Differences in chromatin proteins of resting and growing human lymphocytes.

Two-dimensional polyacrylamide gel electrophoresis comparisons were made for the non-histone “Chromatin fraction II” proteins of normal, phytohemagglutinin-stimulated and acute leukemic lymphocytes. The “Chromatin fraction II” proteins were extracted from the nuclear residue fraction after initial treatment with (a) 0.075 M NaCl containing 0.025 M EDTA, pH 8; (b) 0.01 M Tris-HCl, pH 8; and (c) 0.4 N H₂SO₄. Most of the proteins found earlier in the “Chromatin fraction II” of rodent liver and hepatomas were also found in the human cells. Some changes such as the decrease in amount of protein BA of normal rodent cells were found in the comparison of normal and stimulated human cells. By comparison with normal lymphocytes, the phytohemagglutinin-treated cells had decreased spot densities and sizes for proteins BA and Bv and an increase in densities and sizes of proteins CB, C25, CS and CT. In the acute lymphocytic leukemic cells there was a decrease in spots A24, BA, Bv, CD and CD′ by comparison with the normal lymphocytes. Protein CG′ which was found earlier in the hepatomas was found in acute lymphocytic leukemic cells but not in the control or phytohemagglutinin-treated cells. These studies show that there is a loss in specific Chromatin proteins BA and Bv from the Chromatin of rapidly turning over cells. Concomitantly, increases are observed for the amounts of protein spots CB, C25, CS and CT in the actively growing cell samples.     

Inhibitor of pyrimidine metabolism from tumor tissues

Inhibitors of normal rat liver 5′-nucleotidase and dUMP kinase $\text{in vitro}$ were found in rapidly proliferating tissues, such as Yoshida sarcoma. Two inhibitors were separated from Yoshida sarcoma by zone electrophoresis, gel filtration on Sephadex G-200 and DEAE-cellulose column chromatography. One inhibited both 5′-nucleotidase and dUMP kinase, while the other inhibited only dUMP kinase. These inhibitors were not detectable in normal rat liver. They were induced in regenerating rat liver and present in rapidly proliferating tissues, such as Yoshida sarcoma and Ehrlich ascites tumor and rat marrow cells. These inhibitors were heat labile. One had a large molecular weight (500,000>) and the other a small molecular weight ($\text{Ca}\text{.}$ 50,000).     

Nucleolar localization of protein B23 (375.1) by immunocytochemical techniques

Highly acidic phosphoprotein B23 ($\text{37}\text{5.1}\text{;}\text{M.W. x}\text{10}{}^3\text{/}\text{pI}$) which is in preribosomal RNP particles in nucleoli of Novikoff hepatoma cells (1) was found to be one of the two major silver staining nucleolar proteins (2). An improved isolation method was developed for protein B23 which included 4 M urea/3 M LiCl extraction of nucleoli, dialysis of the extract against 4 M urea/20 mM Tris-malate/pH 5.5 and DEAE-cellulose chromatography. For studies on cellular localization of this protein, highly purified protein B23 was used to produce anti- B23 antibodies in rabbits. The specificity of the anti- B23 antibodies was demonstrated by formation of immunoprecipitin bands with the purified antigen and crude nucleolar extracts from Novikoff hepatoma cells. With the indirect peroxidase immunostaining method, a specific localization of protein B23 was demonstrated in the nucleoli of normal rat liver, thioacetamide-treated rat liver and Novikoff hepatoma cells.     

Carnitine synthesis in rat tissue slices

The ability of rat liver, kidney, muscle, heart and testis tissue to carry out the $\text{in vitro}$ synthesis of carnitine via ε-N-trimethyllysine and γ-butyrobetaine was studied. All tissues formed γ-butyrobetaine from trimethyllysine, but liver and testis also formed carnitine in about 7% and 1% yield respectively. Liver slices formed trimethyllysine from lysine in about 6% yield. These $\text{in vitro}$ studies thus establish that liver has all the enzymes of the carnitine biosynthetic pathway. This tissue appears to be the primary site of carnitine synthesis in the rat as implied from whole animal studies in this and other laboratories.     

S-Adenosylmethionine synthetase isozyme patterns from rat hepatoma induced by N-2-fluorenylacetamide

Isozyme patterns of $\text{S}$-adenosylmethionine synthetase have been measured with and without dimethylsulfoxide in hepatoma of rats induced by $\text{N}$-2-fluorenylacetamide. The isozymes of α- and β-types existing in normal rat liver gradually decreased with the progress of hepatocarcinoma, and the kidney type γ-enzyme appears along with disappearance of both α- and β-enzymes. The liver from rat fetus contains a greater part of γ-type enzyme.     

Stringent control of glycolysis in Escherichia coli.

When $\text{Escherichia}\text{coli}\text{CP78}\text{(}\text{rel}{}^{\mathrm{+}}$) growing on glucose was starved for isoleucine by the addition of valine, the intracellular levels of fructose 6-phosphate, fructose 1,6-bisphosphate and dihydroxyacetone phosphate were abruptly decreased to one-half, but those of glucose 6-phosphate and ATP remained constant. In contrast, this was not the case with CP79($\text{rel}{}^{\mathrm{?}}$). Chloramphenicol released the response observed in CP78. These results suggest that the glycolytic activity is also under the stringent control. Since only glucosephosphate isomerase[EC 5.3.1.9] was significantly inhibited by guanosine 5′-diphosphate 3′-diphosphate among several glycolytic enzymes tested, the enzyme might be responsible for the decrease observed in CP78.     

Partial purification and characterization of citrate synthase from Dictyostelium discoideum.

The effect of thyroidectomy on oxidative metabolism of rat liver, kidney, and brain mitochondria has been examined. The respiration in liver, kidney, and brain mitochondria was affected differentially after thyroidectomy, the common effect in all the tissues being the impairment in state 3 as well as state 4 rates of succinate oxidation. Thyroidectomy did not have any effect on $\text{ADP}\text{O}$ ratios; however, compared to normal, respiratory control indexes were, in general, somewhat higher. Thyroidectomy also did not alter total ATPase activity of liver, kidney, and brain mitochondria, although the basal ATPase activity had decreased significantly under these conditions. The cytochrome content of the mitochondria also showed tissue-specific changes after thyroidectomy; however, no significant changes in the absorption characteristics of the cytochromes were seen. The succinate and glutamate dehydrogenase activities of mitochondria from liver, kidney, and brain were not affected by thyroidectomy, thereby ruling out the possibility that the decrease in substrate oxidation may be due to alterations in the primary dehydrogenase levels. It is concluded that thyroid hormone(s) may have a tissue-specific role in regulating the metabolic functions of mitochondria.     

Effect of methylglyoxal bis (guanylhydrazone) (MGBG) in vivo on the decarboxylation of s-adenosylmethionine and synthesis of spermidine in the rat and guinea pig.

The rates of decarboxylation of $\text{S}$-adenosylmethionine and synthesis of spermidine have been measured in extracts of liver, kidney and brain of the rat and guinea pig after intraperitoneal injection of MGBG, both before and after dialysis. The rate of decarboxylation of $\text{S}$-adenosylmethionine paralleled that of spermidine synthesis in all of the tissues investigated, even when spermidine synthesis was measured using preformed decarboxylated $\text{S}$-adenosylmethionine as substrate instead of $\text{S}$-adenosylmethionine itself. MGBG inhibited CO₂ production and spermidine synthesis to a similar extent in extracts of liver and kidney of both the rat and the guinea pig. After dialysis, a similar increase in both CO₂ production and spermidine synthesis was noted in these extracts. No effects on CO₂ production or spermidine synthesis were noted on extracts of brain of the rat or guinea pig, either before or after dialysis. When MGBG was injected intracisternally, CO₂ production and spermidine synthesis by extracts of brain were inhibited to the same extent, and after dialysis a similar increase in CO₂ production and spermidine synthesis was observed. These results indicate that the effects of MGBG are essentially the same in brain as they are in liver and kidney, and the MGBG injected intraperitoneally does not pass into the brain.     

In vivo lipogenesis and enzyme levels in adipose and liver tissues from pair-fed genetically obese and lean rats

Genetically obese Zucker rats pair-fed to lean controls were similar in body weight and food intake, however, epididymal fat pads were considerably larger than lean controls. $\text{In}$$\text{vivo}$ incorporation of acetate-¹⁴C into adipose tissue lipid was not significantly different, however, $\text{in}$$\text{vivo}$ liver lipogenesis was elevated in the obese rat. Characterization of enzyme profiles in both liver and adipose tissues revealed that enzymes normally associated with lipogenesis were elevated in liver tissue from obese rats. Malic enzyme and citrate cleavage enzyme were both depressed in adipose tissue of obese animals. From these data, it appears that the liver may be prominently involved in the development of excessive blood lipid and enlarged fat cells in the Zucker obese rat.     

[3H]cyclo(Histidyl-Proline) in rat tissues: Distribution,clearance and binding

Cyclo(Histidyl-Proline), a metabolite of TRH, has been demonstrated to have a number of biological activities. The clearance, distribution and binding of the peptide in the rat was studied. Cyclo(His-Pro) was cleared from the circulation biphasically ($\text{tl}\text{2}\text{= 1.25 and 33 min}$). Unmetabolized cyclo(His-Pro) appeared rapidly in urine. Accumulation of [³H]cyclo(His-Pro) in adrenal, liver and kidney was demonstrated. Membrane preparations from adrenal and liver, but not from kidney, brain, pituitary, and other tissues were shown to bind cyclo(His-Pro) specifically.     

Acidic proteins from Saccharomyces cerevisiae ribosomes.

Two dimensional gel electrophoresis of ribosomal proteins from $\text{Saccharomyces}\text{cerevisiae}$ reveals the presence of three spots in the region corresponding to proteins of high acidic character. Washing the ribosomes with 0.4 M NH₄Cl and 50% ethanol, followed by chromatography of the extracted proteins on DEAE-cellulose, indicated the presence of two fractions of acidic proteins; (A and Ax), having very similar molecular weights (12.000–13.000), but phosphorylated to different extents. Fractions A and Ax are immunologically distinct and their immunologic properties differ from acidic proteins found in $\text{Escherichia}\text{coli}$, rat liver, and $\text{Artemia}\text{salina}$ ribosomes.Protein A can be resolved into two bands by electrofocusing, and two dimensional gel electrophoresis. The two components correspond to proteins L44 and L45 according to the standard nomenclature. Proteins Ax seems to correspond to the spot that moves above and to the left of L44 and L45 and is at least three times more phosphorylated than these two proteins.     

Separate identities of ligandin and the h-protein,a major protein to which carcinogenic hydrocarbons are covalently bound

There is a protein moiety in the C3H mouse liver cytosol which gives a line of identity with rat liver ligandin one of three azo dye binding proteins of the liver using anti-rat ligandin. This mouse liver protein has been termed mouse ligandin and is not the $\text{h}$-protein, the major target protein in the mouse liver of methylcholanthrene and its metabolites. Mouse ligandin is identical to a minor methylcholanthrene binding protein species that was found previously to consist of basic proteins II and III. Both mouse ligandin and mouse $\text{h}$-protein contain glutathione S-transferase activity with different substrate specificitles.     

Denaturation of cytochrome P-450 by cyclophosphamide metabolites

Cyclophosphamide (CP) metabolites, acrolein and 4-hydroxy-CP, were found to denature rat liver microsomal cytochrome P-450, whereas another metabolite, phosphoramide mustard, CP $\text{per}\text{se}$ or its analog Ifosfamide had no effect. The denaturation produced by CP metabolites could be blocked by cysteine, suggesting an interaction between CP metabolite(s) and sulfhydryl groups in cysteine and probably in cytochrome P-450. These studies might explain the biochemical basis of the specific depression of various microsomal mixed function oxygenase activities produced by high doses of CP.     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

Cyclic adenosine 3':5'-monophosphate levels in normal and transformed cells of higher plants

Cyclic adenosine 3′:5′-monophosphate (cAMP) concentrations were determined for various normal and transformed (crown-gall) plant tissues grown in sterile culture. No significant differences in cAMP concentrations were found between normal and transformed cells of $\text{Vinca rosea, Helianthus annuus}$, and $\text{Nicotiana tabacum}$, unlike the suppressed synthesis observed in transformed cells of mammalian systems. cAMP concentrations of these tissues in culture averaged 135 nanomolar. No correlation was found between cAMP concentrations and tissue culture generation times.     

Phosphorylation of nuclear matrix proteins in isolated regenerating rat liver nuclei

The phosphorylation of nuclear matrix proteins from normal and regenerating rat liver nuclei was examined using an $\text{in vitro}$ system of isolated nuclei and γ-³²P-ATP. Phosphorylation of the nuclear matrix proteins was 2–3 fold higher than that of the total nuclear proteins in normal nuclei. The level of phosphorylation of the matrix proteins was enhanced an additional three fold at a period in liver regeneration (12 hours) just preceding the onset of DNA synthesis.     

In vitro effects of toxaphene on mitochondrial calcium ATPase and calcium uptake in selected rat tissues

$\text{In vitro}$ effects of toxaphene on Ca²⁺-ATPase activity and ⁴⁵Ca²⁺-uptake were studied in mitochondrial fractions of heart, kidney and liver tissues of rat. Mitochondrial fractions were prepared by the conventional centrifugation method. Ca²⁺-ATPase activity was determined by measuring the inorganic phosphate liberated during ATP hydrolysis. Toxaphene inhibited Ca²⁺-ATPase in a concentration dependent manner in all the three tissues. Substrate activation kinetics, with heart, kidney and liver tissue fractions, revealed that toxaphene inhibited Ca²⁺-ATPase activity non-competetively by decreasing the maximum velocity of the enzyme without affecting the enzyme-substrate affinity. Toxaphene also inhibited mitochondrial ⁴⁵Ca²⁺-uptake in the three selected tissues in a concentration dependent manner. These results indicate that toxaphene is an inhibitor of mitochondrial Ca²⁺-ATPase and calcium transport in heart, kidney and liver tissues of rat.     

The distribution of copper in neonatal mottled mutant mice after exposure to copper and penicillamine

Tissue copper levels of brindled ($\text{Mo}$^br) mice and normal litter-mates after single and repeated dosing with CuCl₂ and/or D-penicillamine are examined, together with a study of the cytosol distribution of copper after CuCl₂ treatment. The results confirm that the mutant mouse kidney is capable of extensive copper accumulation in association with the low MW copper-binding protein. Deficient tissues such as brain, heart and spleen are able to sequester sufficient of the exogenous copper to raise their levels to the normal control level, whereas mutant liver levels, even after copper treatment, remain below normal, indicating that the $\text{Mo}$ gene affects the ability of the liver to retain copper.     

Activation and detoxification of bromobenzene in extrahepatic tissues

Bromobenzene causes hepatic and extrahepatic toxicity in rats. Toxicity is related to the presence of covalently bound material in these tissues. A major bromobenzene metabolite, p-bromophenol, has been shown to give rise to covalently bound material in liver, lung and kidney $\text{in vivo}$, but is not toxic. p-Bromophenol is formed from bromobenzene in liver, lung and kidney microsomes and is subsequently metabolized to 4-bromocatechol and covalently bound material. Bromobenzene-3, 4-oxide generated $\text{in situ}$ by liver microsomes, is detoxified by kidney, liver and lung cytosol. The results suggest that the kidney toxicity caused by bromobenzene is probably not mediated by either bromobenzene-3, 4-oxide or the reactive metabolites of p-bromophenol. In contrast, bromobenzene-3, 4-oxide may play a role in the lung toxicity observed after bromobenzene administration. However, the covalently bound material found in extrahepatic tissues may be derived from both bromobenzene-3, 4-oxide or the reactive metabolites of p-bromophenol, which may be formed directly by these tissues or transported there from the liver.     

Evidence for ceruloplasmin as a copper transport protein.

Rats fed a copper-deficient diet for eight weeks showed a large decrease in cytochrome $\text{c}$ oxidase in heart, spleen, liver, lung, and pancreas but no significant change in kidney and brain. Three injections of human or rat ceruloplasmin over a five day period greatly increased cytochrome $\text{c}$ oxidase activity in spleen, liver, heart and lung. Rats receiving CuCl₂, Cu-histidine, and Cu-albumin produced a smaller and slower increase in cytochrome $\text{c}$ oxidase compared to ceruloplasmin treated animals. In Cu-histidine treated rats, the increase in enzyme activity did not occur until after the plasma ceruloplasmin level reached a maximal value. It is concluded that ceruloplasmin functions as a primary copper transport protein from which copper atoms are transferred to cytochrome $\text{c}$ oxidase and probably other copper containing proteins.