contrast to the acidic N alpha-terminal third, the COOH-terminal two-thirds of the 23,053-Da fragment had a predominantly basic character. The highly acidic character of the N alpha-terminal portion of band 3 accounts for the capacity of this part of the protein to bind glycolytic enzymes in a highly electrostatic fashion, presumably through interaction with their cationic substrate-binding sites.     

Radiation equivalent of genotoxic chemicals: Validation in cultured mammalian cell lines

Published data on mutations induced by ionizing radiation and 6 monofunctional alkylating agents, namely EMS, MMS, ENNG, MNNG, ENU and MNU, in different cell lines (Chinese hamster ovary, Chinese hamster lung V79, mouse lymphoma L5178 and human cells) were analysed so that radiation-equivalent chemical (REC) values could be calculated.REC values thus obtained for a given alkylating agent with different cell lines fall within a narrow range suggesting its validation in cultured mammalian cell systems including human.     

Vitamin A economy of the developing chick embryo and of the freshly hatched chick

1. The changes in the net amounts of retinol, retinyl esters and retinal in both the developing chick embryo and the newly hatched chick were investigated. The embryo requires about 68nmol of the vitamin for its growth, whereas the baby chick requires about 108nmol during the first 7 days after hatching. 2. Retinal was present in the egg in fairly high concentrations at the beginning of the incubation but it virtually disappeared from the extra-embryonic tissue after day 17 of incubation. It was not found in the liver of the embryo or of the newly hatched chick up until day 7.     

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...