[en] Non-enzymatic glycosylation (glycation) seems to have the potential to alter the structure of crystallins and make them susceptible to thiol oxidation leading to disulfide-linked high molecular weight (HMW) aggregate formation. They used streptozotocin diabetic rats during precataract and cataract stages and long-term cell-free glycation of bovine lens crystallins to study the relationship between glycation and lens crystallin aggregation. HMW aggregates and other protein components of the water-soluble (WS) and urea-soluble (US) fractions were separated by molecular sieve high performance liquid chromatography. Glycation was estimated by both [³H]NaBH₄ reduction and phenylboronate agarose affinity chromatography. Levels of total glycated protein (GP) in the US fractions were about 2-fold higher than in the WS fractions and there was a linear increase in GP in both WS and US fractions. This increase was parallelled by a corresponding increase in HMW aggregates. Total GP extracted by the affinity method from the US fraction showed a predominance of HMW aggregates and vice versa. Cell-free glycation studies with bovine crystallins confirmed the results of the animals studies. Increasing glycation caused a corresponding increase in protein insolubilization and the insoluble fraction thus formed also contained more glycated protein. It appears that lens protein glycation, HMW aggregate formation, and protein insolubilization are interrelated

[en] The biological receptor for tumor-promoting phorbol esters has been identified as the Ca²⁺/phospholipid dependent enzyme, protein kinase C. In the red cell, this enzyme is mainly cytosolic but becomes translocated to the membrane if the cellular Ca²⁺ is allowed to rise. Since cellular Ca²⁺ in sickle red cells is high, it was reasoned that this enzyme may become more membrane-bound. In fact, the authors noticed a four-fold increase in the binding of ³H-PDBu by membrane ghosts isolated from sickle red cells compared to normal red cells (pmoles PDBu bound/mg protein; normal = 0.3 vs sickle cell = 1.4). Attempts to assay the enzyme directly as phospholipid-activated ³²P incorporation into the acid-precipitable membrane proteins also indicated a two-fold increase in the radiolabelling of sickle cell membrane ghosts. Autophosphorylation of membrane proteins and analysis of the phosphorylation profile by SDS-PAGE and autoradiography revealed phosphorylation predominantly of bands 3, 4.1 and 4.9 which are known protein kinase C substrates for the red cell enzyme. The increased membrane-associated protein kinase C in sickle red cells may have a bearing on the altered membrane properties reported in this condition

[en] The authors have been interested in human fetal hemoglobin acetylation and have focused on search of an acetyltransferase that can acetylate this hemoglobin. Cord blood ribosomal and postribosomal acetyltransferases have been partially purified by histone-Sepharose 4B affinity chromatography. Analysis of Dextran 40 fractionated cells has indicated that the enzyme is present only in reticulocytes and young erythrocytes, and mature erythrocytes are devoid of this enzyme. Enzymes prepared from the ribosomal and postribosomal sources showed similar temperature, pH, and cation dependence. In vitro acetylation of Hb F₀, Hb A₀ and native γ, β, and α chains with [¹⁴C]acetyl-CoA in the presence of these enzyme preparations failed to show any specificity to any one of the hemoglobins. No significant differences in hemoglobin acetylation were seen between the two types of enzymes. However, acetylation of Hb F₀ and γ chains did produce Hb F/sub Iac/ (Hb F/sub I/ or Hb F/sub Ic/) and γ/sub Iac/ (γ/sub I/ or γ/sub Ic/), respectively. Peptide analyses showed that the γ chain NH₂-terminus and other sites were being acetylated. Thus, site specificity is seen only when the ribosomal enzyme acts on ribosome-bound nascent chains. It is also possible that the presence of free enzyme, acetyle-CoA and potential hemoglobin substrates in the cytoplasm may lead to the formation of some Hb F/sub Iac/ and small amounts of other unidentified acetylated hemoglobins in red cells

[en] A minor component of human fetal hemoglobin (Hb F) is acetylated at the amino-terminus of the γ-globin chains. A similar minor component of Hb F is formed during translation of cord blood mRNA in the rabbit reticulocyte lysate system. The acetylation appeared to be enzymatic. This system contains an acetyltransferase capable of acetylating histones and hemoglobins. The enzyme, partially purified by histone-Sepharose affinity chromatography was capable of incorporating labeled acetyl- group from 1-[¹⁴C-acetyl]-CoA into both human Hb F₀ and HB A₀, but at a lower rate than for histones. Characterization of the labeled products indicated that the α-chains of both hemoglobins were being acetylated presumably at a lysyl-residue, but in the case of Hb F₀ the amino-terminus of the γ-globin chains was acetylated as well. While histone-Sepharose bound more than 95% of the enzyme, Sepharose linked Hb F₀, γ-globin chains, and Hb Bart's bound 14, 5, and 12% of the activity, respectively. Enzyme bound to these resins was not any more active on the hemoglobins than was the enzyme bound to the histone-Sepharose. The histone-Sepharose was also used to detect the enzyme in human cord blood red cells separated by dextran 40 density gradient centrifugation. Activity was found mostly in the young cells, and was directly related to the number of reticulocytes present in any one fraction

[en] Acetyltransferase was isolated by histone-Sepharose affinity chromatography from human cord blood red cells. The enzyme was detected only in very young red cells. The semipurified enzyme and [¹⁴C]acetyl-CoA were used to acetylate isolated Hb F tetramer and α and γ subunits. The in vitro acetylated products were characterized by globin chain separation by CM-cellulose chromatography and tryptic peptide analysis by reverse-phase HPLC. Acetylation of both the γ-chains and the α-chains could occur within the Hb F tetramer. Acetylation also could take place on intact subunits. It appears that some Hb F/sub Ic/ could be formed in the cells by utilizing Hb F or free γ-chains as acetylation substrate