Growth hormone and liver mitochondria. Effects on cytochromes and some enzymes.

A reliable and reproducible assay was developed for measuring mitochondrial α-keto acid decarboxylase activity using ferricyanide as the electron acceptor. This method permitted the functional isolation and investigation of the decarboxylase step of the branched-chain α-keto acid dehydrogenases in rat liver mitochondria. Pyruvate and α-ketoglutarate decarboxylases are known to be separate and distinct enzymes from the branched-chain α-keto acid decarboxylases and were studied as controls. The relative specific activities of rat liver mitochondrial decarboxylases as measured by the ferricyanide assay showed that pyruvate and α-ketoglutarate were decarboxylated twice as rapidly as α-ketoisovalerate and four to ten times as fast as α-keto-β-methylvalerate and α-ketoisocaproate. The three branched-chain α-keto acids individually inhibit pyruvate and α-ketoglutarate decarboxylases. Inactivation of mitochondrial branched-chain α-keto acid decarboxylase activity by freezing and thawing and by prolonged storage resulted in a proportional decrease in decarboxylase activity toward each of the three branched-chain α-keto acids. However, hypophysectomy was found to increase decarboxylase activity with α-keto-β-methylvalerate to four times normal and with α-ketoisovalerate to three times normal, but the activity with α-ketoisocaproate was not changed. Hypophysectomy did not alter mitochondrial decarboxylase activity with pyruvate, α-ketoglutarate, or α-ketovalerate. The finding that hypophysectomy differentially alters the mitochondrial decarboxylase activity with the three branched-chain α-keto acids suggests either that there is more than one substrate-specific enzyme with branched-chain α-keto acid decarboxylase activity or that there is a modification of one enzyme such that the catalytic activity is selectively altered toward the three substrates.     

Caffeoylquinic Acids: Separation Method,Antiradical Properties and Cytotoxicity

Twelve chlorogenic acid derivatives and two flavones were isolated from Moquiniastrum floribundum (Asteraceae, other name: Gochnatia floribunda). Compounds were evaluated in relation to their cytotoxicity and antiradical properties. Cytotoxicity was not observed for compounds, however, chlorogenic acid derivatives showed antiradical activity and were more active than the Trolox standard. Quinic acid esterified with caffeoyl group at C‐4 position showed higher antiradical activity compared to acylation at C‐3 or C‐5 positions. Additional caffeoyl groups esterified in quinic acid increase the antiradical activity observed for 4‐caffeoylquinic acid. Excepted to 3,4‐dicaffeoylquinic acid methyl ester, methyl ester derivatives show higher capacity of trapping radicals than their respective acids. Consequently, the presence of caffeoyl group at C‐4 position of quinic acid is suggested as fundamental to obtain the highest antiradical activity.     

Effect of d-Limonene and related compounds on bile flow and biliary lipid composition in rats and dogs

d-Limonene enhanced bile flow in rats and dogs with a dose response correlation. The choleretic activity was much higher in the metabolites of d-limonene such as p-mentha-1,8-dien-10-ol, p-menth-1-ene-8,9-diol and p-mentha-1,8-dien-6-ol than d-limonene, and this suggested that the choleretic activity of d-limonene was attributable at least in part to its metabolites.The choleretic activities of esters of p-menth-1-ene-8,9-diol with acetic acid, propionic acid, stearic acid, palmitic acid, linoleic acid, benzoic acid, salicylic acid, α-naphthylacetic acid and nicotinic acid were also investigated in rats. Among these compounds, acetate, propionate and nicotinate possessed considerable, but lesser activities than the original diol. In dogs, however, the choleretic activity of p-menth-l-ene-8,9-diol acetate and propionate was much higher than that of original diol, suggesting that the choleretic activity of these esters is attributable to the esters themselves.d-Limonene decreased the ratio of biliary bile salts and phospholipids to cholesterol, whereas p-menth-l-ene-8,9-diol increased it.     

Antiapoptotic and proapoptotic action of various amino acids and analogs in starving MOLT-4 cells.

This study is based on our previous findings showing that certain amino acids may protect hybridoma cells against starvation-induced apoptosis. In the present work we have screened 44 amino acids and analogs for their capacity of modulating apoptosis in human T-lymphoblastic leukemia cell line MOLT-4 exposed to starvation in a nutrient-poor medium. The panel of tested substances was found to contain not only compounds with antiapoptotic activity (e.g., l-glutamine, l-histidine, glycine, l-proline, and l-2-aminopentanoic acid), but also compounds with proapoptotic activity (e.g., l-phenylalanine, l-tryptophan, l-arginine, and l-2-aminohexanoic acid). The apoptosis-modulating effects were dependent on fine details of the structure of the compounds. A switch from antiapoptotic activity to proapoptotic activity was found between 6-aminohexanoic acid and 7-aminoheptanoic acid, as well as between l-2-aminopentanoic acid and l-2-aminohexanoic acid. D-amino acids tested were without effect.     

Ascorbic acid specifically enhances dopamine beta-monooxygenase activity in resting and stimulated chromaffin cells

Ascorbic acid enhancement of norepinephrine formation from tyrosine in cultured bovine chromaffin cells was characterized in detail as a model system for determining ascorbate requirements. In resting cells, ascorbic acid increased dopamine beta-monooxygenase activity without changing tyrosine 3-monooxygenase activity. [14C]Norepinephrine specific activity was increased by ascorbic acid, while [14C]dopamine specific activity was unchanged. Dopamine content, dopamine biosynthesis, tyrosine content, and tyrosine uptake were also unaffected by ascorbic acid. Furthermore, increased norepinephrine formation could not be attributed to changes in norepinephrine catabolism. Enhancement of dopamine beta-monooxygenase activity was specific for ascorbic acid, since other reducing agents with higher redox potentials were unable to increase norepinephrine formation. The specific effect of ascorbic acid on enhancement of norepinephrine formation was also observed in chromaffin cells stimulated to secrete with carbachol, acetylcholine, veratridine, and potassium chloride. In stimulated cells with and without ascorbate, there were no differences in dopamine content, tyrosine uptake, dopamine specific activity, and norepinephrine catabolism. These data indicate that, under a wide variety of conditions, only one catecholamine biosynthetic enzyme activity, dopamine beta-monooxygenase, is specifically stimulated by ascorbic acid alone in cultured chromaffin cells. This model system exemplifies a new approach for determining ascorbic acid requirements in cells and animals.     

The effects of acetylsalicylic acid on phosphoenolpyruvate carboxykinase activity and acinar heterotopy in livers from juvenile and adult rats

Summary Male and female juvenile as well as adult rats were treated with acetylsalicilic acid in order to examine the effect of the drug on over-all activity and activity distribution of phosphoenolpyruvate carboxykinase (PEPCK) in the liver acinus. Upon administration of acetylsalicilic acid PEPCK activity increased in juvenile males and adult females, but was reduced in juvenile females and adult males. The periportal-perivenous activity gradient along the sinusoidal length, which is flatter in untreated juvenile rats compared to the livers of adult rats, was distinctly steepened by acetylsalicilic acid treatment. Acetylsalicilic acid did not affect the gradient in adult rats.     

The effects of acetylsalicylic acid on phosphoenolpyruvate carboxykinase activity and acinar heterotopy in livers from juvenile and adult rats

Male and female juvenile as well as adult rats were treated with acetylsalicylic acid in order to examine the effect of the drug on over-all activity and activity distribution of phosphoenolpyruvate carboxykinase (PEPCK) in the liver acinus. Upon administration of acetylsalicylic acid PEPCK activity increased in juvenile males and adult females, but was reduced in juvenile females and adult males. The periportal-perivenous activity gradient along the sinusoidal length, which is flatter in untreated juvenile rats compared to the livers of adult rats, was distinctly steepened by acetylsalicylic acid treatment. Acetylsalicylic acid did not affect the gradient in adult rats.     

12 alpha-hydroxylase activity in human liver and its relation to cholesterol 7 alpha-hydroxylase activity.

Interruption of the enterohepatic circulation by cholestyramine causes a several-fold increase in bile acid synthesis, reflected in a stimulation of cholesterol 7 alpha-hydroxylase activity; the synthesis of cholic acid being stimulated to a greater extent than chenodeoxycholic acid. It is not known if this preferential increase in cholic acid is due to an increase of the 12 alpha-hydroxylase activity. The present study aimed at investigating the 12 alpha-hydroxylase activity and its relation to cholesterol 7 alpha-hydroxylase activity in liver microsomes of patients with different levels of cholesterol 7 alpha-hydroxylase activity. Liver biopsies were obtained from four gallstone-free patients, and seven untreated and two cholestyramine-treated gallstone patients undergoing cholecystectomy, and four patients with Crohn's disease undergoing intestinal resection. The combined group of cholestyramine-treated and ileum-resected patients had four times higher cholesterol 7 alpha-hydroxylase activity and two times higher 12 alpha-hydroxylase activity than the other patients. A positive correlation was obtained between cholesterol 7 alpha-hydroxylase activity and 12 alpha-hydroxylase activity (r = +0.69; n = 16). These results indicate that the increased ratio between the synthesis of cholic acid and chenodeoxycholic acid during cholestyramine treatment is due to a compensatory increase of the 12 alpha-hydroxylase activity.     

Characteristics of acid lipase and acid cholesteryl esterase activity in parenchymal and non-parenchymal rat liver cells

(1) Parenchymal and non-parenchymal cells were isolated from rat liver. The characteristics of acid lipase activity with 4-methylumbelliferyl oleate as substrate and acid cholesteryl esterase activity with cholesteryl[1-14C]oleate as substrate were investigated. The substrates were incorporated in egg yolk lecithin vesicles and assays for total cell homogenates were developed, which were linear with the amount of protein and time. With 4-methylumbelliferyl oleate as substrate, both parenchymal and non-parechymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 2.5 times higher than for parenchymal cells. It is concluded that 4-methylumbelliferyl oleate hydrolysis is catalyzed by similar enzyme(s) in both cell types. (2) With cholesteryl[1-14C]oleate as substrate both parenchymal and non-parenchymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 11.4 times higher than for parenchymal cells. It is further shown that the cholesteryl ester hydrolysis in both cell types show different properties. (3) The high activity and high affinity of acid cholesteryl esterase from non-parenchymal cells for cholesterol oleate hydrolysis as compared to parenchymal cells indicate a relative specialization of non-parenchymal cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells possess the enzymic equipment to hydrolyze very efficiently internalized cholesterol esters, which supports the suggestion that these cell types are an important site for lipoprotein catabolism in liver.     

Effects of mitochondria and o-methoxybenzoylalanine on 3-hydroxyanthranilic acid dioxygenase activity and quinolinic acid synthesis

The use of o-methoxybenzoylalanine, a selective kynureninase inhibitor, has been proposed with the aim of reducing brain synthesis of quinolinic acid, an excitotoxic tryptophan metabolite. In liver homogenates, however, this compound caused unexpected accumulation of 3-hydroxyanthranilic acid, the product of kynureninase activity and the precursor of quinolinic acid. To explain this observation, we investigated the interaction(s) of o-methoxybenzoylalanine with 3-hydroxyanthranilic acid dioxygenase, the enzyme responsible for quinolinic acid formation. When the purified enzyme or partially purified cytosol preparations were used, o-methoxybenzoylalanine did not affect 3-hydroxyanthranilic acid dioxygenase activity. However, a significant reduction of this enzymatic activity did occur when o-methoxybenzoylalanine was tested in the presence of mitochondria. It is interesting that addition of purified mitochondria to 3-hydroxyanthranilic acid dioxygenase preparations reduced the enzymatic activity and the synthesis of quinolinic acid. In vivo, administration of o-methoxybenzoylalanine significantly reduced quinolinic acid synthesis and content in both blood and brain of mice. Our results suggest that mitochondrial protein(s) interact(s) with soluble 3-hydroxyanthranilic acid dioxygenase and cause(s) modifications in the enzyme resulting in a decrease in its activity. These modifications also allow the enzyme to interact with o-methoxybenzoylalanine, thus leading to a further reduction in quinolinic acid synthesis.     

Glycoprotein biosynthesis: Folic acid effects on glycoprotein:glycosyl transferase activities of rat kidney and liver

Folic acid at 14 μM to 1.4 mM increased the activity of the collagen:glc and fetuin:gal and decreased the activity of the fetuin:NANA glycoprotein:glycosyl transferases of rat liver and kidney in vitro; highest effects were found with 1.4 mM folic acid. 1.4 mM folic acid increased kidney fetuin:gal activity 5-fold and decreased fetuin:NANA activity 3-fold. At 1.4 mM, folinic acid and p-methylaminobenzoic acid were totally inactive toward the transferases, methasquin was moderately active, and homofolic, tetrahydrohomofolic and methotrexate were very active toward the transferases. In all instances, however, the fetuin:gal and collagen:glc transferases were activated while the fetuin: NANA transferase was inhibited. From the data presented, folic acid is viewed as a possible control molecule in the synthesis of glycoprotein.     

Induction and quantification of phenylacyl-CoA ligase enzyme activities in Pseudomonas putida CA-3 grown on aromatic carboxylic acids

Three phenylacyl-CoA ligase activities were detected in extracts of Pseudomonas putida CA-3 cells grown with a variety of aromatic carboxylic acids. The three phenylacyl-CoA enzyme activities measured were phenylpropyl-CoA ligase (acting on both phenylpropanoic acid and cinnamic acid), a phenylacetyl-CoA ligase, and a medium chain length phenylalkanoyl-CoA ligase acting on aromatic substrates with 5 or more carbons in the acyl moiety. The rate of each enzyme activity detected in extracts of P. putida CA-3 cells is dependent on the growth substrate supplied. High rates of phenylpropyl-CoA ligase activity were observed with extracts of cells grown on phenylpropanoic acid, cinnamic acid or medium chain length phenylalkanoic acids with an uneven number of carbons in the acyl moiety. Extracts of P. putida CA-3 cells exhibited high rates of phenylacetyl-CoA ligase activity when grown on phenylacetic acid or medium chain length phenylalkanoic acids with an even number of carbons in the acyl moiety. In addition, high rates of medium chain length phenylalkanoyl-CoA ligase activity, towards phenylvaleric acid and phenylhexanoic acid, were exhibited by extracts of cells grown on all medium chain length phenylalkanoic acids. Low levels of the various phenylacyl-CoA ligase activities were found in extracts of cells grown on benzoic acid and glucose. Benzoyl-CoA ligase activity was not detected in any cell free extracts generated in this study.     

Intracellular monosaccharide and amino acid concentrations and activities and the mechanisms of insulin action.

Current amino acid and monosaccharide transport models are based on an assumption which equates the intracellular chemical activity of a solute with its concentration. This assumption was tested for alpha-aminoisobutyric acid and 3-O-methylglucose in a giant cell, the amphibian oocyte, by using recently developed cryomicrodissection and internal reference phase techniques. We found the following. (i) alpha-Aminoisobutyric acid and 3-O-methylglucose activities were much greater in cytoplasm than was suggested by concentration data; i.e., activity coefficients were higher than in ordinary water solutions. This is attributable to the inaccessibility of considerable water as solvent (solute exclusion). (ii) Solute concentrations varied regionally as follows: nucleus > > animal cytoplasm > vegetal cytoplasm. Insulin increased the nucleus/cytoplasm concentration asymmetry, apparently by increasing cytoplasmic solute exclusion. (iii) Nuclear activity coefficients more closely resembled those of ordinary saline solutions so that nucleus/ extracellular concentration ratios reflected transmembrane activity gradients better than did cytoplasm (or whole cell)/extracellular ratios. (iv) Mediated passive alpha-aminoisobutyric acid and 3-O-methylglucose transport were constituent oocyte membrane properties. Membrane active transport was initiated with time (in the presence of substrate) and by insulin. (v) Increased temperature mimicked insulin in enhancing transmembrane alpha-aminoisobutyric acid activity gradients and increasing the nucleus/cytoplasm concentration asymmetry. These results indicated that concentration data are a misleading measure of cellular amino acid and monosaccharide activity; some consequences of this observation were explored. A model is proposed in which cell water has reduced solvent capacity or is compartmentalized (considered less likely) and is susceptible to physiological modulation. The model accounts for many observations in small cells, suggesting generality of the exclusion phenomenon and a previously unrecognized metabolic control mechanism.     

Studies on bone enzymes. The assay of acid hydrolases and other enzymes in bone tissue.

1. Nine acid hydrolases, cytochrome oxidase, alkaline phenylphosphatase and catalase were demonstrated in 0.25m-sucrose homogenates of newborn-rat calvaria. The acid hydrolases were: acid phenylphosphatase, acid beta-glycerophosphatase, beta-glucuronidase, beta-N-acetylglucosaminidase (beta-N-acetylaminodeoxyglucosidase), acid ribonuclease and acid deoxyribonuclease, showing optimum activity at about pH5; cathepsin, beta-galactosidase and hyaluronidase, with optimum activity at about pH3.6. 2. The main kinetic characters of these enzymes have been studied and methods for their quantitative assay have been worked out. The activities present in bone are given and compared with those found in liver. 3. Acid-phosphatase activity was assayed with phenyl phosphate and beta-glycerophosphate as substrates: activities with these two substrates appeared to be due to two different enzymes. Acid phenylphosphatase is particularly labile and is readily inactivated by various physical or chemical agents.     

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2mM sodium fluoride, whereas for osteoclasts 50–100mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.     

Regulation of phenylalanine ammonia-lyase activity and growth in lettuce by light and gibberellic acid

Abstract The effects of light and gibberellic acid (GA3) on growth and phenylalanine ammonia-lyase (PAL) activity were studied in seedlings of lettuce (Lactuca sativa L.). Using an in vivo assay for PAL it was shown that wounding caused by excising hypocotyls results in an increase in PAL activity with time that can mask the effect of light on the activity of this enzyme. When hypocotyl sections were excised from light-treated seedlings immediately prior to the in vivo assay of PAL, light was shown to cause a marked increase in PAL activity. Experiments with an inhibitor of PAL activity, α-aminooxy-β-phenylpropionic acid (AOPP), confirmed that the volatile radioactive products measured in the in vivo assay resulted from the activity of PAL. Gibberellic acid suppresses the light-induced increase in PAL activity and there is an inverse relationship between GA₃-induced growth and the activity of PAL. Over a wide range of GA₃ concentrations, the activity of PAL is also inversely correlated with growth rate along the length of the hypocotyl section; the upper halves of sections elongate more rapidly and have lower levels of PAL than the lower halves. Despite the strong correlation between growth and PAL activity, experiments with AOPP and t-cinnamic acid show that it is unlikely that elongation is regulated directly by products of PAL activity.     

Activity and mRNA levels of enzymes involved in hepatic fatty acid synthesis and oxidation in mice fed conjugated linoleic acid

The effects of dietary conjugated linoleic acid (CLA) on the activity and mRNA levels of hepatic enzymes involved in fatty acid synthesis and oxidation were examined in mice. In the first experiment, male ICR and C57BL/6J mice were fed diets containing either a 1.5% fatty acid preparation rich in CLA or a preparation rich in linoleic acid. In the second experiment, male ICR mice were fed diets containing either 1.5% linoleic acid, palmitic acid or the CLA preparation. After 21 days, CLA relative to linoleic acid greatly decreased white adipose tissue mass but caused hepatomegaly accompanying an approximate 10-fold increase in the tissue triacylglycerol content irrespective of mouse strain. CLA compared to linoleic acid greatly increased the activity and mRNA levels of various lipogenic enzymes in both experiments. Moreover, CLA increased the mRNA expression of Delta6- and Delta5-desaturases, and sterol regulatory element binding protein-1 (SREBP-1). The mitochondrial and peroxisomal palmitoyl-CoA oxidation rate was about 2.5-fold higher in mice fed CLA than in those fed linoleic acid in both experiments. The increase was associated with the up-regulation of the activity and mRNA expression of various fatty acid oxidation enzymes. The palmitic acid diet compared to the linoleic acid diet was rather ineffective in modulating the hepatic lipid levels or activity and mRNA levels of enzymes in fatty acid metabolism. It is apparent that dietary CLA concomitantly increases the activity and mRNA levels of enzymes involved in fatty acid synthesis and oxidation, and desaturation of polyunsaturated fatty acid in the mouse liver. Both the activation of peroxisomal proliferator alpha and up-regulation of SREBP-1 may be responsible for this.     

Hyaluronic acid is an endogenous inducer of interleukin-1 production by human monocytes and rabbit macrophages

When human peripheral monocytes and rabbit peritoneal macrophages were incubated with hyaluronic acid, the media were found to contain interleukin-1 (IL-1) activity and to stimulate collagenase production by rabbit fibroblasts. A digestion of hyaluronic acid by testicular hyaluronidase decreased the IL-1 inducing activity. Polymixin B, an inhibitor of endotoxin, did not exert any effect towards the action of hyaluronic acid. Hyaluronic acid also stimulated human polymorphonuclear leucocytes to produce IL-1 like activity. These results indicate that hyaluronic acid is an endogenous IL-1 inducer and may play important roles in the pathological and/or physiological changes of connective tissues.     

Synthesis and dual biological effects of hydroxycinnamoyl phenylalanyl/prolyl hydroxamic acid derivatives as tyrosinase inhibitor and antioxidant

We previously reported that caffeoyl-amino acidyl-hydroxamic acid (CA-Xaa-NHOH) acted as both a good antioxidant and tyrosinase inhibitor, in particular when caffeic acid was conjugated with proline or amino acids having aromatic ring like phenylalanine. Here, various hydroxycinnamic acid (HCA) derivatives were further conjugated with phenylalanyl hydroxamic acid and prolyl hydroxamic acid (HCA-Phe-NHOH and HCA-Pro-NHOH) to study the structure and activity relationship as both antioxidants and tyrosinase inhibitors. When their biological activities were evaluated, all HCA-Phe-NHOH and HCA-Pro-NHOH exhibited enhanced antioxidant activity compared to HCA alone. Moreover, derivatives of caffeic acid, ferulic acid, and sinapic acid inhibited lipid peroxidation more efficiently than vitamin E analogue (Trolox). In addition, derivatives of caffeic acid and sinapic acid efficiently inhibited tyrosinase activity and reduced melanin content in melanocytes Mel-Ab cell.