Intracellular turnover of stable and labile soluble liver proteins

The cytosol proteins of mouse or rat liver were separated on the basis of charge and characterized with respect to molecular size, turnover in vivo, and several chemical properties. The basic proteins, which were synthesized and degraded slower than acidic proteins, were generally smaller, as multimers and subunits, than the acidic proteins. Charge and size, therefore, did not appear to be independent characteristics for soluble liver proteins. The amino acid composition of the smaller, basic, stable proteins was very similar to that of the larger, acidic, labile proteins. The charge differences between these two protein fractions could be attributed to the ratio of acidic to basic amino acid residues rather than to carbohydrate, nucleic acid, or phosphate content. The percentage of hydrophobic amino acid residues in the two protein fractions was the same. The acidic labile proteins were more vulnerable to proteolysis and associated with the lysosomal-mitochondrial fractions somewhat more extensively in vitro than the basic, stable proteins. These studies indicate that the information determining the half-lives of soluble enzymes is in the protein moiety of those enzymes and that the factors that correlate with turnover may not be independent variables for soluble mammalian proteins.     

Induction of synthesis of bovine adrenocortical cytochromes P-450scc, P-45011 beta, P-450C21, and adrenodoxin by prostaglandins E2 and F2 alpha and cholera toxin

To further elucidate the mechanisms by which ACTH (adrenocorticotropin) exerts its long-term action to maintain normal levels of adrenocortical cytochromes P-450 and related enzymes, the abilities of cholera toxin and prostaglandins E2 and F2 alpha to induce the synthesis of cytochromes P-450scc, P-45011 beta, and P-450C21 and adrenodoxin have been examined. These effectors stimulate the production of cyclic AMP and thus steroidogenesis in the adrenal cortex. Using bovine adrenocortical cells in primary monolayer culture, we have shown that treatment with cholera toxin results in increased synthesis of cytochromes P-450scc and P-45011 beta and adrenodoxin, similar to the effect observed upon ACTH treatment. Prostaglandins E2 and F2 alpha are less effective at inducing the synthesis of the mitochondrial cytochromes P-450, and do not seem to induce the synthesis of adrenodoxin. Furthermore, cholera toxin was found to be less effective at inducing the synthesis of microsomal cytochrome P-450C21 than ACTH, and no more effective than the prostaglandins. Thus, while it appears that elevation of cyclic AMP levels is a necessary step leading to increased synthesis of adrenocortical forms of cytochrome P-450, the detailed mechanism of this induction will be found to be different for each of the different enzymes.     

Disposition of polar and nonpolar residues on outer surfaces of transmembrane helical segments of proteins involved in proton translocation

"Helical wheel" projections of transmembrane helical segments of membrane proteins involved in proton translocation were constructed. The particular proteins studied were the uncF protein subunit of the Escherichia coli proton-ATPase, the uncE protein subunit of the E. coli proton-ATPase, and cytochrome oxidase subunit III. Clear demarcation of polar and nonpolar regions on surfaces of transmembrane helical segments was seen in the uncF protein and in uncE protein helical segment two, but not in uncE protein helical segment one. The transmembrane segment of cytochrome oxidase subunit III which includes the dicyclohexylcarbodiimide (DCCD)-reactive residue was very similar to E. coli uncE protein helical segment two. The DCCD-reactive residue in both was clearly located on a nonpolar surface.     

Phytohemagglutinin-lnduced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment

Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25? and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25? or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunoflu-orescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.     

Influence of dietary selenium on the mutagenic activity of perfusate and bile from rat liver, perfused with 1,1-dimethylhydrazine

The mutagenic effect of 1,1-dimethylhydrazine (UDMH) was studied in the liver perfusion/cell culture system. Male Wistar rats, fed a selenium-deficient diet with or without selenium supplementation in the drinking water, were used as liver donors. UDMH caused an increased mutation frequency in Chinese hamster V79 cells exposed in the perfusate. The effect was statistically significant with both selenium-deficient and selenium-supplemented livers. With selenium-deficient livers, a significant mutagenic effect was also obtained when V79 cells were treated with bile collected after the administration of UDMH. Bile flow and bile acid excretion were not affected by UDMH treatment of selenium-deficient or selenium-supplemented livers. There was a tendency towards reduced C-oxygenation of N,N-dimethylaniline in microsomes from selenium-deficient livers perfused with UDMH. The lactate/pyruvate ratio in the perfusate was increased by UDMH, the effect being more pronounced with selenium-deficient than selenium-supplemented livers.     

Dipeptide metabolism in the isolated perfused rat kidney

Renal handling of glycyl-proline was studied in the isolated perfused rat kidney. Glycyl-proline disappeared from the perfusate as a function of time. The dipeptide was freely filtered at the glomerulus but only 6% of the filtered load was excreted in the urine as the intact peptide. More than 90% of the filtered dipeptide was reabsorbed as the intact peptide and/or its hydrolytic products. Non-filtration mechanisms were also involved to a significant extent in the clearance of the peptide. Hydrolysis at intratubular, intracellular and peritubular sites all contribute to the disappearance of the dipeptide from the perfusate, though the relative contributions of each mechanism are not known. Significant metabolic conversions, especially the conversion of glycine to serine, were also observed during perfusion.     

31P nuclear magnetic resonance of metabolic changes associated with cyanide intoxication in the perfused rat liver.

Metabolic changes associated with cyanide intoxication were observed for the first time in perfused rat liver using ³¹P nuclear magnetic resonance (NMR) at 60.7 MHz. Well-oxygenated control livers showed strong ATP peaks and little discernable internal orthophosphate (Pi). Perfusion with 2 mM cyanide eliminated the observable ATP peaks and caused internal Pi to increase. Despite clear evidence for ATP hydrolysis, resonances from cytoplasmic ADP were conspicuously absent. Resumption of perfusion with cyanide-free buffer caused a dramatic return of the ATP peaks with a concomitant fall in internal Pi. These metabolic changes are consistent with reversible binding of cyanide to mitochondrial cytochromes and their observation by ³¹P NMR indicates the potential of this method for studying metabolism in whole, perfused rat liver under physiologic conditions.     

Enhanced degradation of cathepsin D synthesized in the presence of the threonine analog beta-hydroxynorvaline

The threonine analog beta-hydroxynorvaline is an inhibitor of asparagine-linked glycosylation. In the presence of the analog human fibroblasts synthesized cathepsin D molecules containing two, one, or no oligosaccharides. The nonglycosylated cathepsin D precursor was but a minor species and was degraded within 45 min of its synthesis, presumably in the lumen of the endoplasmic reticulum. The polypeptides with one or two oligosaccharides were normally segregated into lysosomes and their proteolytic maturation was not affected. The stability of mature glycosylated and nonglycosylated cathepsin D polypeptides within the lysosomes, however, was markedly decreased. The recovery of cathepsin D polypeptides was increased in the presence of inhibitors of cysteine and aspartyl-proteinases. These data suggest that the absence of carbohydrate side chains in cathepsin D results in an enhancement of the degradation rate of the precursor in the endoplasmic reticulum, and the replacement of threonine by beta-hydroxynorvaline in an enhanced degradation of the mature cathepsin D in lysosomes.     

Kinetic analysis of superoxide anion production by activated and resident murine peritoneal macrophages

Using a continuous spectrophotometric assay, we have monitored the formation of superoxide anion (O₂^?) by activated and resident murine peritoneal macrophages. Macrophages elicited by injection with Corynebacterium parvum, as well as resident macrophages from untreated mice, were kept in suspension culture overnight to eliminate short-lived, contaminating neutrophils. Cytochemical analysis of the cultured macrophages disclosed that essentially all of the activated macrophages reduced nitroblue tetrazolium (NBT) dye vigorously. In contrast, only 18% of the resident macrophages demonstrated vigorous NBT reduction; the remainder of the resident macrophages reduced NBT very weakly. Kinetic analysis of macrophage O₂^? formation revealed that activated macrophages exposed to phorbol myristate acetate (PMA) produced O₂^? at a 13-fold greater maximum rate than resident macrophages. The decline in the rate of O₂^? production with time by activated macrophages was also greater than that of resident macrophages. The data indicate that the greater O₂^? production by activated macrophage populations is due to (i) the presence of an increased percentage of macrophages that respond to PMA with vigorous O₂^? production, and (ii) an increased maximum rate of O₂^? formation by these macrophages.     

Rate control of phosphorylation-coupled respiration by rat liver mitochondria

Liver mitochondria provided with an oxidizable substrate, ATP, oxygen, and an ADP-generating system (soluble F₁-ATPase) were used to reevaluate the rate-controlling step(s) intrinsic to all of the processes of mitochondrial oxidative phosphorylation. The quantity termed “control strength” (C), previously defined as the fractional change in flux through a (system) induced by a fractional change in the concentration of an individual enzyme in the system, has been used to evaluate rate-influencing steps in this overall process by carefully defining the dimensions of the “system” under analysis. If the system is defined by a suspension of mitochondria provided with substrates, plus an extrinsic ADP-generating process (ATPase), the value of C of the latter for the overall process of phosphorylation-linked respiration is near 1.0 until the capacity of the mitochondria to phosphorylate ADP is approached, after which C for the soluble ATPase becomes zero as the maximum capacity for phosphorylation is attained. Carboxyatractyloside was found only marginally to inhibit respiration stimulated by ATPase, even when a large percentage of adenine nucleotide translocase molecules were immobilized. The relative lack of effect of carboxyatractyloside on phosphorylating respiration is explained by the readjustment of the concentration of one of the substrates (ADP) and an inhibitor (ATP), which results from inhibition of adenine nucleotide translocase. The residual blunted inhibition of respiration is explained by product inhibition of the ADP-regenerating ATPase, and not necessarily to any intrinsically mitochondrial intermediate process. The system being evaluated can be redefined to include only the processes intrinsic to mitochondria. This can be achieved by providing exactly comparable substrate concentrations to the mitochondria under comparable incubation conditions. Under these conditions, the adenine nucleotide translocase is the principal, if not the only, rate-controlling step in the overall process of oxidative phosphorylation until a new rate-limitation is attained (ATP synthesis). These data are consistent with the conclusion that, at intermediate rates of phosphorylation-coupled respiration, the extramitochondrial $\text{ATP}\text{ADP}$ ratio regulates this process through its kinetic effects on the catalytic properties of the adenine nucleotide translocase.     

Rapid preparation of a relatively stable, partially purified lipoprotein lipase from perfused rat heart

Rat hearts, extensively washed with cold 0.15 M NaCl solution, were perfused with 5 ml of 0.15 M NaCl containing 16 U of heparin and 10% glycerol to release endothelium-bound lipoprotein lipase. Approximately 100 mU of enzyme activity could be released from each heart (weighing about 1.7 g). Several hearts could be sequentially perfused with the same heparin solution to enrich it in lipase activity. When compared with other equally rapid and frequently used sources of rat lipoprotein lipase (such as heart acetone powder or postheparin plasma), our enzyme preparation had a much higher specific activity suggesting that a greater purification level had been already achieved in a single step. In addition, this lipoprotein lipase preparation contained only trace amounts of lipids, was stable for an hour at 37 degrees C and retained 75% of its activity after 10 days at 4 degrees C. The described procedure is a quick way to prepare a soluble, partially purified and relatively stable lipoprotein lipase that may be useful especially for the in vitro preparation of triacylglycerol-rich lipoprotein remnants.     

Phospholipid composition of lamellar bodies formed by fetal rabbit lung type II cells in organ culture

Lung tissue obtained from fetal rabbits of 23 days gestational age was maintained in organ culture to study the in vitro formation of lamellar body phospholipids. During the culture period, the epithelium of the prealveolar ducts of the explants differentiated to form type II pneumonocytes. After 8 days in culture, the explants were harvested, homogenized, and two lamellar body fractions were isolated by sucrose density gradient centrifugation. The lamellar body fraction which best retained the distinct multilamellar structure was recovered at the interface between a solution of buffer without sucrose and buffer containing 0.41 m sucrose. The phospholipid compositions of both lamellar body fractions were similar to those reported for lamellar bodies and surfactant isolated from fetal rabbit lung, with the exception of a slightly higher phosphatidylethanolamine content. The disaturated phosphatidylcholine content of the lamellar body fractions, expressed as a percentage of total lipid phosphorus, was not influenced by the presence of palmitate in the medium.     

Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster.

In situ hybridization of ¹²⁵I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ. This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead.     

Effect of acetaldehyde and cyanamide on the metabolism of formaldehyde by hepatocytes, mitochondria, and soluble supernatant from rat liver

Formaldehyde can be metabolized primarily by two different pathways, one involving oxidation by the low-Km mitochondrial aldehyde dehydrogenase, the other involving a specific, glutathione-dependent, formaldehyde dehydrogenase. To estimate the roles played by each enzyme in formaldehyde metabolism by rat hepatocytes, experiments with acetaldehyde and cyanamide, a potent inhibitor of the low-Km aldehyde dehydrogenase were carried out. The glutathione-dependent oxidation of formaldehyde by 100,000g rat liver supernatant fractions was not affected by either acetaldehyde or by cyanamide. By contrast, the uptake of formaldehyde by intact mitochondria was inhibited 75 to 90% by cyanamide. Acetaldehyde inhibited the uptake of formaldehyde by mitochondria in a competitive fashion. Formaldehyde was a weak inhibitor of the oxidation of acetaldehyde by mitochondria, suggesting that, relative to formaldehyde, acetaldehyde was a preferred substrate. In isolated hepatocytes, cyanamide, which inhibited the oxidation of acetaldehyde by 75 to 90%, produced only 30 to 50% inhibition of formaldehyde uptake by cells as well as of the production of 14CO2 and of formate from [14C]formaldehyde. The extent of inhibition by cyanamide was the same as that produced by acetaldehyde (30-40%). In the presence of cyanamide, acetaldehyde was no longer inhibitory, suggesting that acetaldehyde and cyanamide may act at the same site(s) and inhibit the same formaldehyde-oxidizing enzyme system. These results suggest that, in rat hepatocytes, formaldehyde is oxidized by cyanamide- and acetaldehyde-sensitive (low-Km aldehyde dehydrogenase) and insensitive (formaldehyde dehydrogenase) reactions, and that both enzymes appear to contribute about equally toward the overall metabolism of formaldehyde.     

Purification of rat liver monoamine oxidase by octyl glucoside extraction and reconstitution

Catalytically active isoenzymes of rat liver monoamine oxidase have been copurified from the outer mitochondrial membrane by a novel method involving repetitive solubilization with octyl-β-d-glucopyranoside followed by reconstitution into lipid vesicles. As analyzed using sodium dodecyl sulfate-gel electrophoresis, the purified enzyme migrates as a single band of protein of molecular weight 60,000. The preparation is capable of metabolizing 576 nmol serotonin and 777 nmol β-phenylethylamine/min/mg protein. Apparent K_m values and sensitivity to the inhibitor clorgyline are very similar for the purified and outer mitochondrial membrane-bound enzyme when determined with the substrates β-phenylethylamine, serotonin, and tyramine.     

Possible involvement of cyclooxygenase products in the actions of platelet-activating factor and of lipoxygenase products in the vascular effects of epinephrine in perfused rat liver

Platelet-activating factor (PAF) stimulates glycogenolysis and induces vasoconstriction in perfused rat liver. The effect of PAF was rapid but transient and it was blocked by indomethacin and bromophenacyl bromide which suggests a role of cyclooxygenase metabolites in its action. The homologous desensitization of glycogenolysis produced by PAF and the sensitivity of its actions to inhibitors of cyclooxygenase and phospholipase A2 markedly differentiate the mechanism of action of this agent with that of alpha 1-adrenergic agents, vasopressin or angiotensin II. No effect of PAF in isolated hepatocytes was observed which suggest that cells other than hepatocytes could be involved in its action in perfused liver. In addition nordihydroguaiaretic acid and bromophenacyl bromide abolished the vascular effect (but not the glycogenolysis) produced by epinephrine which suggest a role for lipoxygenase products in this effect.     

Studies on the pathway of methane formation from procarbazine,a 2-methylbenzylhydrazine derivative,by rat liver microsomes

The oxidative metabolism of procarbazine, its azo, hydrazone, and two azoxy derivatives, and methylhydrazine by hepatic microsomes from phenobarbital-pretreated rats was investigated to elucidate the pathway of metabolism that resulted in methane formation from procarbazine. When incubated with microsomal reaction mixtures fortified with NADPH, all of the compounds, except the azoxy isomers, were metabolized to yield methane. A lag phase in methane formation was noted for procarbazine, but not for the other compounds. Kinetic and inhibition studies utilizing methimazole and ethylhydrazine precluded methylhydrazine as an intermediate in methane formation from procarbazine. When the azo derivative was oxidatively metabolized in the presence of liver microsomes, no hydrazone tautomer was detected. Upon monitoring the production of the azo and hydrazone metabolites formed during microsomal metabolism of procarbazine, the azo derivative was formed in sufficient quantities to account for the majority of the methane produced. In addition, small amounts of hydrazone were also detected. It was concluded that both the azo and hydrazone metabolites of procarbazine contribute to methane formation from the terminal methyl group of the hydrazine with the azo derivative being the predominant source and the hydrazone derivative being a minor source of methane. Consideration of the chemical and enzymatic pathways of procarbazine oxidation and the implication of a methyl radical intermediate in methane formation are discussed.     

Mass-spectrometric study of the transesterification of O,N-bis(trifluoroacetyl) n-butyl esters of some hydroxylated amino acids into their O-carbethoxy, N-trifluoroacetyl, n-butyl ester derivatives during the N-carbethoxy derivatization of histidine

The transesterification of O-TFA, N-TFA, n-butyl ester derivatives of some hydroxylated amino acids was studied by gas—liquid chromatography and combined gas—liquid chromatography—mass spectrometry. Changes in elution patterns and fragmentation of the two different O-derivatives are discussed.     

Plasma catecholamines: effects of footshock level and hormonal modulators of memory storage

Plasma levels of norepinephrine (NE) and epinephrine (EPI) were measured in male Sprague-Dawley rats before and at several times after training injections of agents known to enhance or to impair later retention performance for a one-trial inhibitory (passive) avoidance task. Two days before testing, each animal was surgically prepared with a chronic tail artery catheter that allows for repeated blood sampling in unhandled rats. Exposure to a single, intense training footshock (3.0 mA, 2.0 sec duration) resulted in an immediate but transient increase in plasma levels of EPI and to a lesser extent NE. Plasma levels of both catecholamines did not differ between unshocked controls and animals that received a weak training footshock (0.6 mA, 0.5 sec duration). An injection of EPI at a dose that enhances retention performance (0.1 mg/kg, sc) resulted in increments in plasma EPI levels of 0.8-1.9 ng/ml from 5 to 40 min after injection. An injection of EPI (0.5 mg/kg, sc) at a dose that produces retrograde amnesia resulted in increments in plasma EPI ranging from 3.7 to 4.5 ng/ml during the 40 min after injection. Plasma NE levels were not significantly altered following an EPI injection. A single injection of adrenocorticotropin (ACTH, 0.3 or 3.0 IU per rat) did not alter the plasma catecholamine responses to training with a weak footshock. Similarly, the synthetic ACTH analog, Organon 2766 (125 or 250 mg/Kg) did not affect plasma catecholamines in untrained (unshocked) rats.These results demonstrate that significant increments in plasma levels of NE and EPI occur shortly after inhibitory avoidance training. Furthermore, an injection of EPI that enhances retention of an inhibitory avoidance task mimics the magnitude, though not the temporal characteristics, of the endogenous adrenal medullary response to a training footshock. Other hormonal treatments (ACTH and Organon 2766) which enhance memory storage do not affect plasma levels of NE and EPI.     

Measurement of a folate binding protein from rat liver cytosol by radioimmunoassay

A radioimmunoassay has been developed for the folate binding protein from rat liver cytosol with a molecular weight of 150,000 which was recently purified to homogeneity (Suzuki, N., and Wagner, C., 1980, Arch. Biochem. Biophys.199, 236–248). This method has indicated that the binding protein (FBP-CII) is found primarily in the liver. A significant amount of FBP-CII was also found in the kidney and much reduced levels in spleen, serum, brain, lung, and heart. No FBP-CII could be detected in small intestine, skeletal muscle, or testes. Small amounts of cross-reacting material were found in the livers of mouse, dog, chick, and humans. Levels of FBP-CII were not decreased in the livers of folate-deficient rats. Assays of rat fetal liver and kidney 2 days prior to birth showed much lower levels which increased rapidly at birth. These data are consistent with the FBP-CII fulfilling a role as a folate storage protein in rat liver.