H.Bruce Bosmann

Platelet adhesiveness and aggregation: II. Surface sialic acid, glycoprotein:N-acetylneuraminic acid transferase, and neuraminidase of human blood platelets☆

Abstract 1. 1. Human blood platelets were demonstrated to have 2.4·10 6 molecules of sialic acid per platelet surface in a neuraminidase susceptible linkage by biochemical and electrokinetic methods. 2. 2. The platelets were found to contain a fetuin:sialyl glycoprotein:glycosyl transferase; several pieces of evidence indicated that both a sialyl transferase and endogenous acceptors were located on the outer surface of the platelet plasma membrane. 52% of the transferase was recovered in the plasma membrane fraction of the platelet with a 54.3-fold purification. The enzyme has a pH optimum of 7.5, erquires Mn 2+ for activity and has high activity with either fetuin minus sialic acid or prothrombin minus sialic acid as acceptor. 3. 3. The fetuin:sialyl transferase utiling fetuin minus sialic acid and CMP-sialic acid as Substrates a and b, respectively, gave the following kinetic constants using the Cleland bisubstrate model: K a = 675 μ M, K b = 90 μ M, K ia = 18.5 μ M, K ib = 2.5 μ M and V 1 = 500 pmoles/mg protein per 5 min. 4. 4. At 1·10 −2 M the fetuin:sialyl transferase was inhibited by the following compounds which affect platelet adhesion by the indicated percentage: D -glucosamine, 80; aspirin, 95; UDP, 94; galactose, 37; glucose, 0; uridine, 0; and L -fucose, 0. 5. 5. The fetuin:sialyl transferase was dependent on Mn 2+ for activity; Co 2+ , Mg 2+ , Ba 2+ and Ca 2+ could substitute somewhat for the Mn 2+ . The following divalent cations inhibited the reation: Hg 2+ , Cu 2+ , Pb 2+ , and Cd 2+ . 6. 6. The platelets contained low but measurable neuraminidase activity; this activity was purified 20-fold and could release sialic acid residues from intact platelets as demonstrated by decreased platelet electrophoretic mobility after incubation of intact platelets with the platelet neuraminidase.

The use of urinary N-acetyl-β-glucosaminidase in human renal toxicology I. Partial biochemical characterization and excretion in humans and release from the isolated perfused rat kidney

Abstract Selected biochemical properties and the human excretory pattern of urinary N-acetyl-β-glucosaminidase were characterized. A renal origin of the urinary enzyme was demonstrated by comparison of the release from the isolated perfused rat kidney with excretion from the in situ organ. The rate of activity excretion from both the perfused and in situ organ was 22% of the total organ content per day. The basal enzyme release was into tubular luman only, and contralumenal release into the renal lymphatic system was essentially zero. The human urinary activity exhibited unusual properties which indicated that the rate of excretion is directly proportional to the corresponding rate of release by the tubular epithelium. In urine, 0.2% of the activity is lost per hour at 37°C, and the activity is not denatured by physiologic variations in urine pH and osmolarity. The urinary activity is not associated with cells, is almost entirely soluble and exhibits no latency upon addition of Triton X-100. A cortico-medullary activity gradient exists in the human kidney. The rate of activity excretion in 30 human subjects was constant under maximal variations in urine flow rate. The 3-day excretion in 21 subjects fluctuated around a fairly constant mean rate, which was characteristic of each individual, and subsequent 3-day collections up to 3 weeks later were identical. The rate of excretion was independent of body mass, lean body mass, and sex. The pattern of N-acetyl-β-glucosaminidase excretion fluctuated identically with that of several other acid hydrolases. It is concluded that, with certain restrictions, N-acetyl-β-glucosaminidase excretion can be employed as a marker of the rate of release of at least several acid hydrolases from the human kidney.

Elevated glycosidases and proteolytic enzymes in cells transformed by RNA tumor virus.

Abstract Activities of 9 glycosidases (α-fucosidase and β-xylosidase were absent in all cells) were found to be significantly elevated in MSV-3T3 and RSV-3T3 cells compared to 3T3 cells; acid phosphatase and β-glucuronidase levels were similar in the normal and RNA virus transformed cells. Trypsin-like and cathepsin-like activity were also elevated in the cells transformed by the RNA tumor viruses. The results are discussed with respect to the phenomena of sublethal autolysis.

Platelet adhesiveness and aggregation: The collagen:Glycosyl, polypeptide:N-acetylgalactosaminyl and glycoprotein:Galactosyl transferases of human platelets

Abstract Four glycoprotein:glycosyl transferases were identified, purified, and characterized from human platelets. The enzymes present were collagen:glc, collagen:gal, polypeptide:galNAc, and glycoprotein:gal; the fetuin:glcNAc transferase was absent. Each of the 4 transferases was found to be almost exclusively bound to the platelet plasma membrane. Incubation of platelet homogenates without exogenous acceptors yielded no transfer of monosaccharide, indicating the complete lack of endogenous acceptors. These results lead to the interesting speculation that the transferases may not be responsible in the mature platelet for glycoprotein synthesis at all, but rather that they may function for intercellular adhesion and the primary step in hemostasis, the adhesion of collagen to platelets.

Mechanism of action of disodium cromoglycate--mast cell calcium ion influx after a histamine-releasing stimulus.

Abstract Rat peritoneal mast cells release histamine and accumulate 45 Ca in a dose-dependent manner when concentrations of compound 48/80 ranging from 0·1 to 1·0 μg/ml are incubated with suspensions of the cells for 5 min at 37°. Influx of 45 Ca stimulated by compound 48/80 can be inhibited to varying degrees by prior addition of disodium cromoglycate. Inhibition was dependent on the concentration of both disodium cromoglycate and compound 48/80. The electrokinetic properties of intact rat mast cells are described; disodium cromoglycate caused a plasma membrane alteration possibly related to Ca 2+ influx. Cromoglycate increased mast cell electrophoretic mobility but decreased the electrophoretic mobility of rat erythrocytes. The net electrophoretic mobility was a function of terminal sialic acid residues, ionic strenght, and pH. Binding of disodium cromoglycate to Ca 2+ could not be demonstrated by a variety of sensitive physical techniques. The data support the theory that secretion of mast cell histamine is coupled to Ca 2+ influx. It is suggested that disodium cromoglycate prevents mast cell histamine release by a plasma membrane alteration which prevents an increase in membrane permeability to Ca 2+ stimulated by compound 48/80.

Camptothecin effects on DNA synthesis in murine leukemia cells

Abstract The effects of camptothecin on DNA synthesis were investigated using L1210 and L5178Y cells in culture. After addition of the drug, incorporation of labeled thymidine into cell DNA was gradually inhibited; at a 10 μg/ml drug level, 1 h was required to inhibit thymidine incorporation by 95%. Upon removal of drug, this inhibition was slowly reversed over a period of several hours; but the drug-treated cells were incapable of subsequent division. Cells in the S phase of the cell cycle were most sensitive to camptothecin.

Diazepam receptor characterization: Specific binding of a benzodiazepine to macromolecules in various areas of rat brain

Diazepam is an effective anti-anxiety drug or mild tranquilizer [l] which is also used as a muscle relaxant [2] , a presurgery medication [3], an anti-epileptic agent and in alcohol withdrawal. It is a widely prescribed drug [4]. The toxicity of diazepam is remarkably low, as is its physical dependence [S] . Despite its widespread use, very little is known about the molecular basis of its action. Recent investigations, concerning macromolecules that bind hormones or drugs, have been carried out in an attempt to correlate such binding with pharmacological actions. If a binding macromolecule is found for a drug, it is usually designated as a receptor, and it is assumed that an endogenous or naturally occurring ligand exists for the receptor. In the present study we found evidence for a highly specific, saturable binding of [3H]diazepam to a purified synaptosomal fraction from rat forebrain and present some of the characteristics of this binding. (A note of caution should be interjected since there is a great deal of difference between a binding entity, a receptor and a receptor site. In the past, glass, plastics, and other inert materials have been shown to be effective ‘binders’ of ligands.) Such data should lead to a better understanding of the molecular action of diazepam. While this work was nearing completion, Squires and Braestrup [6] reported on benzodiazepine receptors in rat brain. In our report, using a higher specific activity of [3H]diazepam and a more purified binding fraction, we give amplified details of the diazepambinding site. 2. Materials and methods

Mitochondrial Autonomy: Incorporation of Monosaccharides into Glycoprotein by Isolated Mitochondria

Isolated intact mitochondria selectively incorporate monosaccharides from nucleotide diphosphate monosaccharides into protein. Fucose, mannose, glucose, and galactose were incorporated by the mitochondria into glycoprotein; xylose was not. Structural integrity of the mitochondria was not necessary for the incorporation of monosaccharide into glycoprotein; mitochondria broken by homogenization also incorporated monosaccharide. The monosaccharides incorporated into glycoprotein were localized in the inner mitochondrial membranes, the same membranes which contain the protein into which leucine is incorporated by the isolated mitochondria.

Membrane marker enzymes: Isolation, purification, and properties of 5′-nucleotidase from rat cerebellum

A 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) from rat cerebellum was purified 855-fold by (NH4)2SO4 fractionation and column chromatography on Sephadex G-100 and G-200. The enzyme was activated by divalent cations with Mg2+, Cp2+, and Mn2+ being the most efficient activators. The 5′-nucleotidase had an optimum pH of 6.8 ± 0.2. The relative rates of hydrolysis of 5′-AMP, 5′-UMP, 5′-CMP, 5′-GMP and 5′-IMP were 100, 46, 35, 17 and 9, respectively; there was no hydrolysis of 2′- or 3′-nucleoside monophosphates. Values for the Michaelis constant were 5′-AMP, 0.08 mM; 5′-UMP, 0.82 mM; 5′-CMP, 0.92 mM; 5′-GMP, 1.41 mM; and 5′-IMP, 2.1 mM. Using 5′-AMP as substrate the 5′-nucleotidase was inhibited by NaF, HgCl2, CuCl2, p-chloromercuribenzoate, and p-hydroxymercuribenzoate. The 5′-nucleotidase was inhibited by 5 · 10−3M thymidine, guanosine and inosine but not by uridine or adenosine. The concentrations of ATP, UTP, ITP, CTP and GTP required to inhibit the hydrolysis of 5′-AMP by 50% were 10, 68, 240, 250 and 720 μM, respectively.

Cell surface glycosyl transferases and acceptors in normal and RNA- and DNA-virus transformed fibroblasts

Abstract Cell surface glycosyl transferases and acceptors were found to be present in normal and oncogenic virus transformed fibroblasts. In cells from confluent cultures, the reaction was elevated 2- to 4-fold in MSV-3T3, RSV-3T3 and PY-3T3 cells compared to normal 3T3 cells, but in cells from sparse cultures, the reaction was essentially equivalent in the normal and transformed cells. Using exogenous acceptors, elevated surface levels of some glycoprotein:glycosyl transferases were demonstrated in the RNA- and DNA-virus transformed cells. The results may be important in phenomena such as cell:-cell adhesion and contact inhibition.