Marko Goličnik

Combined gas chromatographic/mass spectrometric analysis of cholesterol precursors and plant sterols in cultured cells

We developed a powerful gas chromatographic/mass spectrometric method allowing quantitative analysis of 11 structurally similar cholesterol precursors and plant sterols (squalene, desmosterol, 7-dehydrocholesterol, lathosterol, zymosterol, dihydro-lanosterol, lanosterol, FF-MAS, T-MAS, campesterol, sitosterol) from cultured human hepatocytes in a single chromatographic run. Deuterium labelled cholesterol, sitosterol and lathosterol were used as internal standards. Care was taken to select ions for the detection that gave the most appropriate discrimination in the assay. Replicate analyses gave a coefficient of variation less than 6%. Recovery experiments were satisfactory for 7-dehydrocholesterol, campesterol, desmosterol, lathosterol, zymosterol and cholesterol with less than 7% difference between expected and found levels. For other sterols, the difference between expected and found levels varied between 10 and 16%. It is concluded that this method is suitable for studies on the effect of different inhibitors and stimulators of cholesterol synthesis in cultured cells. Additionally, the method is relevant also for clinical applications since abnormally increased late cholesterol intermediates in patients are representations of the inherited disorders linked to different enzyme defects in the post-squalene cholesterol biosynthesis.

A Trojan horse transition state analogue generated by MgF3− formation in an enzyme active site

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. β-Phosphoglucomutase catalyses the isomerization of β-glucose-1-phosphate to β-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using 19F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF3− mimics the transferring PO3− moiety. Here we present a detailed characterization of the metal ion–fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of β-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.

Determination of reference genes for circadian studies in different tissues and mouse strains

BackgroundCircadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration.ResultsIn the present study the expression of ten candidate reference genes (Actb, Eif2a, Gapdh, Hmbs, Hprt1, Ppib, Rn18s, Rplp0, Tbcc and Utp6c) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and Crem KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (Actb) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons.ConclusionsGenetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology.

The Interplay of cis-Regulatory Elements Rules Circadian Rhythms in Mouse Liver

The mammalian circadian clock is driven by cell-autonomous transcriptional feedback loops that involve E-boxes, D-boxes, and ROR-elements. In peripheral organs, circadian rhythms are additionally affected by systemic factors. We show that intrinsic combinatorial gene regulation governs the liver clock. With a temporal resolution of 2 h, we measured the expression of 21 clock genes in mouse liver under constant darkness and equinoctial light-dark cycles. Based on these data and known transcription factor binding sites, we develop a six-variable gene regulatory network. The transcriptional feedback loops are represented by equations with time-delayed variables, which substantially simplifies modelling of intermediate protein dynamics. Our model accurately reproduces measured phases, amplitudes, and waveforms of clock genes. Analysis of the network reveals properties of the clock: overcritical delays generate oscillations; synergy of inhibition and activation enhances amplitudes; and combinatorial modulation of transcription controls the phases. The agreement of measurements and simulations suggests that the intrinsic gene regulatory network primarily determines the circadian clock in liver, whereas systemic cues such as light-dark cycles serve to fine-tune the rhythms.

CREM modulates the circadian expression of CYP51, HMGCR and cholesterogenesis in the liver

We show for the first time that isoforms of the cAMP response element modulator Crem, regulate the circadian expression of Cyp51 and other cholesterogenic genes in the mouse liver. In the wild type mice the expression of Cyp51, Hmgs, Fpps, and Sqs is minimal between CT12 and CT16 and peaks between CT20 and CT24. Cyp51, Fpps, and Sqs lost the circadian behavior in Crem-/- livers while Hmgcr is phase advanced from CT20 to CT12. This coincides with a phase advance of lathosterol/cholesterol ratio, as detected by GC-MS. Overexpression of CREMtau and ICER has little effect on the Hmgcr proximal promoter while they influence expression from the CYP51 promoter. Our data indicate that Crem-dependent regulation of Cyp51 in the liver results in circadian expression of this gene. We propose that cAMP signaling might generally be involved in the circadian regulation of cholesterol synthesis on the periphery.

Explicit reformulations of time-dependent solution for a Michaelis–Menten enzyme reaction model

The exact closed-form solution to the Michaelis-Menten equation is expressed in terms of the Lambert W(x) function. However, the utility of this solution is limited because the W(x) function is not widely available in curve-fitting software. Based on various approximations to the W(x) function, different explicit equations expressed in terms of the elementary functions are proposed here as useful shortcuts to fit time depletion of substrate concentration directly to progress curves using commonly available nonlinear regression computer programs. The results are compared with those obtained by fitting other algebraic equations that have been proposed previously in the literature.

Kinetic analysis of beta-phosphoglucomutase and its inhibition by magnesium fluoride.

The isomerization of beta-glucose-1-phosphate (betaG1P) to beta-glucose-6-phosphate (G6P) catalyzed by beta-phosphoglucomutase (betaPGM) has been examined using steady- and presteady-state kinetic analysis. In the presence of low concentrations of beta-glucose-1,6-bisphosphate (betaG16BP), the reaction proceeds through a Ping Pong Bi Bi mechanism with substrate inhibition (kcat = 65 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.7 microM, Ki = 122 microM). If alphaG16BP is used as a cofactor, more complex kinetic behavior is observed, but the nonlinear progress curves can be fit to reveal further catalytic parameters (kcat = 74 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.8 microM, Ki = 122 microM, K(alphaG16BP) = 91 microM for productive binding, K(alphaG16BP) = 21 microM for unproductive binding). These data reveal that variations in the substrate structure affect transition-state affinity (approximately 140,000-fold in terms of rate acceleration) substantially more than ground-state binding (110-fold in terms of binding affinity). When fluoride and magnesium ions are present, time-dependent inhibition of the betaPGM is observed. The concentration dependence of the parameters obtained from fitting these progress curves shows that a betaG1P x MgF3(-) x betaPGM inhibitory complex is formed under the reaction conditions. The overall stability constant for this complex is approximately 2 x 10(-16) M(5) and suggests an affinity of the MgF3(-) moiety to this transition-state analogue (TSA) of < or = 70 nM. The detailed kinetic analysis shows how a special type of TSA that does not exist in solution is assembled in the active site of an enzyme. Further experiments show that under the conditions of previous structural studies, phosphorylated glucose only persists when bound to the enzyme as the TSA. The preference for TSA formation when fluoride is present, and the hydrolysis of substrates when it is not, rules out the formation of a stable pentavalent phosphorane intermediate in the active site of betaPGM.

Exact and Approximate Solutions for the Decades-Old Michaelis-Menten Equation: Progress-Curve Analysis through Integrated Rate Equations.

The Michaelis‐Menten rate equation can be found in most general biochemistry textbooks, where the time derivative of the substrate is a hyperbolic function of two kinetic parameters (the limiting rate V, and the Michaelis constant KM) and the amount of substrate. However, fundamental concepts of enzyme kinetics can be difficult to understand fully, or can even be misunderstood, by students when based only on the differential form of the Michaelis‐Menten equation, and the variety of methods available to calculate the kinetic constants from rate versus substrate concentration “textbook data.” Consequently, enzyme kinetics can be confusing if an analytical solution of the Michaelis‐Menten equation is not available. Therefore, the still rarely known exact solution to the Michaelis‐Menten equation is presented here through the explicit closed‐form equation in terms of the Lambert W(x) function. Unfortunately, as the W(x) is not available in standard curve‐fitting computer programs, the practical use of this direct solution is limited for most life‐science students. Thus, the purpose of this article is to provide analytical approximations to the equation for modeling Michaelis‐Menten kinetics. The elementary and explicit nature of these approximations can provide students with direct and simple estimations of kinetic parameters from raw experimental time‐course data. The Michaelis‐Menten kinetics studied in the latter context can provide an ideal alternative to the 100‐year‐old problems of data transformation, graphical visualization, and data analysis of enzyme‐catalyzed reactions. Hence, the content of the course presented here could gradually become an important component of the modern biochemistry curriculum in the 21st century. Biochemistry and Molecular Biology Education Vol. 39, No. 2, pp. 117–125, 2011

Multi-step analysis as a tool for kinetic parameter estimation and mechanism discrimination in the reaction between tight-binding fasciculin 2 and electric eel acetylcholinesterase

The mechanism of action of a potent peptidic inhibitor fasciculin 2 (Fas2) on electric eel acetylcholinesterase (eleelAChE) has been examined in a three-level analysis. Classical steps included equilibration experiments for the evaluation of high affinity binding constant and the existence of residual hydrolytic activity in a solution of completely Fas2 saturated enzyme. The two rate constants for the association (k(on)) and the dissociation (k(off)) of Fas2 with free enzyme were determined by the time course of residual enzyme activity measurements. In the third step, with a nonclassical progress curve analysis, we found that the Fas2-enzyme complex exhibited hydrolytic activity in a butyrylcholinesterase-like kinetics. The switch appears to be a consequence of steric obstruction, but also the consequence of subtle rapid conformational changes around catalytic site, upon slow single-step binding of large Fas2 molecule at the peripheral site. An unusual unilateral effect of bound Fas2 is reflected by acylation-independent association and dissociation rates and might indeed be due to inability of small acylation agent to influence the binding of a large opponent.

Explicit analytic approximations for time-dependent solutions of the generalized integrated Michaelis-Menten equation.

Various explicit reformulations of time-dependent solutions for the classical two-step irreversible Michaelis-Menten enzyme reaction model have been described recently. In the current study, I present further improvements in terms of a generalized integrated form of the Michaelis-Menten equation for computation of substrate or product concentrations as functions of time for more real-world, enzyme-catalyzed reactions affected by the product. The explicit equations presented here can be considered as a simpler and useful alternative to the exact solution for the generalized integrated Michaelis-Menten equation when fitted to time course data using standard curve-fitting software.