The primary properties?

Science is concerned not just with objects but also with their various aspects, such as their colors, temperatures, sizes, and shapes. These aspects, or properties, are generally thought to be of at least two kinds: primary properties, such as shape, size, and motion and secondary ones, such as temperature and color. However, there is little agreement on just what the difference is between these two kinds of properties. An argument has recently been put forth that assumes the two kinds of properties differ only in that the secondary ones are dependent on the conditions under which they are observed. This paper suggests, however, that since the primary properties depend on conditions, too, any argument based on this assumption is flawed. There are two main traditional accounts of the distinction. The first account of the distinction-the ideas of primary qualities resemble the properties that cause these ideas, whereas those of the secondary ones do not resemble them-relies on what to many is an implausible assumption, that some of our ideas resemble features of the world and we can speak intelligibly about such resemblances. This paper suggests that we cannot, and hence we have no more reason to believe that the ideas of the primary qualities resemble aspects of the world than we do that those of the secondary qualities do so. The second traditional account-primary properties are those a body has, however small it might be, whereas secondary properties are a consequence just of relations between bodies-fares better than the other two accounts. However, the notion of force posed a serious problem for this second basis for the distinction, one that has, astonishingly, hardly found its way into the philosophical literature.

Guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate and adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate. New nucleotide affinity labels which react with rabbit muscle pyruvate kinase.

Three new reactive nucleotide analogues with bromo-keto substituents adjacent to a thiophosphate have been synthesized. Guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate (GMPS-BDB), reacts covalently with rabbit muscle pyruvate kinase with complete inactivation and incorporation of 1.8 mol of reagent/mol of enzyme subunit. By contrast, the mono-keto compound, guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (GMPS-BOP), causes no loss of pyruvate kinase activity. When the analogous adenosyl nucleotide derivatives are incubated with pyruvate kinase, the di-keto compound, adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate (AMPS-BDB), rapidly effects inactivation, whereas the mono-keto compound, adenosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (AMPS-BOP), causes no loss of activity. Complete protection against inactivation by GMPS-BDB is provided by phosphoenolpyruvate in the presence of K+ and Mn2+ and the amount of reagent incorporated (0.9 mol/reagent/mol subunit) is reduced to half that observed in the absence of protectants. Gas-phase sequencing of the tryptic peptides purified from inactive GMPS-BDB or AMPS-BDB-modified enzyme gave the cysteine-labeled peptides: C151DENILWLDYK161, and N162IC164K165 as the two major peptide products, with a smaller amount of N43TGIIC48TIGPASR55. Reaction in the presence of the protectants PEP, K+, and Mn2+ yielded Cys164 as the only labeled residue, indicating that inactivation is primarily due to modification of Cys151. We propose that GMPS-BDB (or AMPS-BDB), which may exist in enolized form in aqueous solution, functions as a reactive analogue of phosphoenolpyruvate and GDP (ADP) to target Cys151 in the active site of pyruvate kinase.

Cysteinyl peptides labeled by dibromobutanedione in reaction with rabbit muscle pyruvate kinase.

The bifunctional reagent 1,4-dibromobutanedione (DBBD) reacts covalently with pyruvate kinase from rabbit muscle to cause inactivation of the enzyme at a rate that is linearly dependent on the reagent concentration, giving a second order rate constant of 444 min-1 M-1. The individual substrates phosphoenolpyruvate (with KCl), ADP, or ATP in the presence of divalent metal cation provide marked protection against inactivation suggesting that reaction occurs in the region of the active site. The limited incorporation of DBBD into pyruvate kinase was measured by reduction of the carbonyl groups of the enzyme-bound reagent using [3H]NaBH4. When pyruvate kinase was reacted with 120 microM DBBD at pH 7.0 for 50 min in the absence of protectants, 1.8 mol of tritium/mol of subunit was incorporated, whereas in the presence of phosphoenolpyruvate with KCl, only 1.0 mol of tritium was incorporated per mole of subunit. Modified peptides were isolated from tryptic digests of pyruvate kinase. Reaction of enzyme in the presence of substrate (showing no activity loss) yielded a single peptide, Asn-Ile-X1-Lys, where X1 corresponds to Cys164 of the known amino acid sequence of muscle pyruvate kinase. In the absence of protectants, reaction for 10 min (when the enzyme retained substantial activity) yielded Asn-Ile-X1-Lys as the major labeled peptide, whereas reaction for 50 min (when the enzyme was 88% inactivated) yielded predominantly Asn-Ile-X1-Lys cross-linked to X2-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys, where X2 corresponds to Cys151. Because activity loss correlates with the appearance of the cross-linked peptides but not with formation of Asn-Ile-X1-Lys, inactivation is likely caused by the reaction leading to the cross-link between Cys151 and Cys164. The distance between the alpha-carbons of these residues in the crystal structure is 15.5 A, whereas only 12.0 A can be spanned by the two side chains linked by a dioxobutyl group, suggesting either that pyruvate kinase undergoes a conformational change in forming the cross-link or that local rapid fluctuations in structure occur in solution to the extent of 3.5 A in this region of pyruvate kinase.

Cysteinyl peptides of rabbit muscle pyruvate kinase labeled by the affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate.

The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) reacts covalently with rabbit muscle pyruvate kinase, incorporating 2 mol of reagent/mol of enzyme subunit upon complete inactivation. Protection against inactivation is provided by phosphoenolpyruvate, K+, and Mn2+ and only 1 mol of reagent/mol of subunit is incorporated [DeCamp, D.L., Lim, S., & Colman, R.F. (1988) Biochemistry 27, 7651-7658]. We have now identified the resultant modified residues. After reaction with 8-BDB-TA-5'-TP at pH 7.0, modified enzyme was incubated with [3H]NaBH4 to reduce the carbonyl groups of enzyme-bound 8-BDB-TA-5'-TP and to introduce a radioactive tracer into the modified residues. Following carboxymethylation and digestion with trypsin, the radioactive peptides were separated on a phenylboronate agarose column followed by reverse-phase high-performance liquid chromatography in 0.1% trifluoroacetic acid with an acetonitrile gradient. Gas-phase sequencing gave the cysteine-modified peptides Asn162-Ile-Cys-Lys165 and Cys151-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys161, with a smaller amount of Asn43-Thr-Gly-Ile-Ile-Cys-Thr-Ile-Gly-Pro-Ala-Ser-Arg55. Reaction in the presence of the protectants phosphoenolpyruvate, K+, and Mn2+ yielded Asn-Ile-Cys-Lys as the only labeled peptide, indicating that inactivation is caused by modification of Cys151 and Cys48.

Identification of the site of acetyl-S-enzyme formation on avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase.

Avian liver mitochondrial hydroxymethylglutaryl-CoA synthase contains an active-site cysteine involved in forming the labile acetyl-S-enzyme intermediate. Identification of and assignment of function to this cysteine have been accomplished by use of an experimental strategy that relies upon generation and rapid purification of the S-acetylcysteine-containing active-site peptide under mildly acidic conditions that stabilize the thioester adduct. Automated Edman degradation techniques indicate the peptide's sequence to be Arg-Glu-Ser-Gly-Asn-Thr-Asp-Val-Glu-Gly-Ile-Asp-Thr-Thr-Asn-Ala-Cys-Tyr. The acetylated cysteine corresponds to position 129 in the sequence deduced from cDNA data for the hamster cytosolic enzyme [Gil, G., Goldstein, J.L., Slaughter, C.A., & Brown, M.S. (1986) J. Biol. Chem. 261, 3710-3716]. The acetyl-peptide sequence overlaps that reported for a tryptic peptide that contains a cysteine targeted by the affinity label 3-chloropropionyl-CoA [Miziorko, H. M., & Behnke, C. E. (1985) J. Biol. Chem. 260, 13513-13516]. Thus, availability of these structural data allows unambiguous assignment of the acetylation site on the protein as well as a refinement of the mechanism explaining the previously observed affinity labeling of the enzyme.

Crystallization and preliminary X-ray data for the general acyl-CoA dehydrogenase.

The general acyl-CoA dehydrogenase from pig liver mitochondria has been crystallized in a form suitable for detailed three-dimensional x-ray structure analysis. Crystals grown from Tris buffer and polyethylene glycol solution diffract to high resolution and have the space group C2221, a = 128.2, b = 136.1, and c = 106.3 A. A measured crystal density of 1.178 g/cm3 and a crystal volume/unit of molecular mass, Vm = 2.5 A3/dalton, suggest that the asymmetric unit contains two monomers of the tetrameric dehydrogenase molecule.