Increased concentration of CTP synthetase in hepatoma 3924A: immunological evidence

CTP synthetase (UTP:L-glutamine ligase, EC 6.3.4.2) was purified 370-fold from rapidly growing rat hepatoma 3924A. A major band was demonstrated by acrylamide gel electrophoresis which corresponded to this enzymic activity. It was estimated that the enzyme was 90% pure. Antibodies were produced in rabbit using this purified hepatoma enzyme. The specificity of the anti-serum was proved by the absence of the reaction between control serum and CTP synthetase. The amount of anti-serum required to inactivate completely the cytosolic CTP synthetase of hepatoma 3924A was 11-fold of that required for normal liver which is in good agreement with the 11-fold increase in CTP synthetase activity in this hepatoma. These results demonstrate that the liver and hepatoma 3924A CTP synthetases were immunologically similar or identical and that the markedly increased enzymic activity in hepatoma 3924A reflected an increase in the enzyme protein amount. These studies provide further evidence that in the neoplastic transformation a reprogramming of gene expression takes place which is manifested in the emergence of increased concentrations of CTP synthetase which should provide selective advantages to cancer cells by increasing the capacity for this rate-limiting step in $\text{de novo}$ CTP biosynthesis.     

Effect of glucocorticoids on the production of lactate dehydrogenase, malate dehydrogenase and alpha-fetoprotein by Morris hepatoma 7777 in vitro.

Non-AFP-producing Morris hepatoma 7777 were treated with glucocorticoids in order to compare the responses for AFP production and for lactate and malate dehydrogenases. Steroid hormone treatment did not affect the production of AFP. However, there was an approximate tripling of levels of both LDH and MDH (cytosolic plus mitochondrial).     

Mammary glucose 6-phosphate dehydrogenase. Molecular weight studies

Glucose 6-phosphate dehydrogenase was isolated from lactating rat mammary glands by a procedure extended and modified from one previously described. The sedimentation coefficient, S_20,W, was 10.3 in 0.01 m potassium phosphate, pH 6.9, containing 0.1 m NaCl at three protein concentrations between 0.51 and 1.45 mg/ml. The partial specific volume, v?, was 0.735 ml/g as determined by equilibrium sedimentation centrifugation in H₂O and D₂O containing buffers at pH(D) 6.5 containing 0.01 m potassium phosphate and 0.1 m NaCl. In the same buffer, but with 2.0 m NaCl, the apparent partial specific volume, φ′, was 0.756 ml/g. Equilibrium sedimentation of the enzyme at an initial concentration of 0.8 mg/ml was performed in 0.01 m potassium phosphate, pH 6.5, containing 1.0 mm EDTA, 7.0 mm mercaptoethanol, and various concentrations of NaCl between 0 and 2.0 m and with or without 0.1 mm NADP⁺. Weight-average and Z-average molecular weights were calculated and, from these values, the molecular weights of the monomer and dimer were derived. Under these conditions, the enzyme existed principally as a dimer, of molecular weight approximately 235,000, at low salt concentration, and as a monomer, of molecular weight approximately 120,000 in 1.0 m and 2.0 m NaCl. The subunit molecular weight was found to be 64,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Equilibrium sedimentation in 6 m guanidine hydrochloride gave a subunit molecular weight of 62,000 (assuming v? was unaltered) or 58,000 or 54,000 (assuming v? is decreased by 0.01 or 0.02, respectively, in 6 m guanidine). We conclude that rat mammary glucose 6-phosphate dehydrogenase has a molecular weight similar to that of glucose 6-phosphate dehydrogenases isolated from various other mammalian sources with the notable exception of human erythrocyte glucose 6-phosphate dehydrogenase which, like the microbial glucose 6-phosphate dehydrogenases thus far examined, has a significantly lower molecular weight.     

Biochemical studies of growing mouse oocytes: preparation of oocytes and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities.

Oocytes at several stages of growth were isolated by enzymatic digestion of ovaries from infant mice. These oocytes were free of follicle cells and were obtained in sufficient numbers to permit direct biochemical analysis of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) activities. Both enzymes increase in total activity as the oocyte grows. However, while the specific activity of G6PD remains constant up to an oocyte diameter of 80–85 μm, the specific activity of LDH increases four-fold. The specific activity of G6PD and especially LDH declines in oocytes over 80–85 μm in diameter, suggesting that the synthesis of these enzymes is almost completed at this stage of growth.     

Preparation of 1-acyl-2-succinyl glycero-3-phosphorylcholine and evidence against its involvement in succinate dehydrogenase action

1-Acyl-2-succinyl glycero-3-phosphorylcholine (GPC) was synthesized and its properties described. Although 1-acyl-2-succinyl GPC is a good substrate for succinate dehydrogenase, experiments on the incorporation of [2,3-¹⁴C]succinate into mitochondrial lipids gave no evidence to indicate that it is an intermediate in the enzymic oxidation of succinate to fumarate, as has been suggested earlier.     

Specific maltose labeling in the reducing glucose moiety.

Radioactive maltose with label in the reducing glucose moiety was prepared using a glucosyltransferase enzyme to catalyze exchange of [6-³H]glucose into unlabeled maltose. The enzyme was isolated from spinach by ammonium sulfate precipitation followed by DEAE column chromatography. A 77% yield of [6-³H]maltose was obtained after a reaction of 100 nmol of maltose with 0.0147 nmol of [6-³H]glucose was catalyzed by the most active column peak. The product was exclusively labeled in the reducing glucose moiety as indicated by the label occurring only in sorbitol following sodium borohydride reduction and sulfuric acid hydrolysis. Between 88.3 and 96.0% of the tritium in the synthesized preparation was present as [6-³H]maltose by Dowex 1-X4 chromatography. This column separates [6-³H]maltose-[U-¹⁴C]maltose mixtures and [6-³H]glucose-[U-¹⁴C]glucose mixtures apparently as a result of an isotope effect.     

Altered interaction between binding and catalytic subunits of a cyclic AMP-stimulated protein kinase from hepatoma cells.

Binding activity obtained from an established line of hepatoma tissue culture (HTC) cells has a lower apparent affinity for cyclic AMP at physiological pH than has the analogous binding activity from rat liver. However, the apparent binding affinity of HTC preparations can be reversibly increased by adding NaCl or guanidine · HCl. In the presence of such activating substances, a macromolecular inhibitory activity has been chromatographically separated from the cyclic AMP-binding activity. Removal of this inhibitory component causes the apparent affinity of the cyclic AMP-binding activity from HTC cells to increase and resemble that observed with liver preparations. Before treatment with salt, the inhibitory activity seems to be physically associated with the binding activity. Adding the isolated inhibitory component back to a suitably activated binding preparation from HTC cells results in a decrease in the apparent affinity for cyclic AMP. The isolated inhibitory component is devoid of cyclic AMP-binding and cyclic AMP phosphodiesterase activities and has an apparent minimal molecular weight of about 30,000 by gel filtration. It possesses protein kinase activity and seems to be identical to the catalytic subunit of a cyclic AMP-stimulated protein kinase on the basis of chromatographic properties and sensitivities to heat and low pH. This catalytic subunit represents only a minor portion of total cellular protein kinase activity and is also present in liver extracts. However, the binding activity from liver is not inhibited significantly under conditions where the binding from HTC cells is affected by the catalytic subunit. The difference in this inhibitory response between liver and HTC preparations appears to reflect differences in the cyclic AMP-binding proteins themselves.     

Questioning of reported evidence for guanosine tetraphosphate synthesis in a ribosome system from mouse embryos.

In 1974, Irr, Kaulenas and Unsworth reported that ppGpp is synthesized by cytosolic ribosomes from mouse embryos and proposed a role for ppGpp in the process of differentiation. This proposal is being challenged because ribosomes of mouse embryos from various stages of development and of mouse embryoid bodies were completely inactive in ppGpp formation.     

Increased glycosylation of glomerular basement membrane collagen in diabetes

Basement membrane was purified from glomeruli isolated from normal and streptozotocin-diabetic rats. After extraction of non-collagen protein with 8M urea, the extent of glycosylation in glomerular basement membrane collagen was determined with a specific colorimetric reaction that detects carbohydrate in ketoamine linkage with proteins. The level of glycosylation of glomerular basement membrane collagen purified from diabetic rats was significantly greater than that in non-diabetic animals. Increased basement membrane glycosylation may alter structure-function relationships of the capillary filtration barrier.     

Electrophoretic concentration of macromolecules

An apparatus for concentrating macromolecules and removing macro molecules from nonionic solutes electrophoretically has been developed. Typical applications and techniques are outlined. Concentrations from 24- to 50-fold were achieved in a short period of time. The nondisruptive nature and versatility of the system is shown by the concentration of bovine serum albumin and three different enzymes.     

Reversal of immunological tolerance by aclacinomycin through inhibition of suppressor cell activity

The elimination of suppressor cells by aclacinomycin, which could be the mechanism by which immune responses are enhanced after its administration, was studied in mice in which tolerance had been induced by the injection of high doses of sheep red blood cells (SRBC). We observed that tolerance could not be induced in aclacinomycin-treated mice, and that aclacinomycin inhibited the expression of tolerance to SRBC. This drug also diminished the capacity of spleen cells from SRBC-tolerant mice to inhibit the response of normal animals upon adoptive transfer, indicating that suppressor cells had been eliminated from the tolerant spleen cell population. The efficiency of the elimination of suppressor cells for DTH reactions appears greater than that of suppressor cells for plaque-forming cell responses.     

Chimeric drift in allophenic mice: Analysis of changes in red blood cell and white blood cell populations in C57BL/6 ↔ A mice

Fifteen allophenic mice of the type C57BL/6 ? A were quantitatively analyzed for changes in their peripheral white blood cell composition and hemoglobin composition with age. It was found that $\text{7}\text{15}$ or 47% of the mice showed significant changes, termed “chimeric drift,” in one or the other of these parameters. The seven mice showing chimeric drift were classified as unstable chimeras, as opposed to the eight apparently stable chimeras. Chimeric drift was observed in the direction of either parental type, or back and forth, and was found to be independent of the coat color, age, or sex of the mouse. There was an excellent correlation of peripheral white blood cell and hemoglobin compositions of the stable chimeras. However, the unstable chimeras often showed a marked discordance of these two markers.     

Evidence for a convalent intermediate between alpha-glucosidase and glucose

A stable enzyme-glucose intermediate has been obtained in the short-term reaction between α-methyl-

-glucosidase and α-methyl-

-¹⁴C-glucopyranoside. A rapid-flow technique was employed in which phenol was used to terminate the reaction and to trap the product. It is believed that a covalent linkage is involved because (a) continued washing of the denatured protein failed to remove the radioactivity and (b) the radioactivity was retained by a tryptic peptide isolated by gel filtration. Treatment of the labeled protein with 2 $\text{N}$ HCl at room temperature released over 80% of the radioactivity as a compound with the same chromatographic mobility as glucose. No radioactive product was formed when bovine serum albumin replaced the enzyme, nor when glucosylamine, a potent glucosidase inhibitor, was present with the enzyme.     

Subunit interactions in liver alcohol dehydrogenase: A partial alkylation study.

The transient kinetics of aldehyde reduction by NADH catalyzed by liver alcohol dehydrogenase consist of two kinetic processes. This biphasic rate behavior is consistent with a model in which one of the two identical subunits in the enzyme is inactive during the reaction at the adjacent protomer. Alternatively, enzyme heterogeneity could result in such biphasic behavior. We have prepared liver alcohol dehydrogenase containing a single major isozyme; and the transient kinetics of this purified enzyme are biphasic.Addition of two [¹⁴C]carboxymethyl groups per dimer to the two “reactive” sulfhydryl groups (Cys46) yields enzyme which is catalytically inactive toward alcohol oxidation. Alkylated enzyme, as initially isolated by gel filtration chromatography at pH 7·5, forms an NAD⁺-pyrazole complex. However, the ability to bind NAD⁺-pyrazole is rapidly lost in pH 8·75 buffer; therefore, our alkylated preparations, as isolated by chromatography at pH 8·75, are inactive toward NAD⁺-pyrazole complex formation. We have prepared partially inactivated enzyme by allowing iodoacetic acid to react with liver alcohol dehydrogenase until 50% of the NAD⁺-pyrazole binding capacity remains; under these reaction conditions one [¹⁴C]carboxymethyl group is added per dimer. This partially alkylated enzyme preparation is isolated by gel filtration and has been aged sufficiently to lose NAD⁺-pyrazole binding ability at alkylated subunits. When solutions of native liver alcohol dehydrogenase and partially alkylated liver alcohol dehydrogenase containing the same number of unmodified active sites are allowed to react with substrate under single turnover conditions, partially alkylated enzyme is only half as reactive as native enzyme. This indicates that some molecular species in partially alkylated liver alcohol dehydrogenase that react with pyrazole and NAD⁺ during the active site titration do not react with substrate. These data are consistent with a model in which a subunit adjacent to an alkylated protomer in the dimeric enzyme is inactive toward substrate. In addition, NAD⁺-pyrazole binding at the protomers adjacent to alkylated subunits is slowly lost so that 75% of the enzyme-NAD⁺-pyrazole binding capacity is lost in 50% alkylated enzyme. These data supply strong evidence for subunit interactions in liver alcohol dehydrogenase.Binding experiments performed on partially alkylated liver alcohol dehydrogenase indicate that coenzyme binding is normal at a subunit adjacent to an alkylated protomer even though active ternary complexes cannot be formed. One hypothesis consistent with these results is the unavailability of zinc for substrate binding at the active site in subunits adjacent to alkylated protomers in monoalkylated dimer.     

Mammalian cells with defective mitochondrial functions: a Chinese hamster mutant cell line lacking succinate dehydrogenase activity

A mutant cell line derived from Chinese hamster fibroblasts is described which is defective in oxidative energy metabolism. Glucose is continuously required in the medium. As a result of a block in the Krebs cycle, these cells are auxotrophs for carbon dioxide and asparagine. Several experiments support our conclusion that the mutant cells lack appreciable levels of succinate dehydorgenase activity. Other components of the electron transport chain appear to be fully functional, although there is the possibility that electron transport and oxidative phosphorylation are uncoupled.     

Individual differences in the maternal behavior of male mice: No evidence for a relationship to circulating testosterone levels

Maternal behavior toward newborn pups and endogenous levels of testosterone (T) in peripheral plasma were measured in individual adult male mice. Separate groups of animals that either retrieved, ignored, or killed pups were found not to differ with respect to plasma T levels, body weights, or relative weights of testes, seminal vesicles, and adrenals. Furthermore, animals do not exhibit changes in T following tactile or nontactile interactions with pups.     

A secondary attachment site for bacteriophage lambda in trpC of E. coli.

We have determined the nucleotide sequence of a secondary lambda attachment site in trpC. Direct sequence analysis of lambdatrp transducing phage DNA fragments carrying the two prophage attachment sites reveals a 6 nucleotide homology in the crossover region which is a subset of the 15 nucleotide core sequence in the primary lambda attachment site: GCTTTTTTATACTAA. This 6 nucleotide sequence is also present in the intact trpC genome at the attachment site, as shown by analysis of trpC mRNA spanning this region.     

The cytoplasmic distribution and characterization of poly(A)+RNA in oocytes and embryos of Drosophilia.

Using the presence of poly(A) tracts as a marker for mRNA, we have examined the distribution of this class of RNA between polysomes and free RNP particles. This has been done in mature oocytes and in embryos aged for various times from fertilization through to hatching of a larva. The proportion of ribosomes that are in polysomes to those that are not has been calculated. In mature oocytes, 58% of the poly(A)⁺ RNA and 72% of the ribosomes are not in polysomes. By 1 hr, this drops to 51% of the poly(A)⁺ RNA and 48% of the ribosomes. By 7 hr, a plateau is reached: 30% of each are not in polysomes. The poly(A)⁺ RNA in the cytoplasm of oocytes and 1-hr embryos is found in particles with an average size of 50S and a range of 30–70S. The poly(A)⁺ RNA ranges in size from 7 to 40S, with an average size of 22S. The polyA from this RNA is 50–200 nucleotides long with an average of 115 nucleotides. These data have allowed us to calculate that 1–2% of the total RNA is poly(A)⁺ RNA.     

A clonal analysis of tergite development in Drosophila of ultraabdominal and paradoxical genotypes.

The developmental parameters of homeotic second abdominal anlage cells in flies with Ultraabdominal and paradoxical genotypes are compared with those of normal second abdominal anlage cells through the use of induced mitotic recombination to mark the clonal descendants of single anlage cells. Homeotic and normal second abdominal anlage cells show the same pattern of mitotic activity during development. The homeotic second abdominal anlage cells with Ultraabdominal genotype proliferate to the same extent as normal anlage cells during hemitergite formation. However, the proliferation of homeotic second abdominal anlage cells with paradoxical genotype is decreased due to the failure of some daughter cells either to divide or to differentiate normally. The number of anlage cells in a homeotic second abdominal histoblast with Ultraabdominal genotype is slightly smaller and more variable than that in a normal second abdominal histoblast. The number of anlage cells in a homeotic second abdominal histoblast with paradoxical genotype is much smaller and much more variable than that in a normal second abdominal histoblast. These results are discussed in relation to mechanisms governing cell determination. In addition, some aspects of pattern formation in incomplete homeotic second abdominal hemitergites are presented and discussed.     

FSH receptors in isolated Sertoli cells: changes in concentration of binding sites at the onset of sexual maturation

Previous evidence has shown that isolated rat Sertoli cells have the capacity to metabolize C₁₉ and C₂₁ steroids and the steroidogenic activity is age-dependent and stimulated maximally by FSH in rats between 10 and 17 days of age. The purpose of the present study was to determine whether these age-related variations in sensitivity of Sertoli cells to FSH could be related to differences in FSH receptor concentrations. Sertoli cells were isolated at different ages from rats which had been irradiated in utero. Protein and DNA measurements of Sertoli cells from rats 6 to 65 days old indicated that protein content per Sertoli cell remained constant while DNA content progressively decreased up to 40 days of age. For quantitation of the FSH receptor, the 23,000 x g pellets of Sertoli cells were incubated with purified rat FSH which had been iodinated by the chloramine-T method. Sertoli cells isolated from rats 6, 10, 16 or 60 days of age, exhibited age-related differences in FSH binding activity: the concentration of FSH binding sites in Sertoli cells from 10 and 16 day old rats was significantly higher than in cells from 6 and 60 day old rats. This temporal pattern in FSH receptor concentration parallels the steroidogenic capacity and the FSH sensitivity of the Sertoli cells at the onset of sexual maturation.