Identification of geranylgeranyl-modified proteins in HeLa cells.

Previous studies have shown that animal cells contain isoprenoid-modified proteins and that one of these proteins, lamin B, contains a thioether-linked farnesyl group that is attached to cysteine. In the present study, a novel isoprenoid-modification was identified by labeling HeLa cells with [3H]mevalonic acid and analyzing proteolytic digests of the total cell protein. Radioactive fragments were purified from these digests and treated with Raney nickel.

Human lamin B contains a farnesylated cysteine residue.

We recently showed that HeLa cell lamin B is modified by a mevalonic acid derivative. Here we identified the modified amino acid, determined its mode of linkage to the mevalonic acid derivative, and established the derivative's structure. A cysteine residue is modified because experiments with lamin B that had been biosynthetically labeled with [3H]mevalonic acid or [35S]cysteine and then extensively digested with proteases yielded 3H- or 35S-labeled products that co-chromatographed in five successive systems.

Taking the mystique out of the diagnosis and treatment of craniomandibular (TMJ) disorders.

The temporomandibular joint (TMJ) is of great importance in the assessment, diagnosis and treatment of many craniofacial disorders. Frequently overlooked by medical practitioners as a source of patient discomfort, the key to understanding the joint and its role in abnormal conditions is the complex nature of its structure. Returning to biological fundamentals provides an essential foundation on which to build techniques for diagnosis and ultimately treatment regimens.

Competitive inhibition of phospholipase A2 in vesicles.

Kinetic studies with phospholipase A2 are complicated by the fact that binding of the enzyme to the interface precedes catalytic turnover. This difficulty can be overcome by monitoring interfacial catalysis in the scooting mode where the enzyme does not leave the interface. The kinetics of inhibition by transition-state analogues shows that specific competitive inhibition is the result of competition between inhibitor and substrate for the binding to the active site of the enzyme in the interface.

Human plasma lecithin-cholesterol acyltransferase. Inhibition of the phospholipase A2-like activity by sn-2-difluoroketone phosphatidylcholine analogues.

Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the sn-2-fatty acid of lecithin to cholesterol, forming lysolecithin and cholesteryl ester. We have recently proposed a covalent catalytic mechanism for LCAT in which lecithin cleavage proceeds via the formation of a transition state tetrahedral adduct between the oxygen atom of the catalytic serine residue and the sn-2-carbonyl carbon atom of the substrate (Jauhiainen, M., Ridgway, N.

Bacterial N-succinyl-L-diaminopimelic acid desuccinylase. Purification, partial characterization, and substrate specificity.

The enzyme N-succinyl-L-diaminopimelic acid desuccinylase from Escherichia coli has been purified 7,100-fold to apparent homogeneity. The enzyme is part of the diaminopimelic acid-lysine pathway in bacteria and catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to produce L-diaminopimelic acid and succinate. The enzyme exists as a mixture of dimeric and tetrameric species of identical subunits of molecular weight approximately 40,000.

Fluoro ketone containing peptides as inhibitors of human renin.

The pentapeptide BOC-Phe-Phe-difluorostatone-Leu-Phe-NH2 has been prepared and found to be a potent inhibitor of human renin. This compound contains a difluoromethylene ketone group that exists predominantly in the hydrated form in water. The difluorostatone-containing peptide is 7-fold and 22-fold more potent than the analogous statine- and statone-containing peptides, respectively.

X-ray diffraction analysis of the inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone.

The inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone has been studied. A covalent adduct is formed that deacylates slowly with a half-life of 23 h. X-ray diffraction analysis at 1.

Substituted isatoic anhydrides: selective inactivators of trypsin-like serine proteases.

Derivatives of isatoic anhydride were prepared and tested as inhibitors of serine proteases. A number of isatoic anhydrides with positively charged substituents irreversibly inactivated several trypsin-like enzymes and preferentially inactivated trypsin over chymotrypsin. Further selectivity was obtained by introduction of an aromatic group on the N-1 position of isatoic anhydride.

Fluoro ketone inhibitors of hydrolytic enzymes.

The use of fluoro ketones as inhibitors of hydrolytic enzymes has been investigated. The acetylcholine analogues 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone are inhibitors of acetylcholinesterase with Ki values of 16 X 10(-9) M and 1.6 X 10(-9) M, respectively.

Inactivation of chymotrypsin by 5-benzyl-6-chloro-2-pyrone: 13C NMR and X-ray diffraction analyses of the inactivator-enzyme complex.

The inactivation of chymotrypsin by 5-benzyl-6-chloro-2-pyrone has been studied. Chloride analysis of the inactivated enzyme suggests that chlorine is no longer present in the complex. 13C NMR spectroscopy of chymotrypsin inactivated with 5-benzyl-6-chloro-2-pyrone-2,6-13 C2 shows the presence of two new resonances from the protein-bound inactivator.

Mechanism of inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone.

The mechanism of inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone has been studied. Chloride analysis of the inactivated enzyme suggests that the complex does not contain intact chloropyrone or an acid chloride. 13C NMR studies of the enzyme inactivated with 13C-enriched chloropyrones show that (1) the pyrone ring is no longer intact, (2) C-6 becomes a carboxylate group and C-2 becomes esterified to the enzyme, probably to serine-195, and (3) a double bond is present adjacent to the serine ester.

The effect of proximal cavity design on the cementation of Class II inlays.

This article indicates that the gingival opening of a two-surface cast restoration after cementation is minimized by the use of a preparation with proximal grooves.

Bio-organic chemistry and cytochrome P-450-dependent catalysis.

This review presents current ideas, models and experimental data relating to the precise chemistry that links the transition metal active centre of cytochrome P-450 systems, the unactivated alkane substrate and the triplet atmospheric dioxygen molecule. Aspects considered include the hypervalent transition metal, the reductive activation of the dioxygen molecule by two electrons as an intermediate in the four-equivalent oxidase mechanism, and the details of carbon-hydrogen and carbon-carbon fragmentation. Studies of the microbial camphor 5-exo hydroxylase system are used to exemplify the principles discussed.

Chemical mechanisms for cytochrome P-450 oxidation: spectral and catalytic properties of a manganese-substituted protein.

Bacterial cytochrome P-450 induced by camphor (P-450cam) is reconstituted with manganese-protoporphyrin IX, yielding an enzyme that displays unique spectral properties relative to previously characterized manganese-porphyrin systems. The nitric oxide complex of the manganese(II)-protein shows a hyper-metalloporphyrin spectrum suggestive of thiolate ligation to the porphyrin-bound manganese ion. In the presence of iodosobenzene as a source of active oxygen, manganese-substituted cytochrome P-450cam serves as a catalyst for the epoxidation of an enzyme-bound olefin substrate.

Stereochemistry and deuterium isotope effects in camphor hydroxylation by the cytochrome P450cam monoxygenase system.

Bacterial cytochrome P450cam catalyzes the hydroxylation of camphor to yield 5-exo-hydroxycamphor in vivo and in a reconstituted system with oxygen, pyridine nucleotide, flavoprotein dehydrogenase, and putidaredoxin. Product is also formed when the ferric form of the hemoprotein is mixed with the exogenous oxidants iodosobenzene, m-chloroperbenzoic acid, and hydrogen peroxide. In this paper we show that when the P450cam-dependent hydroxylation reactions are studied with camphor analogues containing deuterium at either the 5-exo or 5-endo position, a very small intermolecular isotope on the overall reaction velocity is observed and a significant intramolecular isotope effect is documented.

Interaction of 5-bromocamphor with cytochrome P-450 cam. Production of 5-ketocamphor from a mixed spin state hemoprotein.

Camphor is stereospecifically hydroxylated by the soil bacterium Pseudomonas putida at the 5-exo position by a cytochrome P-450 mixed function oxidase system consisting of a flavoprotein reductase; putidaredoxin, an iron-sulfur oxidation-reduction transport-effector protein; and the P-450 hemoprotein. We have studied the interaction of a substrate analog of camphor, 5-exo-bromocamphor, with this cytochrome P-450 mixed function oxidase system in order to probe the molecular mechanisms of electron transport and catalytic substrate oxygenation. 5-exo-Bromocamphor was found to bind tightly in a 1:1 complex with P-450 with a dissociation constant of 2.

Binding of xylenol orange to transferrin. Demonstration of metal-anion linkage.

The iron transport protein, transferrin, binds two metal ions and, concomitantly, two carboxylate anions. The metal ion indicators, xylenol orange and semi-xylenol orange are carboxylate anions which exhibit a characteristic visible spectrum when attached to a metal. We prepared the ternary complexes VO2+-transferrin-xylenol orange and VO2+-transferrin-semi-xylenol orange.