Electrophoretic separation of human embryonic globin demonstrates “α-thalassemia” in human leukemia cell line K562

Human embryonic, fetal, and adult globin chains ($\text{ζ, ε,}{}^{\mathrm{A}}\text{γ,}{}^{\mathrm{G}}\text{γ, β, α}$) can be separated by electrophoresis on Triton Acid urea gels. K562, a human leukemia cell line, was induced with hemin, labelled with [³H]-leucine, and globin synthesis analyzed. All globins except β were produced. $\text{ε > ζ;}{}^{\mathrm{G}}\text{γ:}{}^{\mathrm{A}}\text{γ=70:30; non-α:α=>2:1}$. Thus, hemin-induced K562 synthesized embryonic and fetal globin chains, and had globin synthetic imbalance, with “α-thalassemia.”     

Base-paired interaction, in vitro, between hen globin 9S mRNA and eukaryotic ribosomal RNAs.

Hen globin 9S mRNA complexes efficiently with mouse sarcoma 18S rRNA, and to a lesser extent with 28S rRNA, but not with tRNA. The mRNA-18S rRNA complex is dissociated under conditions that lead to disruption of hydrogen bonds, and exhibits a biphasic thermal denaturation curve with Tms at $\text{ca.}$ 39° and 58° in 0.15 $\text{M}$ NaCl-0.03 $\text{M}$ Tris-HCl, pH 7.5. Hen globin mRNA also interacts with 18S rRNAs from various other eukaryotes, and the melting profile and Tm of the complex formed with hen 18S rRNA is very similar to that of the complex formed with mouse sarcoma 18S rRNA.     

Cycloheximide inhibition of insulin control of liver glycogen synthase b into a conversion.

Cycloheximide given to insulin-treated alloxan diabetic rats results in the inhibition of insulin-induced liver glycogen synthase $\text{b}\text{into}\text{a}$ conversion without affecting the level of synthase $\text{b}$. The effect of cycloheximide, believed to elevate cAMP in liver of normal rats, is independent of cAMP levels of the insulin-treated diabetic rat. The inhibition of insulin-mediated synthase $\text{b}$ to $\text{a}$ conversion by cycloheximide does not appear to be the result of a cycloheximide-induced cAMP-dependent phosphorylation of synthase $\text{a}$ to $\text{b}$ and suggests that insulin control of synthase $\text{b}$ and $\text{a}$ interconversions is dependent upon cycloheximide-sensitive protein synthesis.     

Hemoglobin Constant Spring: hemoglobin synthesis in heterozygous and homozygous states.

Thirteen adult and one newborn heterozygotes, and three homozygotes for hemoglobin Constant Spring were examined for globin chain synthesis. Reticulocytes from venous blood were incorporated with [³H]-leucine in an incubation mixture for 3 hours. Globin prepared from the radioactive, washed red cells was fractionated by CM-cellulose chromatography in 8 M urea and the total radioactivity of each globin chain was determined. The mean of $\text{α}\text{β}$ ratio in the heterozygotes was 1.34 ± SD 0.08, which is significantly different from that of 1.07 ± SD 0.03 in eleven normal controls. The $\text{α}\text{β+γ}$ ratio in the heterozygous neonate was also 1.39. The $\text{α}\text{β}$ ratios in the three homozygotes were around 1.6. The α-Constant Spring chain appears to be over produced, but it may be unstable or labile, not fully available for conjugation with the non alpha chains.     

Requirement of different ionic concentrations for optimal translation of α and β globin mRNA in Krebs II ascites cell-free system

The translation of rabbit α globin mRNA in a Krebs II ascites cellfree system was more dependent upon the K⁺ concentration than rabbit β globin mRNA. The optimal KCl concentration was approximately 70 mM for the synthesis of the α chain and between 80 and 90 mM for that of the β chain. With CH₃ CO₂K the optimum concentration for α chain synthesis was also 70 mM but the optimum for the β chain synthesis was not sharp any more and ranged from 70 mM to over 110 mM. In the range of the optimal Mg²⁺ concentration for the α and β globin chain synthesis the $\text{α}\text{β}$ ratio decreased when the Mg²⁺ concentration increased. In the presence of DTT and EDTA the optimal KCl concentration for both α and β globin chain synthesis decreased.     

Liver microsomal electron transport systems. II. The involvement of cytochrome b5 in the NADH-dependent hydroxylation of 3,4-benzpyrene by a reconstituted cytochrome P-448-containing system

An enzyme system in $\text{rat liver microsomes}$ which catalyzes the NADH-dependent $\text{hydroxylation}$ of 3,4-benzpyrene has been reconstituted. The essential components of this NADH-mediated $\text{electron transport chain}$ are cytochrome b₅, NADH-cytochrome b₅ reductase, lipid, and cytochrome P-448.     

Intramolecular catalysis. VII: The nature of side chain shielding of the 12alpha-hydroxyl group of steroids

A series of 12α-hydroxy steroids with varying side chains was prepared, and their 24-hour acetylation yields were compared, l2α-Hydroxy-5β-pregnan-20-one ($\text{lb}$) was prepared from 3α, 12α-diacetoxy-5β~pregnan-20-one ($\text{2}$) and also by side chain degradation of 12α-acetoxy-5β-cholanoic acid ($\text{5d}$). 21-Benzyl-5β-pregnan-12α-ol ($\text{1g}$) was synthesized by hydrogenation of the 21-benzylidine derivative of ketone $\text{1b}$. 23-Pheny1-5β-norcholan-12α-ol ($\text{1k}$) was obtained by the Grignard reaction of 2-phenyl-ethylmagnesium bromide and ketone $\text{1b}$, dehydration, hydrogenation and hydride reduction; a similar sequence produced 20-methyl-5β-pregnan-12α-ol ($\text{lm}$). The acetylation results (Table 11) imply that branching at C-20 may be more significant for 12α-hydroxyl reactivity than side chain length or type. An additional compound with an unbranched side chain, 21-nor-5β-cholan-12α-ol ($\text{14}$), was synthesized by a Grignard reaction on the 21-bromo intermediate $\text{11b}$. Acetylation rates determined by glc indicate (Table 111) That compounds with unbranched side chains have 12α-hydroxyl groups about ten times as reactive as their analogs with 20-methyl groups.     

Cytochromes in the rat kidney brush border membrane.

Spectrophotometric studies of the brush border membrane fraction of the rat kidney as compared with those of its mitochondria and microsomes were carried out. Occurrence of cytochromes has been demonstrated in the brush border membranes. Either in the brush border membranes and in the mitochondria evidence for the presence of cytochromes of the types $\text{a}\text{,}\text{b}$ and $\text{c}$ was found, whereas in the microsomes only cytochrome $\text{b}$ was demonstrated.     

Contribution of microsomal cytochrome b5 electron transport system coupled with Δ5-3β-hydroxysteroid dehydrogenase to androgen synthesis from progesterone

Cytochromes P-450 and $\text{b}$₅ were observed in the microsomal fraction of interstitial tissue of rat testes. Microsomal cytochrome $\text{b}$₅ was reduced by the NADH coupled with the activities of Δ⁵-3β-hydroxysteroid dehydrogenase with Δ⁵-Δ⁴ isomerase through conversion of pregnenolone to progesterone. Activities of NADPH-supported 17α-hydroxylase and C-17-C-20 lyase which converted progesterone to androstenedione were stimulated by either the presence of NADH or the oxidative reaction by the dehydrogenase upon Δ⁵-3β-hydroxysteroids. Androstenedione production enhanced by the reaction of the dehydrogenase was decreased by addition of the antibody against NADH-cytochrome $\text{b}$₅ reductase which was purified from rat hepatic microsomes, suggesting the active participation of cytochrome $\text{b}$₅ in the androgen synthesis.     

Effects of anti-NADPH-cytochrome c reductase and anti-cytochrome b5 antibodies on the hepatic and pulmonary microsomal metabolism and covalent binding of the pulmonary toxin 4-ipomeanol.

An antibody prepared against purified rat liver NADPH-cytochrome $\text{c}$ reductase inhibited both the pulmonary and hepatic microsomal covalent binding of 4-ipomeanol as well as the respective NADPH-cytochrome $\text{c}$ reductase activities, findings which are consistent with previous studies which indicated the participation of cytochrome P450 in the metabolic activation of the toxin. An antibody prepared against purified rat liver cytochrome b₅, which strongly inhibited both the rat hepatic and pulmonary NADH-dependent cytochrome $\text{c}$ reductases, and was inactive against the respective NADPH-dependent cytochrome $\text{c}$ reductases, had little effect on metabolic activation of 4-ipomeanol by hepatic microsomes, but strongly inhibited both the NADH-supported and the NADPH-supported pulmonary microsomal metabolism and covalent binding of the compound. These results suggest that metabolic activation of 4-ipomeanol involves a two-electron transfer in which transfer of the second electron via cytochrome b₅ is rate-limiting in lung microsomes.     

Separation of solubilized alpha and beta adrenergic receptors by affinity chromatography.

Isoelectric focusing in the presence of Nonidet P-40 splits human chromatographically pure γ globin chains into two bands of isoelectric points 6.95 and 6.85, respectively. The comparison of the relative proportions of the two bands with the ratios between the G_γ and A_γ non allelic chains of human fetal hemoglobin suggests that the band at pI 6.95 corresponds to G_γ and the band at pI 6.85 corresponds to the A_γ chain; the latter is the only band present in a patient with Greek type hereditary persistence of fetal hemoglobin, producing only A_γ chains. Fluorography of electrofocusing-separated radioactive γ globin chains synthesized by thalassemic reticulocytes indicates that the relative $\text{G}{}_{\mathrm{\gamma }}\text{A}{}_{\mathrm{\gamma }}$ synthetic ratios are similar to the relative amounts of G_γ and A_γ chains accumulated in the erythrocytes, suggesting that the activities for the G_γ and A_γ mRNAs decay at roughly similar rates.     

Synthesis of a complementary DNA to rat liver albumin mRNA.

NADPH reduces both liver microsomal cytochrome P-450 and cytochrome $\text{b}{}_5$. In the presence of CO, ferrous cytochrome P-450 can slowly transfer electrons to amaranth, an azo dye. This reaction is followed by the reoxidation of cytochrome $\text{b}{}_5$ which proceeds at essentially the same rate as does cytochrome P-450 oxidation. It is suggested that cytochrome $\text{b}{}_5$ directly reduces cytochrome P-450 in rat liver microsomes.     

The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5

The role of cytochrome $\text{b}$₅ in the $\text{p}$-nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome $\text{b}$₅. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome $\text{b}$₅ reductase, and Triton X-100 in addition to cytochrome $\text{b}$₅. The omission of cytochrome $\text{b}$₅ from the complete system entirely abolished the activity. These results clearly show that cytochrome $\text{b}$₅ is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome $\text{b}$₅ in such a way that the second electron is transferred from cytochrome $\text{b}$₅ and thus exhibits the demethylase activity.     

Antenna function of a chlorophyll ab protein complex of Photosystem I

The functional role of a chlorophyll $\text{a}\text{b}$ complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, K_P-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll $\text{a}\text{b}$ complex and the other lacking the complex. K_P-700 for the former preparation is half of that of the preparation which has the chlorophyll $\text{a}\text{b}$ complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll $\text{a}\text{b}$ complex. Experiments involving reconstitution of the chlorophyll $\text{a}\text{b}$ complex with the antenna-depleted PS I preparation indicate a substantial recovery of the K_P-700 rate. These results demonstrate that the chlorophyll $\text{a}\text{b}$ complex functions as a light-harvesting antenna in PS I.     

Regulation of muscle phosphorylase activity by carnosine and anserine

Carnosine (β-alanyl-L-histidine) activates rabbit muscle phosphorylase $\text{a}$ in the presence and absence of AMP and phosphorylase $\text{b}$ in the presence of AMP in a biphasic manner with a maximal activation at about 50mM carnosine and with phosphorylase $\text{b}$ showing a greater degree of activation than phosphorylase $\text{a}$. Anserine (β-alanyl-L-N^π-methyl-histidine) activates phosphorylase $\text{a}$ to a lesser extent than carnosine up to a concentration of 90mM, whereas with phosphorylase $\text{b}$ a weak activation below 30mM and a concentration-dependent inhibition above this concentration occurs. These effects are specific for the dipeptides and are not shown by their constituent amino acids. Carnosine and anserine activate phosphorylase $\text{a}$ in the presence of the allosteric inhibitors ATP, D-glucose and caffeine, and the inhibition of phosphorylase $\text{b}$ by anserine is also observed in the presence of these inhibitors.     

Induction by ouabain of hemoglobin synthesis in cultured friend erythroleukemic cells

Induction of erythroid differentiation in ouabain-resistant murine erythroleukemia cells by ouabain is reported. Ouabain induction results in the appearance of hemoglobin-containing cells 12–24 hr earlier than induction of the same clone by dimethyl sulfoxide. The levels of globin mRNA after ouabain induction are similar in amount to the globin mRNA levels observed after induction by dimethyl sulfoxide. The concentration of ouabain required to induce hemoglobin synthesis depends upon the K⁺ ion levels in the culture medium. Lowering the extracellular K⁺ ion concentration 2–4 fold reduced by 10–40 fold the ouabain concentration necessary for the induction of hemoglobin synthesis. In low K⁺ medium (1.8 mM), ouabain is an effective inducer of hemoglobin synthesis at a concentration of 0.02 mM. This K⁺ effect is specific for ouabain induction, since induction by other inducers, such as dimethyl sulfoxide and dimethyl acetamide, does not exhibit this marked sensitivity to the levels of K⁺ ions in the culture medium. These results suggest that the binding of ouabain to the plasma membrane enzyme, $\text{Na}\text{K}$ ATPase, is required for the induction of erythroid differentiation by ouabain. A small but significant proportion of wild-type, ouabain-sensitive cells also can be induced by ouabain, below ouabain concentrations that are toxic to these cells. The observation that the binding of ouabain to the $\text{Na}\text{K}$ ATPase induces hemoglobin synthesis suggests that changes in the intracellular concentration of K⁺ ions may be involved in the control of erythroid differentiation in Friend erythroleukemic cells.