Parviz Azari

Zinc transferrin: Enhancement of nucleic acid synthesis in phytohemagglutinin-stimulated human lymphocytes

Abstract Zinc transferrin, when added to serum-free cultures of phytohemagglutinin-stimulated human lymphocytes, causes an increase in deoxyribonucleic acid synthesis over that seen with phytohemagglutinin alone, as judged by the uptake of tritiated thymidine. This effect is not seen with zinc acetate, zinc albumin, or zinc ovotransferrin. Zinc transferrin also has a similar effect on ribonucleic acid synthesis. Furthermore, transferrin-bound zinc is specifically taken up by stimulated lymphocytes, maximal uptake occurring approximately 14 hr after the addition of phytohemagglutinin. These results indicate a function for serum transferrin in zinc metabolism, and, moreover, a role for the zinc transferrin complex in lymphocyte metabolism.

A simple and rapid procedure for preparation of large quantities of pure ovotransferrin

Abstract Large quantities of ovotransferrin from chicken egg-white were purified by a simple and rapid procedure employing carboxymethylcellulose. The advantages of this procedure involve the direct isolation of ovotransferrin from other egg-white proteins without resorting to the preliminary crystallization of other proteins or to the conventional (NH 4 ) 2 SO 4 fractionation steps. An entirely new step in the purification involves the crystallization of iron ovotransferrin from (NH 4 ) 2 SO 4 . The ovotransferrin obtained by the reported procedure was found to be homogeneous and suitable for prospective structural studies in relation to its metal binding activity. The amino acid composition of the homogeneous preparation was determined, and it was comprised of 601 residues per molecule of ovotransferrin.

Effect of iron transferrin on nucleic acid synthesis in phytohemagglutinin-stimulated human lymphocytes.

Abstract In serum-free cultures of phytohemagglutinin-stimulated human lymphocytes, iron transferrin causes enhanced uptake of both tritiated thymidine and tritiated uridine over that seen with only phytohemagglutinin. This effect is specific for the iron transferrin complex, no enhancement produced by either free iron(III) or apotransferrin. Iron bound to transferrin is quantitatively taken up by stimulated lymphocyte cultures, while under similar conditions only 10% of transferrin-bound zinc is incorporated. The relative specificity of action of iron and zinc on nucleic acid synthesis is discussed.

Amino acid composition of normal and cataractous human lens proteins.

Abstract The most significant change observed in the amino acid composition of cataractous lens proteins was in the content of cysteine (-SH) and half-cystine (-S-S-) residues. Normal lenses showed approximately 16 residues of cysteine and one half-cystine, where-as fully cataractous lenses showed approximately two residues of cysteine and 13 residues of half-cystine per 1000 amino acid residues. The content of tyrosine, histidine and tryptophan in normal and cataractous lenses was not significantly different. The autoxidation of -SH groups appears to be a major chemical change in the cataractogenic process.

Isolation and characterization of covalently linked, high molecular weight proteins from human cataractous lens

Biogel A-50 m chromatography and sucrose gradient centrifugation were employed for the isolation of covalently-linked high molecular weight proteins from the cataractous human lenses. The nuclear and nuclear plus cortical cataracts showed approximately 16% high-molecular weight proteins, as compared to 3% for posterior subcapsular cataract, as well as normal lenses. The high molecular weight proteins were heterogeneous by sucrose gradient centrifugation and showed a molecular weight range of 2–33 million daltons.

Role of sulfhydryl groups in the formation of a hereditary cataract in the rat

Abstract The predominant chemical change observed in the lens proteins during formation of the hereditary rat cataract was the oxidation of cysteine sulfhydryl (-SH) groups to cystine disulfide (-S-S-) groups. The process was accompanied by the formation of high mol. wt protein aggregates composed of various species of lens proteins. The process could be simulated, to a large extent, by in vitro exposure of normal rat lens to pure oxygen under pressure.

Physicochemical characterization of α-crystallins from bovine lenses: Hydrodynamic and conformational properties

A detailed investigation of hydrodynamic and conformational behavior has been made of the HMα-crystallin and α-crystallins of bovine lens. Results from this study indicated that HMα (high-molecular-weight α-crystallin) and α (low-molecular-weight α-crystallin) possess considerable size and charge heterogeneities in their native structures and subunit polypeptides, respectively. Sedimentation velocity showed a heterogeneous polydisperse system of HMα with an average sedimentation coefficient of about 50 S and a more homogeneous system of α-crystallin of 20 S. Viscosity and circular dichroism studies pointed to a compact and globular shape of dominant β-sheet conformation for α-crystallin, yet a highly asymmetrical and aggregated form for HMα. The conformational stability of α-crystallin was investigated in the presence of various denaturants. The evidence presented shows that hydrogen bonding is the main force in maintaining the quaternary structure of compact native α-crystallin. Conformational flexibility of α-crystallin demonstrated in the equilibrium unfolding study indicated a multistep transition that made the extraction of thermodynamic data from the heat denaturation study difficult. Temperature perturbation on α-crystallin suggested the possible involvement of hydrophobic interaction in the aggregation process, leading to the formation of HMα from α-crystallin. The comparison of conformational properties between HMα and α-crystallin strongly indicated that HMα is a denatured form of α-crystallin.

Action of periodate on ovotransferrin and its metal complexes

Abstract Short-time exposure of ovotransferrin to periodate oxidation at pH 8.5 and 5 resulted in the destruction of three and five tyrosine residues, respectively, per mole of the protein and an almost complete loss in its chromogenic activity. In contrast, the Fe-ovotransferrin complex retained its full chromogenic activity in the presence of periodate and showed no destruction of tyrosine residues. One mole of tryptophan was also destroyed per mole of ovotransferrin, as well as its iron complex. The histidines, methionines, and carbohydrate content (total hexose and glucosamine) of the modified proteins were the same as for the native protein. The modified derivatives were indistinguishable from native OT in electrophoretic mobility, sedimentation in the ultracentrifuge, and antigenic property. A value of 11 moles of tryptophan per mole of ovotransferrin is also reported in this study, which is considered to be a more accurate value than has been reported previously.

Iodination of ovotransferrin and its iron complex. Extent of involvement of tyrosine phenolic groups in the iron binding

Abstract Short-time iodination of metal-free ovotransferrin indicated that the tyrosine groups involved in the iron-binding activity are indistinguishable from other structural tyrosines. Modification of a minimum of 14 tyrosine residues per molecule of protein was required to achieve a complete loss of metal-binding activity. In contrast, a maximum modification of 10 tyrosine residues in iron-ovotransferrin complex could be produced with no loss of iron-binding activity. The difference in the extent of modification of tyrosines, therefore, indicated the involvement of four tyrosines in the binding of two atoms of iron. A minimal modification of histidine residues was also found, which was limited to one residue per molecule of both ovotransferrin and its iron complex. The possible participation of two tryptophan residues in the iron-binding activity is also suggested in the present study.

Physicochemical characterization of γ-crystallins from bovine lens—Hydrodynamic and biochemical properties

A detailed hydrodynamic study has been made on the γ-crystallin of the bovine lens. Sedimentation study indicates that γ-crystallin shows a nearly gaussian peak throughout the course of sedimentation at high speed, using a synthetic boundary cell. The diffusion and sedimentation coefficients are 10.3×10−7 cm2/sec and 2.51 S, respectively. The weight-average molecular weight of the unfractionated γ-crystallin calculated from sedimentation equilibrium is 21,800. The four major subfractions of γ-crystallin show similar hydrodynamic properties with an intrinsic viscosity of 2.50 ml/g and a Stokes radius of 21 Å. The distinct electrophoretic mobilities exhibited by the four subfractions show gel-concentration dependence and similar slopes in the Ferguson plot, indicative of being charge isomers of the same molecular species. Amino acid analysis of these four subfractions corroborated the conclusions that these γ-crystallin polypeptides are closely related and comprise a multigene family of crystallins. Based on the sedimentation and intrinsic viscosity data, γ-crystallin can be modeled as a prolate ellipsoid with an axial ratio of approximately 3.0 and a hydration factor of 0.27 g water per gram protein. The circular dichroism data for γ-crystallins showed a minimum at about 217 nm, characteristic of a β-sheet conformation. These structural characteristics are in good accord with those derived from X-ray diffraction data for γ-crystallin II.