John M. Lowenstein

Nitric Oxide Regulates Exocytosis by S-Nitrosylation of N-ethylmaleimide-Sensitive Factor

Nitric oxide (NO) inhibits vascular inflammation, but the molecular basis for its anti-inflammatory properties is unknown. We show that NO inhibits exocytosis of Weibel-Palade bodies, endothelial granules that mediate vascular inflammation and thrombosis, by regulating the activity of N-ethylmaleimide-sensitive factor (NSF). NO inhibits NSF disassembly of soluble NSF attachment protein receptor (SNARE) complexes by nitrosylating critical cysteine residues of NSF. NO may regulate exocytosis in a variety of physiological processes, including vascular inflammation, neurotransmission, thrombosis, and cytotoxic T lymphocyte cell killing.

Tricarballylate and hydroxycitrate: Substrate and inhibitor of ATP: Citrate oxaloacetate lyase☆

Citrate cleavage enzyme shows atypical kinetics at low chloride concentrations, but normal kinetics at high chloride concentrations. Tricarballylate is a substrate for citrate cleavage enzyme. The reaction with tricarballylate can be demonstrated both by the disappearance of CoA and, in the presence of hydroxylamine, by the appearance of a hydroxamate. This indicates that tricarballylyl-CoA is formed in the reaction. When hydroxamate formation is used to assay activity, the apparent Km for tricarballylate is about three times greater than that for citrate, while the maximum reaction velocity with tricarballylate is about 90% of that observed with citrate. One of the stereoisomers of hydroxycitrate is a powerful inhibitor of citrate cleavage enzyme.

AN ANTIVIRAL MECHANISM OF NITRIC OXIDE : INHIBITION OF A VIRAL PROTEASE

Abstract Although nitric oxide (NO) kills or inhibits the replication of a variety of intracellular pathogens, the antimicrobial mechanisms of NO are unknown. Here, we identify a viral protease as a target of NO. The life cycle of many viruses depends upon viral proteases that cleave viral polyproteins into individual polypeptides. NO inactivates the Coxsackievirus protease 3C, an enzyme necessary for the replication of Coxsackievirus. NO S-nitrosylates the cysteine residue in the active site of protease 3C, inhibiting protease activity and interrupting the viral life cycle. Substituting a serine residue for the active site cysteine renders protease 3C resistant to NO inhibition. Since cysteine proteases are critical for virulence or replication of many viruses, bacteria, and parasites, S-nitrosylation of pathogen cysteine proteases may be a general mechanism of antimicrobial host defenses.

Crystal structures of HINT demonstrate that histidine triad proteins are GalT-related nucleotide-binding proteins.

Histidine triad nucleotide-binding protein (HINT), a dimeric purine nucleotide-binding protein from rabbit heart, is a member of the HIT (histidine triad) superfamily which includes HINT homologues and FHIT (HIT protein encoded at the chromosome 3 fragile site) homologues. Crystal structures of HINT-nucleotide complexes demonstrate that the most conserved residues in the superfamily mediate nucleotide binding and that the HIT motif forms part of the phosphate binding loop. Galactose-1-phosphate uridylyltransferase, whose deficiency causes galactosemia, contains tandem HINT domains with the same fold and mode of nucleotide binding as HINT despite having no overall sequence similarity. Features of FHIT, a diadenosine polyphosphate hydrolase and candidate tumour suppressor, are predicted from HINT-nucleotide structures.

Ammonia Production in Muscle: The Purine Nucleotide Cycle

Experiments are reported which throw new light on the problem of ammonia production by muscle and, probably, by other tissues.

[34] Measurement of rates of lipogenesis with deuterated and tritiated water

Publisher Summary This chapter discusses the measurement of rates of lipogenesis with deuterated and tritiated water. The tritium method described is applied to rat liver, but it can be used for most other tissues without modification. At the end of the experiment the liver is frozen rapidly by being pressed between blocks of aluminum which have been cooled in liquid nitrogen. The livers are stored in a liquid nitrogen refrigerator. Tissue treated in this manner can be used for metabolite analyses and measurements of fatty acid synthesis. The frozen liver is powered in the frozen state using a stainless steel pestle and mortar cooled with Dry Ice or liquid nitrogen. The resulting powder is weighed, and is dispersed in 19 volumes chloroform–methanol (2:1, by volume), and the suspension is shaken gently for 24 hours. The deuterium content of the methyl esters of long-chain fatty acids is determined by mass spectrometry. The isotope ratios can be measured from the recorded mass spectra with a 12-in. Gerber variable scale, Model TP 007100B.

On a possible role of IMP in the regulation of phosphorylase activity in skeletal muscle

In skeletal muscle, the transition from rest to exercise is associated with an accumulation of IMP. Concentrations of 2-4 mM IMP can be reached in perfused hind leg and of l-2 mM IMP in hind leg in situ [ 1,2]. During exercise the total tissue content of AMP rises only slightly above the resting value of 0.1 pmol/g wet wt, or about 0.2 mM intracellularly. The free concentration of AMP, calculated assuming equilibrium with ATP, creatine phosphate and creatine through the creatine kinase and myokinase reactions, is <l PM during rest and rises to several PM during exercise [ 1,2]. Thus in exercising muscle the concentration of IMP can exceed that of free AMP by a factor of 100 or more. Activation of phosphorylase b by IMP was first reported in 1938 [3]. A physiological role for IMP in regulating the activity of phosphorylase b was ruled out on the grounds that the activation of the enzyme by IMP was weak when compared to its activation by AMP. Moreover, conversion of phosphorylase b to phosphorylase a seems to obviate the need for activation of phosphorylase during exercise. However, a report by Piras and Staneloni [4] showed that in rat skeletal muscle the conversion of phosphorylase b toa proceeds for only about 10 s after the start of strong exercise; thereafter phosphorylase a is converted back to the b form, even though exercise is continuing. We have confirmed this observation, finding that phosphorylase is largely converted back to the b form within 20 s after the start of exercise. Accumulation of IMP has reached a virtual maximum at this time. The large excess of IMP over free AMP that accumulates during exercise suggests that although IMP is a weaker activator of phosphorylase b than AMP, it

3-β-Hydroxysterol synthesis by the liver☆

Incorporation of 3H from [3H]H2O was used to measure 3-β-hydroxysterol synthesis in rat liver. The amount of 3H incorporated has been related to the amount of carbon incorporated by measuring the incorporation of [14C]glucose and correcting for the dilution in specific activity of the labeled carbon by endogenous carbon compounds. 3-β-Hydroxysterol is inhibited in starvation and by (−)-hydroxycitrate.

Activation of phosphoinositide-specific phospholipase Cδ from rat liver by polyamines and basic proteins

Phospholipase C from rat liver with a molecular weight of 87,000 (PLC delta) is stimulated by polyamines, basic proteins, and basic polyamino acids. The activation occurs in both the presence and the absence of detergents. Half-maximum activation by spermine is observed at 0.15 mM, with optimum effects between 0.2 and 0.5 mM. Spermine inhibits above 0.5 mM. Half-maximum activation by spermidine and putrescine is observed at 0.9 and 6 mM, respectively, with optimum effects at 2 and 5 mM, respectively. These polyamines also inhibit at higher concentrations. Neomycin activates the enzyme with an optimum concentration of 10 microM, but maximum activation is less than with polyamines. Half-maximum activation by histone 2B occurs at 0.5 micrograms/ml (36 nM), with maximum stimulation at 1.5 micrograms/ml. Other histones, protamine, melittin, poly-L-ornithine, poly-L-lysine, poly-D-lysine, and poly-L-arginine, activate optimally at 3-10 micrograms/ml. Myelin basic protein and lysozyme activate optimally at 50-100 micrograms/ml. Typical activations are three- to eightfold, but under some conditions the enzyme shows little or no activity in the absence of basic activators. The basic activators lower the salt concentration required for maximal activity. In the case of the detergent-micelle assay, histone shifts the optimum NaCl concentration from 350 to 200 mM for PIP2, from 260 to 100 mM for PIP, and from 150 to 0 mM for PI. Histone potentiates the activation by Ca2+, but does not shift the optimum Ca2+ concentration. The optimum salt and Ca2+ concentrations are linked, such that a decrease in the concentration of one decreases the optimum concentration of the other. Activation by histone is diminished by MgCl2 in a concentration-dependent manner.

Citrate cleavage enzyme in livers of obese and nonobese mice.

The specific activity of the citrate cleavage enzyme is 3.3 times greater in livers of obese mice than in livers of their nonobese litter mates. The difference persists during starvation. The specific activity of the acetate activating enzyme is approximately the same in the livers of both types of animals. It is proposed that a high activity of citrate cleavage enzyme is one of the factors responsible for obesity.