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Biomedical subjects

A H Merrill

Publications and source records attributed to A H Merrill.

At least 127 records · Page 7Linked to original sources
PubMed1986

Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst.

The neutrophil oxidative burst is characterized by increased cellular O2 consumption due to the activation of a membrane-associated superoxide-generating NADPH-oxidase. The response is triggered by a variety of stimuli, including opsonized zymosan, formylmethionylleucinephenylalanine (FMLP), arachidonate, short-chain diacylglycerols, and phorbol myristate acetate (PMA). We herein demonstrate that incubation of cells with sphinganine or sphingosine blocks or reverses activation by these agonists. The inhibition is reversible, does not affect cell viability, and does not affect another complex cell function, phagocytosis. Inhibitory concentrations of sphinganine did not significantly affect cytoplasmic calcium levels or FMLP-generated calcium transients. Structural requirements for inhibition of the oxidative burst include a long aliphatic chain and an amino-containing head-group, and there is modest specificity for the native (erythro) isomer of sphinganine. Inhibition involves stimulus-induced activation mechanisms rather than a direct effect on the NADPH oxidase, since sphinganine did not inhibit NADPH-dependent superoxide generation in isolated membranes containing the active enzyme. Activation by FMLP, diacylglycerol, PMA, opsonized zymosan, and arachidonate was blocked by the same concentrations of sphinganine, indicating that these agonists share a common inhibited step. Three lines of evidence indicate that this step involves protein kinase C. First, in a micelle system and in platelets, long-chain bases are inhibitors of this enzyme (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). Second, sphinganine blocks PMA-stimulated incorporation of 32PO4 into neutrophil proteins. Third, sphinganine inhibits the binding of [3H]phorbol dibutyrate to its cellular receptor, known to be protein kinase C. We suggest that long-chain bases function as physiologic modulators of cellular regulatory pathways involving protein kinase C.

Arachidonic Acid↗
PubMed1986

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets.

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.

Amines↗
PubMed1986

Inhibition of phorbol ester-dependent differentiation of human promyelocytic leukemic (HL-60) cells by sphinganine and other long-chain bases.

The effects of long-chain (sphingoid) bases on the phorbol ester-dependent differentiation of HL-60 cells were investigated since these molecules are potent inhibitors of protein kinase C (Hannun, Y. A., Loomis, C. R., Merrill, A. H., Jr., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). After 24 h, low concentrations of sphinganine (1-5 microM blocked both cell adherence and the inhibition of growth in response to phorbol 12-myristate 13-acetate, as measured by cell number and acid phosphatase activity. Sphinganine and sphingosine decreased adherence by 50% at 1-3 microM; other long-chain bases were effective in parallel to their inhibition of protein kinase C. Sphinganine decreased the binding of [3H]phorbol dibutyrate by the phorbol receptor of HL-60 cells, protein kinase C, and inhibited the response of HL-60 cells to dioctanoylglycerol, a cell permeable activator of this enzyme. Long-chain base uptake by HL-60 cells was demonstrated with [3-3H]sphinganine and within 1-3 days much had been converted to ceramides. By day 3, most of the cells had recovered the ability to adhere and exhibited macrophage characteristics, whereas cells in suspension did not differentiate. The level of free sphinganine in HL-60 cells was determined to be 12.3 +/- 1.2 pmol/10(6) cells. These results establish that sphingoid bases inhibit protein kinase C in HL-60 cells and may function physiologically as negative effectors of this enzyme.

Acid Phosphatase↗
PubMed1986

Use of cytochrome P-450scc to measure cholesterol-lipid interactions.

The interaction of cholesterol with phospholipids has been studied with a variety of techniques; however, the possible consequences of such interactions in vivo have not been demonstrated. In this study, the cholesterol-dependent absorbance spectrum of cytochrome P-450scc was used to monitor cholesterol availability in both micellar and vesicular environments. By use of this approach, in conjunction with titration of putative cholesterol binding species, a tight, approximately equimolar complex of cholesterol and digitonin was demonstrated. Sphingomyelin (SM) (both the synthetic N-palmitoyl and bovine brain forms) gave sigmoidal titration curves, suggesting a cooperative interaction between this lipid and cholesterol. The interaction of bovine brain glycerolipids and cholesterol was weaker than that of SM and showed no cooperativity. The importance of the phospholipid head group in these interactions was established by the differences in the ability of synthetic 1-palmitoyl-2-oleoylphosphatidylcholine, -phosphatidylethanolamine, and -phosphatidylserine to affect cholesterol availability. Comparison of these results with those of the bovine brain phospholipids indicates that the acyl chain composition of these molecules is also important to these interactions. Titrations of SM in phospholipid vesicles containing cytochrome P-450scc and different types of phosphatidylcholine established that the SM-cholesterol interactions also occur in a bilayer membrane. This study demonstrates that the association of cholesterol with cytochrome P-450scc is inhibited by concentrations of SM commonly found in biological membranes. Therefore, such cholesterol-lipid interactions can potentially affect the function of membrane enzymes.

Adrenal Cortex↗
PubMed1986

Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s).

The de novo biosynthesis of sphinganine and sphingosine was studied using LM cells incubated with [14C] serine in serum-free media. Most of the radiolabeled long-chain bases were initially found in dihydroceramides (as sphinganine) and the proportion appearing in complex sphingolipids (as sphingosine) increased over time. Since free long-chain bases were not detected (although formation of 3-ketosphinganine, the first condensation product of serine and palmitoyl-CoA, could be demonstrated in vitro), it appears that the first step is rate-limiting for dihydroceramide biosynthesis. The kinetics suggested that after N-acyl-sphinganines were formed they were dehydrogenated to N-acylsphingosines. No evidence was found for the formation in vivo or in vitro of the putative intermediates of the direct biosynthesis of sphingosine from sphinganine (i.e. 3-ketosphingosine and free sphingosine). The conversion of N-acylsphinganines to N-acyl-sphingosines was confirmed by incubating cells with [14C] serine followed by unlabeled serine, which resulted in a rapid increase in the sphingosine-to-sphinganine ratio in amide-linked sphingolipids during the chase. These findings are most consistent with a pathway for long-chain base biosynthesis in which N-acyl-sphinganines are first synthesized by LM cells and the 4-trans-double bond is added to this or subsequent products, as opposed to the most cited pathway wherein sphingosine is made directly from sphinganine.

Acylation↗
PubMed1986

Differences in the long chain (sphingoid) base composition of sphingomyelin from rats bearing Morris hepatoma 7777.

The long chain bases of sphingomyelin from Morris hepatoma 7777 and host and control livers were analyzed by capillary gas liquid chromatography. Sphingosine (18:1) was the major long chain base of control livers (66.5%) and hepatomas (65.6%), but hepatomas also had a high percentage (9.3 vs 4.4) of the 16:1 homolog. Host liver had the most unusual long chain base composition, with ca. equal 16:1 (24.4%) and 18:1 (21.4%) and high amounts of 20-carbon bases (9.2% 20:0 and 15.3% 20:1). These differences may be related to the aberrant fatty acid metabolism known to occur in tumor-bearing animals. Such large perturbations in the long chain base composition of hepatic sphingomyelin are unprecedented and could have a major impact on the properties of host membranes.

Animals↗
PubMed1986

Activities of the hepatic enzymes of vitamin B6 metabolism for patients with cirrhosis.

Patients with cirrhosis and other hepatic diseases frequently exhibit lower concentrations of plasma pyridoxal 5'-phosphate (PLP), which is derived primarily from liver. To determine the biochemical basis for this abnormality, the enzymes of vitamin B6 metabolism--pyridoxal kinase, pyridoxine (pyridoxamine) 5'-phosphate oxidase, PLP phosphatase(s), and pyridoxal oxidase(s)--were analyzed in liver. The activities of the two biosynthetic enzymes, pyridoxal kinase and pyridoxine (pyridoxamine) 5'-phosphate oxidase were similar for both. The phosphatase activities were significantly higher (mean +/- SD of 9.55 +/- 8.03 versus 3.97 +/- 2.36 nmol X min X mg protein, p less than 0.05) for cirrhotics. Pyridoxal oxidase activities appeared slightly lower for cirrhotics. There was considerable variation in many indices of liver function, which suggests that the defects contributing to altered vitamin B6 metabolism may be complex and individualistic. These analyses have shown that cirrhotics are capable of apparently normal PLP synthesis and that increased hepatic dephosphorylation may be responsible for low levels of plasma PLP.

Adult↗
PubMed1986

Immunoglobulins associated with elevated riboflavin binding by plasma from cancer patients.

Plasma from 182 patients with different malignant diseases was tested for riboflavin binding by immunoglobulins, which have been recently identified as major carriers of this micronutrient. A wide range of binding (5.9 to 130 pmole/ml plasma) was observed, and significant elevations were found for patients having breast cancer (21.2 +/- 1.9, P less than 0.05) and melanoma (25.7 +/- 1.9, P less than 0.001) compared to controls (15.5 +/- 1.9). The proteins responsible for a majority of the higher binding were identified as immunoglobulins, based on their elution from gel filtration columns and the removal of 57-88% of the non-albumin binding by treating of plasma with Protein A-agarose. The binding was only weakly related to the total concentration of immunoglobulins (r = 0.11 by linear regression analysis), however, and is apparently due to a subclass that is elevated in some types of cancer. Elevated levels of these immunoglobulins may contribute to the lower urinary levels and clearance of riboflavin in cancer.

Breast Neoplasms↗
PubMed1985

Increases in serum sphingomyelin by 17 beta-estradiol.

The effects of estrogens on plasma sphingomyelin and the hepatic activity of the initial enzyme of sphingomyelin synthesis were examined using immature chicks. After three days of 17 beta-estradiol administration, serum sphingomyelin, total phospholipids, and cholesterol doubled, and triacylglycerol levels increased 7.5 fold. The sphingomyelin content and percentage of total phospholipids of liver were unaffected by estrogen treatment. The specific activity of serine palmitoyltransferase (EC 2.3.1.50) was unchanged, but the total activity appeared slightly higher due to increased liver weights. The higher sphingomyelin may, therefore, be due less to increased levels of biosynthetic enzymes than to factors such as the substrate (i.e., fatty acid) supply or decreased clearance of plasma sphingomyelin. These results are similar to earlier findings with key enzymes of cholesterol and glycerolipid biosynthesis and suggest that the three lipid pathways may be coordinated during estrogen treatment and enhanced very-low density lipoprotein (VLDL) synthesis.

Acyltransferases↗
PubMed1985

Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible.

Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of [2-14C]riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect [2-14C]riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient.

Autoradiography↗
PubMed1985

Activities of serine palmitoyltransferase (3-ketosphinganine synthase) in microsomes from different rat tissues.

Serine palmitoyltransferase [EC 2.3.1.50] catalyzes the first unique reaction of sphingolipid biosynthesis. To determine whether or not different rat tissues are capable of initiating this pathway, its activity was determined for microsomes from rat liver, lung, brain, kidney, intestine, spleen, muscle, heart, pancreas, testes, ovary, and stomach. Serine palmitoyltransferase was found in every tissue, and, when compared to the microsomal glycerol 3-phosphate acyltransferase, the activities correlated directly with their sphingomyelin levels as a percentage of total phospholipids. This suggests that the activities were comparable to expected cellular needs for long-chain bases, if the initial enzymes of glycerolipid and sphingolipid biosynthesis influence the phospholipid composition of cells by determining the relative partitioning of fatty acyl-CoA's toward these two lipid classes. Serine palmitoyltransferase activities were also determined using different fatty acyl-CoA's and were consistently greatest with CoA thioesters of saturated fatty acids with 16 +/- 1 carbon atoms. This suggests that the predominance of 18-carbon long-chain bases in vivo is due to the higher activity of this enzyme with palmitoyl-CoA. Together, these findings indicate a role for serine palmitoyltransferase in regulating both the type and amount of long-chain bases found in tissues.

Acyl Coenzyme A↗
PubMed1984

Activities of the initial enzymes of glycerolipid and sphingolipid synthesis in lung microsomes from rats exposed to air or 85% oxygen.

Lungs of adult rats exposed to 85% oxygen undergo extensive cellular reorganization; therefore, to investigate changes in lipid metabolism the initial enzymes of glycerolipid and sphingolipid synthesis were measured in lung microsomes. After 1 week of O2 treatment, the specific activity of the glycerol 3-phosphate acyltransferase increased to nearly twice that of the controls and remained elevated for the 3 weeks of study. Serine palmitoyl-transferase activities were approximately the same for both groups. These results suggest that in addition to cellular proliferation caused by hyperoxia there are also selective changes in glycerolipid synthesis, which may explain the decreased sphingomyelin content of lung and lamellar bodies.

Acyltransferases↗
PubMed1984

Enzymology of long-chain base synthesis by liver: characterization of serine palmitoyltransferase in rat liver microsomes.

Serine palmitoyltransferase [palmitoyl-CoA:L-serine C-palmitoyltransferase (decarboxylating) EC 2.3.1.50] catalyzes the initial and committed step in the biosynthesis of the long-chain bases of sphingolipids. A simple assay, based upon the incorporation of [3H]serine into the chloroform-soluble product 3-ketosphinganine, has been developed and demonstrated to be valid for analyzing this enzyme in rat liver microsomes. More than 75% of the serine palmitoyltransferase of rat liver was associated with the microsomal subfraction. The dependencies of activity on the incubation time, pH, temperature, other assay components (e.g., dithiothreitol, EDTA, and pyridoxal 5'-phosphate), and the concentrations of microsomal protein, L-serine, and palmitoyl-CoA were investigated. The requirement of pyridoxal 5'-phosphate for activity was established by formation of the apoenzyme by dialysis against cysteine, and recovery of full activity upon reconstitution with the coenzyme. Activities with fatty acyl-CoA's of varying alkyl chain length were distributed nearly symmetrically around a maximum at 16 carbons (palmitoyl-CoA) for the fully saturated substrates. Less activity was obtained with the CoA thioesters of cis-unsaturated fatty acids, but trans-9-hexadecenoyl-CoA yielded essentially the same activity as palmitoyl-CoA. Hence, this enzyme is capable of initiating the synthesis of the major long-chain bases, as well as compounds that may constitute the unidentified bases reported in analyses of mammalian sphingolipids.

Acyl Coenzyme A↗
PubMed1984

Metabolism of vitamin B-6 by human liver.

The enzymes that metabolize vitamin B-6 were analyzed in liver biopsy samples from five patients without hepatic disease by using methods optimized for small samples. Pyridoxal kinase (EC 2.7.1.35) activities were 11.2 +/- 3.6 nmol/minute per gram of tissue and 0.16 +/- 0.05 nmol/minute per milligram of soluble protein (mean +/- SD); a clear dependence of the activity on zinc as the divalent cation was observed. Pyridoxine (pyridoxamine) 5'-phosphate oxidase (EC 1.4.3.5) activities, when using N-(5'-phosphopyridoxyl)-[3H]tryptamine as the substrate, were 0.64 +/- 0.22 pmol/minute per milligram of protein and 47 +/- 19 pmol/minute per gram of tissue. The activities were 63 +/- 18% lower when riboflavin 5'-phosphate was omitted from the assay; hence, it appears the oxidase is only partially saturated with its cofactor. The pyridoxal 5'-phosphate hydrolase(s) activities at alkaline pH were 282 +/- 183 nmol/minute per gram of tissue and 4.0 +/- 3.2 nmol/minute per milligram of particulate protein. Pyridoxal was rapidly oxidized to pyridoxic acid (28.1 +/- 19.8 nmol/minute per gram of tissue and 0.37 +/- 0.24 nmol/minute per milligram of soluble protein) by soluble enzyme(s), and the rate was unaffected by pyridine nucleotides. These experiments constitute the first quantitative analyses of the enzymes responsible for metabolizing vitamin B-6 in human liver, and provide data for interpreting the pharmacokinetics of B-6 utilization by humans, as well as methods for investigating diseases with aberrant metabolism of this nutrient.

Alcohol Oxidoreductases↗
PubMed1984

Utilization of different fatty acyl-CoA thioesters by serine palmitoyltransferase from rat brain.

Serine palmitoyltransferase (EC 2.3.1.50) catalyzes the first unique reaction of sphingolipid biosynthesis. Activities were determined with different fatty acyl-CoA substrates to describe the range of long-chain bases that could be made by rat brain microsomes. The activities were greatest with palmitoyl-CoA and palmitelaidoyl-CoA, followed by fully saturated homologs differing from these by only one carbon atom, and diminished considerably as the alkyl-chain length increased or decreased, or with the presence of a cis-double bond. These characteristics explain the predominance of long-chain bases with 18 carbon atoms in brain sphingolipids, and account for the minor variants such as the C17- and C20-long chain bases.

Acyl Coenzyme A↗
PubMed1984

Comparison of serine palmitoyltransferase in Morris hepatoma 7777 and rat liver.

Serine palmitoyltransferase (EC 2.3.1.50) catalyzes an initial and regulatory reaction of sphingolipid biosynthesis, the formation of a homologue of 3-ketosphinganine from L-serine and a fatty acyl coenzyme A thioester. We have demonstrated that this enzyme exists in microsomes from Morris hepatoma 7777 and, moreover, found that its specific activity was 199 +/- 23 pmol/min/mg of microsomal protein, which was significantly higher than that for microsomes from host (69 +/- 4.2 pmol/min/mg; S.D.) or control (43 +/- 4.1 pmol/min/mg) rat livers when assayed under optimal conditions. The activities varied with tumor weight. For comparison, the activities of microsomes from regenerating liver were also higher; whereas fetal and neonatal rat livers had substantially lower activities. Assays conducted without added pyridoxal 5'-phosphate revealed that the enzyme in microsomes isolated from the tumor was more readily depleted of this cofactor than was that in control microsomes; this phenomenon was most pronounced with the larger tumors. The other properties of the enzyme resembled those for liver. On the basis of these experiments, we propose that the elevated proportions of sphingomyelin and other sphingolipids found in hepatomas and regenerating rat liver are related to increases in long-chain base synthesis by serine palmitoyltransferase. Since the activity is apparently more sensitive to the availability of pyridoxal 5'-phosphate in the hepatoma, this reaction could become limiting under more severe vitamin B6 deficiencies.

Acyltransferases↗