IT is generally accepted that red cells possess enzymatic reducing mechanisms that maintain hemoglobin in its active state.1 , 2 It is also well established that the reduction is dependent on the regeneration of reduced pyridine nucleotides.3 4 5 Treating red cells with nitrite and incubating them with glucose, lactate, or other substrates that allow the production of reduced pyridine nucleotides promotes methemoglobin reduction.1 , 2 , 6 , 7 Under normal conditions, NADH (reduced nicotinamide adenine dinucleotide) accounts for most of the methemoglobin-reducing capacity of the erythrocyte.2 , 8 9 10 11 An NADH dehydrogenase — variously named methemoglobin reductase or cytochrome b5 reductase — has been proved to be a component of the . . .

A case of congenital enzymopenic methemoglobinemia associated with severe mental retardation is described. The deficiency of cytochrome b5 reductase activity in the erythrocytes and the leukocytes of the propositus is demonstrated by kinetic measurement and by disc gel electrophoresis. Analysis of cultured amniotic fluid cells during a second pregnancy of the mother revealed an almost complete deficiency of the enzyme. The absence of cytochrome b5 reductase activity in the blood from the aborted fetus confirmed the prenatal diagnosis. The data presented support the opinion that in pregnancies at risk for the severe form of congenital enzymopenic methemoglobinemia, prenatal diagnosis is warranted.

A sensitive, precise enzymic/spectrophotometric method for determining cytochrome b5 in small amounts of blood is described. Mean values for healthy individuals, ages 20 to 70 years, were 0.26 (SD 0.03) mumol per liter of erythrocytes or 0.87 (SD 0.14) nmol per gram of hemoglobin. We believe the assay is preferable to methods described hitherto, primarily because of its high sensitivity.

Replicative activity of isolated chromatin from late passage cultured mouse cells has been compared to the activities of chromatin preparaions from dividing and quiescent early passage cells. Rates of endogenous DNA synthesis are similar for chromatin from growing or resting cells but this activity is stimulated 2.5-fold in senescent cell chromatin. Chromatin from growing young cells copies exogenously added single stranded DNA at the highest efficiency. Chromatin of senescent cells copies this template at a lower rate and resting young cell chromatin replicates single stranded DNA at the lowest efficiency. Similar relative rates are obtained when activated DNA is copied by the various chromatin preparations. Total activity of DNA polymerase extracted by salt from chromatin is similar for dividing and quiescent young cells but the proportion of DNA polymerase beta is higher in the latter. Elevated activities of DNA polymerases are extracted from chromatin of old cells. It is concluded, therefore, that chromatin-directed replication is differently arrested in non-dividing senescent cells and in quiescent early passage cells. The possible regulatory mechanisms of DNA replication in quiescence and aging are discussed.

FEBS LettersVolume 99, Issue 2 p. 265-269 Full-length articleFree Access Nuclear protein from cultured hepatoma cells preferentially inhibits copying of denatured DNA by isolated chromatin A. Kaftory, A. Kaftory Unit of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, IsraelSearch for more papers by this authorP. Weisman-Shomer, P. Weisman-Shomer Unit of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, IsraelSearch for more papers by this authorM. Fry, M. Fry Unit of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, IsraelSearch for more papers by this author A. Kaftory, A. Kaftory Unit of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, IsraelSearch for more papers by this authorP. Weisman-Shomer, P. Weisman-Shomer Unit of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, IsraelSearch for more papers by this authorM. Fry, M. Fry Unit of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, IsraelSearch for more papers by this author First published: March 15, 1979 https://doi.org/10.1016/0014-5793(79)80969-2Citations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume99, Issue2March 15, 1979Pages 265-269 ReferencesRelatedInformation

Total proteins of replicating and stationary phase early and late passage cultured chick fibroblasts have been labeled in vitro with radioactive isotopes of iodine. Protein preparations labeled with 125I and 131I were mixed together and the DNA binding proteins (DBP) isolated by chromatography on single stranded DNA covalently linked to Sepharose. Proportions of the various DBP were measured after electrophoresis of the double labeled DBP preparations on sodium dodecyl sulfate-polyacrylamide gels. Three major DBP were present at different proportions in actively dividing or resting early and late passage cells. The relative amounts of DBP of 92,000 and 175,000 daltons were identical in exponentially growing young and senescent fibroblasts. Both cultures were also similarly devoid of a 63,000 dalton protein. Comparison of the distribution of DBP in replicating and highly dense, stationary young cells revealed that the relative amounts of the species of molecular weights 92,000 and 175,000 were significantly reduced in resting cells whereas the proportion of the 63,000 dalton species was greatly increased in these cultures. By clear contrast, the DBP profile of resting, senescent cells which cease to divide at relatively low densities, was indistinguisable from that of actively dividing young or senescent fibroblasts. Thus, while arrest of growth of young cells is accompanied by distinct change in the proportions of several DBP, cessation of proliferation of old cultures does not involve such change.

The over-all rates of protein synthesis, degradation and net accumulation were estimated in rapidly growing young and slowly doubling old cultures of chick fibroblasts. We find that not only the rate of protein synthesis is reduced in senescent cultures, but the average rate of protein degradation is also slowed down considerably. This decrease in the rate of protein breakdown in aging cells stands in contrast with the previously observed acceleration of this process by other conditions (such as serum deprivation or overcrowding) that lead to the cessation of cellular growth. Though the retarded protein degradation may contribute to the acculation of abnormal proteins in senescent cells we find that the breakdown of grossly abnormal puromycin peptides proceeds equally rapidly in young and old cultures. The protein content of senescent cells increases by 1.8-fold as compared to young cells, while the average cell volume is increased even more (almost 5-fold). By contrast, consideration of the over-all balance of protein metabolism in these cells indicates that the average concentration of metabolically turning-over proteins is somewhat higher in senescent than in young fibroblasts.

Chromatin prepared from S phase hepatoma tissue culture (HTC) cell incorporates in vitro about 11-14 pmoles [3H]dTMP into DNA in 30 min. Single-stranded DNA added to this chromatin stimulates DNA synthesis more than 40-fold whereas activated DNA enhances it about 60-fold. By contrast, stimulation of DNA synthesis by activated DNA in a crude nuclear extract exceeds the stimulation exerted by denatured DNA by a factor of 7. Stimulation of DNA synthesis by denatured DNA is not due to stabilization of either the chromatin or the product of the endogenous reaction. On the other hand, we find that poly(dC) and poly (dT) enhance DNA synthesis by serving as templates which are copied by chromatin in a true complementary fashion. It seems therefore, that eukaryotic cell chromatin is able to copy single-stranded DNA at a high efficiency. Chromatin of G1 arrested cell copies exogenous templates at a considerably reduced rate. The enzyme responsible for the copying of denatured DNA is tentatively identified as DNA polymerase alpha on the basis of its sensitivity to sulfhydril group blocking, its requirements for ions and failure to copy the ribo strand of oligo(dT) poly(A).