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Biomedical subjects

M L Karnovsky

Publications and source records attributed to M L Karnovsky.

At least 73 records · Page 4Linked to original sources
PubMed1982

Identification of a rat liver microsomal polypeptide involved in the transport of glucose 6-phosphate. Labeling with 4,4'-diisothiocyano-1,2-diphenyl[3H]ethane-2,2'-disulfonic acid.

Transport of glucose-6-P in intact rat liver microsomes is inhibited by 4,4'-diisothiocyano-1,2-diphenyl[3H]ethane-2,2'-disulfonic acid ([3H]H2DIDS). The concentration of [3H]H2DIDS that inhibits transport activity by 50% is 35 microM. Glucose-6-P protected against the inhibition of transport activity caused by [3H]H2DIDS; mannose-6-P, 2-deoxyglucose-6-P, galactose-6-P, fructose-6-P, or glycerol-2-P did not. [3H]H2DIDS-treated microsomes were solubilized in sodium dodecyl sulfate and the microsomal polypeptides were separated by polyacrylamide gel electrophoresis. Labeled polypeptides were identified by autoradiography. Treatment of microsomes with concentrations of [3H]H2DIDS (50-100 microM) that resulted in maximal inhibition of transport activity allowed the identification of two microsomal polypeptides that contained most of the incorporated radioactivity. Their molecular weights were 54,000 and 59,000. The labeling of the former, but not the latter polypeptide was saturable and correlated linearly with the level of inhibition of transport activity. Concomitantly with its protective effect on translocase activity, the presence of glucose-6-P during the reaction with [3H]H2DIDS resulted in a significant increase in the amount of label incorporated into this peptide. This stimulation of labeling was specific for glucose-6-P. These results implicate the 54,000-dalton polypeptide as an obligatory component of the rat liver microsomal glucose-6-P translocase.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

The composition of sleep-promoting factor isolated from human urine.

A sleep-promoting substance, Factor S, has been extracted and purified from large volumes of human urine. Urinary Factor S is a small glycopeptide; amino acid-amino sugar analyses of the purified material revealed a substance composed of glutamic acid, alanine, diaminopimelic acid, and muramic acid in molar ratios of 2:2:1:1. The active glycopeptide resembles bacterial peptidoglycans but the composition suggests that it is not simply of adventitious origin. Other reasons for this conclusion are also given. Infusions into the lateral ventricle of the brain of about 5 pmol/kg of body weight induce a 50% increase in slow wave sleep in rabbits. The excess sleep is normal as judged by electrophysiological and behavioral criteria; it resembles the deep sleep that occurs when animals are allowed to sleep following prolonged sleep deprivation.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1982

Sleep-promoting effects of muramyl peptides.

A muramyl peptide that induces excess slow-wave sleep in rats, rabbits, and cats has recently been isolated from human urine. We now report that synthetic acetylmuramyl-L-alanyl-D-isoglutamine ("muramyl dipeptide") and its lysyl derivative (acetylmuramyl-L-alanyl-D-isoglutaminyllysine) can mimic the somnogenic effects of the natural peptide. Both compounds are also pyrogenic and may cause other disturbances of autonomic function. The pyrogenic effects of intravenously administered muramyl dipeptide can be suppressed by previous treatment with acetaminophen without blocking the sleep-promoting effects.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1982

Glycogen accumulation in polymorphonuclear leukocytes, and other intracellular alterations that occur during inflammation.

Neutrophils isolated from the blood were compared to those from inflammatory exudates in the peritoneal cavity of guinea pigs. Inflammatory neutrophils were shown to have 10-fold more glycogen than blood neutrophils. This was also reflected in the morphology of these cells. The large accumulations of glycogen in inflammatory neutrophils exists in ordered arrays of beta-granules. Other morphological changes including accumulations of lipid droplets and a decrease in the number of lysosomal granules also accompany the change from blood neutrophils to inflammatory neutrophils. These results show that there are major metabolic differences in the two types of neutrophils.

Animals↗
PubMed1981

cis-Polyunsaturated fatty acids induce high levels of superoxide production by human neutrophils.

Arachidonate stimulates the production of large quantities of superoxide by human neutrophils: 93.8 +/- 12.5 S.D. nmol of O2(-)/min/10(7) cells. This rate is comparable to that observed with the most effective neutrophil-stimulating agents previously reported. Other cis-unsaturated fatty acids are also capable of eliciting this response, the order of effectiveness being: arachidonate greater than gamma-linolenate greater than linoleate greater than oleate. Linolelaidate, myristate, and palmitate are ineffective. These data are discussed in relation to recent reports concerning the oxidation of arachidonic acid by human neutrophils and by a cell-free system that generates superoxide and hydrogen peroxide.

Arachidonic Acid↗
PubMed1981

Superoxide production by an unusual aldehyde oxidase in guinea pig granulocytes. Characterization and cytochemical localization.

An aldehyde oxidase extracted from guinea pig granulocytes with isotonic KCl catalyzes the oxidation of a variety of aliphatic aldehydes and 2-OH-pyrimidine. The stoichiometry of the oxidation of 2-OH-pyrimidine is consistent with the reaction 2-OH-pyrimidine + OH- + O2 leads to uracil + H2O2. Between 75 and 90% of the peroxide produced results from dismutation of superoxide formed as an intermediate. The Km for 2-OH-pyrimidine is approximately 1.65 mM and the maximum velocity is 22.9 +/- 5.5 S.D. nmol of superoxide/min/10(7) cells. This same maximum velocity is observed for the substrates isobutyraldehyde, propionaldehyde, and acetaldehyde. Unlike other aldehyde oxidases, this enzyme is inactive with purines as substrates and is insensitive to antimycin A, menadione, and Triton X-100. The enzyme is inhibited by cyanide, methanol, and arsenite. The apparent molecular weight is approximately 340,000 +/- 25,000 and the pH optimum is in the range of 7.5 to 9.0. Electron cytochemistry reveals an association of this oxidase with the phagosome membrane. The potential significance of this oxidase is discussed in relation to microbicidal mechanisms.

Aldehyde Oxidase↗
PubMed1981

Studies on zonation in the epididymis of the guinea pig. I. Ultrastructural and biochemical analysis of the zone rich in large lipid droplets (zone II).

In the epididymis of the guinea pig, zone II exhibits striking histological features that distinguish it readily from the other six regions of the epididymis. At the light microscope level, the pseudostratified epithelium of zone II is characterized by tall principal cells that are densely packed with large, intensely staining granules or droplets ranging up to 8 mu in diameter. At the electron microscope level, the principal cells exhibit numerous large lipid droplets and abundant agranular endoplasmic reticulum, which is frequently arranged in concentric whorls around one or more of the droplets. Quantitative biochemical studies comparing zone II with zones I and III show that zone II contains 2.5 - 3-fold more cholesterol and a significantly greater amount of cholesterol ester than the other two zones. These data indicate that the epididymal duct of the guinea pig includes a clearly defined region of epithelial cells possessing ultrastructural and biochemical characteristics consistent with steroidogenic activity. The potential significance of these observations to the epididymal physiology of the guinea pig and epididymal physiology in general is discussed.

Animals↗
PubMed1981

Oxidative decarboxylation of free and peptide-linked amino acids in phagocytizing guinea pig granulocytes.

The oxidative decarboxylation of amino acids by a system consisting of myeloperoxidase-hydrogen peroxide-chloride has been demonstrated previously by others and the process has been considered to be part of the microbicidal armamentarium of some phagocytic leukocytes. We were able to translate these earlier observations, made on model systems, to intact guinea pig granulocytes. We could demonstrate differences in the cellular handling of peptide-linked amino acids as particles, compared with free amino acids. Specific inhibitors were used to explore two routes of oxidative decarboxylation: (a) the myeloperoxidase-catalyzed direct decarboxylation-deamination reaction, and (b) oxidation of alpha-keto acids after transamination of amino acids. These inhibitors were cyanide, azide, and tapazole for the former pathway, and amino-oxyacetate for the latter. Amino-oxyacetate profoundly inhibited the decarboxylation of free 14C-amino acids (alanine and aspartate) in both resting and stimulated cells, but had only a minimal effect on 14CO2 production from ingested insoluble 14C-protein. On the other hand, the peroxidase inhibitors cyanide, azide, and tapazole dramatically inhibited the production of 14CO2 from ingested particulate 14C-protein, but had only small effects on the decarboxylation of free amino acid. Soluble, uniformly labeled 14C-protein was not significantly converted to 14CO2 even in the presence of phagocytizable polystyrene beads. These observation suggest that the amino acids taken up by phagocytosis (e.g., as denatured protein particles) are oxidatively decarboxylated and deaminated in the phagosomes by the myeloperoxidase-hydrogen peroxide-chloride system; soluble free amino acids that enter the cytoplasm by diffusion or transport are oxidatively decarboxylated after transamination by the normal cellular amino acid oxidative pathway.

Amino Acids↗
PubMed1980

The determination and localization of sialic acid in guinea-pig granulocytes.

When intact guinea-pig granulocytes (polymorphonuclear leucocytes) disrupted by sonication or with detergent were treated with neuraminidase from Vibrio cholerae, 3.1--3.2 nmol of sialic acid/10(7) cells was released. By using a chromatographic procedure for the specific determination of total cell sialic acid, this releasable portion was found to constitute 70% of the total sialate. All of the neuraminidase-releasable sialic acid of the cells could be removed by enzymic treatment of intact cells with neuraminidase. It thus seemed likely that the neuraminidase-releasable sialic acid is all on the cell surface. To make sure that the result was not due to entry of neuraminidase into the cells, the enzyme was bound covalently to Sepharose 6B, and intact polymorphonuclear leucocytes were treated with the bound enzyme. All of the neuraminidase-releasable sialic acid could still be removed, though more slowly. The cells remained intact and only 1.5--2% of the bound enzyme was released from the Sepharose during incubation. Freed enzyme could have been responsible, at the very most, for release of 18% of the sialic acid. Fractionation studies showed that the nucleus and cytoplasm contain low amounts of sialic acid and that the neuraminidase-releasable sialic acid distributes in a manner similar to the distribution of 5'-nucleotidase, an unambiguous marker for the plasma membrane in these cells. Thus neuraminidase-releasable sialate constitutes a clear marker for the membrane of polymorphonuclear leucocytes. Most of the neuraminidase-insensitive sialate was present in the granule fraction. Removal of sialic acid from intact polymorphonuclear leucocytes did not affect their ecto-AMPase, -ATPase and -p-nitrophenyl phosphatase activities.

Adenosine Monophosphate↗
PubMed1980

Metabolism of purines in macrophages. Effect of functional state of the cells.

Ecto-5'-nucleotidase is known to be diminished markedly in activated compared to control mouse macrophages. The level of three purine nucleoside metabolizing enzymes, adenosine deaminase (EC 3.5.4.4), purine nucleoside phosphorylase (EC 2.4.2.1), and adenine phosphoribosyltransferase (EC 2.4.2.7) were measured in the sonicates of different populations of mouse peritoneal macrophages. Levels of adenine phosphoribosyltransferase and purine nucleoside phosphorylase in macrophages that were elicited with sodium caseinate or activated in vivo by prior intravenous injection of Listeria monocytogenes were eight times higher than those in resident cells. Levels of adenosine deaminase also tended to increase and were two times higher in elicited cells than in resident cells. The Km of each enzyme was the same in each cell population. The findings suggest that the levels of the ecto-5'-nucleotidase and of the intracellular enzymes are coordinated.

Adenine Phosphoribosyltransferase↗
PubMed1980

Oxidation of glucose by mouse peritoneal macrophages: a comparison of suspensions and monolayers.

Macrophages, when maintained in vitro, take up glucose from the medium and oxidize it to CO2. The rate of oxidation of glucose varies considerably, depending on the physical state of the cell preparation. Cells in suspension oxidize glucose at a level six-fold that of cells in monolayers. The differences cannot be attributed to change in the rats of transport of glucose. On the other hand, an increase in intracellular glycogen (about three-fold) and free glucose plus glucose-6-P (many-fold) was found in the cells prepared as monolayers. During subsequent incubation with glucose-14C, this could be the cause of an isotope dilution effect and could explain the lower production of 14CO2 by the adherent cells. Since oxidation of glucose-1-14C to 14CO2 is used by many investigators to indicate the functional state of macrophages, we suggest close attention be paid to the system used, i.e., monolayers vs. suspensions.

Animals↗
PubMed1980

Comparative aspects of oxidative metabolism of neutrophils from human blood and guinea pig peritonea: magnitude of the respiratory burst, dependence upon stimulating agents, and localization of the oxidases.

We have compared the subcellular sites of H2O2 and presumably also superoxide-(O2-) production, and certain aspects of metabolic responses (O2 consumption, O2- production) of stimulated neutrophils from human blood and those elicited into guinea pig peritonea. Stimulation was accomplished with either opsonized zymosan or phorbol-12-myristate-13-acetate (PMA). Striking quantitative differences were observed between these cell types with regard to the increased respiration and O2- production observed during stimulation. These differences were most apparent when opsonized zymosan served as the stimulating agent. They were minimized when the soluble stimulating agent, PMA, was used. With either stimulus, the subcellular sites of H2O2 production were the same for both types of neutrophils, i.e., the plasmalemma and phagosomal membranes. No H2O2 production could be detected cytochemically in the absence of stimulation. Treatment of both unstimulated human blood and elicited guinea pig peritoneal neutrophils with the nonpenetrating, covalently linking reagent, p-diazobenzenesulfonic acid, failed to diminish O2- production upon subsequent stimulation, in contrast to a previous report. These data are discussed in terms of the possible cytological arrangements of the respiratory enzyme(s), and the different modes of stimulation of neutrophil metabolism by various agents. Ancillary data on elicited mouse peritoneal neutrophils are presented.

Animals↗