Engelbert Buxbaum

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The long-term (34 weeks) effect of streptozotocin induced diabetes was assessed in Wistar rats. Na ⁺ /K ⁺ -ATPase activity was measured by ouabain inhibitable ⁸⁶ Rb ⁺ -uptake into erythrocytes. No difference in the rate of Rb ⁺ -uptake, the K m for Rb ⁺ or the K i for ouabain was detected between normal and diabetic rats. Thus, the change in Na ⁺ /...

Denaturing, discontinuous electrophoresis in the presence of SDS has become a standard method for the protein scientist. However, there are situations where this method produces suboptimal results. In these cases electrophoresis in the presence of positively charged detergents like cetyltrimethylammonium bromide (CTAB) may work considerably better....

Amino acids are carboxylic acids with an amino-group in the α-position. With the exception of glycine, all amino acids are chiral; usually the L-form is used in living organisms. Twenty-two different amino acids are encoded in genes. Polycondensation of amino acids leads to peptides and proteins. The different side-chains of the various amino acids...

Cells interact not only with each other, but also with the extracellular matrix, which consists of proteoglycan, collagen, elastin, and multiadhesive proteins such as laminin. Both cell/cell and cell/matrix interactions require cell adhesion molecules, some of which occur in cell junctions. Disintegrins and matrix specific metalloprotease (MMP) dis...

All enzymes in a metabolic pathway operating in steady state have the same flux and maintain concentrations of all intermediates. The system will respond to perturbation in such a way that the perturbation is counteracted. This results in homeostasis. Cells can change the flux through a pathway by changing enzyme concentration, switching enzymes on...

Transport from the ER through Golgi-apparatus to plasma membrane, secretory vesicles, or lysosomes occurs in specialised vesicles. Proteins in the plasma membrane, some with bound ligand, are recycled in clathrin-coated vesicles, which after uncoating fuse with the endosome. From there proteins can return to the plasma membrane or move on to the ly...

In addition to their substrates, enzymes also bind substances that reduce their activity. Such binding can be reversible resulting in inhibition, or irreversible resulting in inactivation of the enzyme. We distinguish four types of inhibition: competitive The inhibitor binds only to the free enzyme, not to the enzyme substrate complex. The EI-compl...

Before proteins can be studied, they need to be isolated from all other proteins present in a cell or organism. Differences in size, shape, charge, stability, or binding properties between proteins are utilised, using chromatographical, electrophoretic, or precipitation methods. Before membrane proteins can be purified, they need to be solubilised...

Mitochondria contain their own DNA, they divide throughout interphase and are distributed randomly between daughter cells during cell division. Most mitochondrial proteins are encoded in the nucleus and produced in the cytosol. They are then imported into the mitochondria via the TOM/TIM transport system, which recognises them by a specific localis...

Many important diseases including morbus Alzheimer, Prion diseases, type II diabetes, or some cancers involve protein misfolding. In all these diseases, called amyloidoses, intrinsically disordered proteins fold into \(\upbeta\)-helices. One misfolded protein molecule auto-catalytically converts other protein molecules, leading to aggregation. Intr...

Enzyme and substrate form an ES-complex, which reacts further to enzyme and product. This process requires time, so each enzyme molecule can only handle a certain number of substrate molecules per unit time, called the turnover-number k cat. This number multiplied with the number of enzyme molecules is the limiting reaction velocity, V max, reached...

Our bodies have efficient defence systems against invading pathogens and against cancer cells. We distinguish cellular from humoral and innate from acquired systems. Humoral defences consist of antibacterial polypeptides, Pathogen-associated molecular pattern (PAMP)-receptors, complement systems, and antibodies. Antibacterial polypeptides include e...

This book serves as an introduction to protein structure and function. Starting with their makeup from simple building blocks, called amino acids, the 3-dimensional structure of proteins is explained. This leads to a discussion of how misfolding of proteins causes diseases like cancer, various encephalopathies, or diabetes. Enzymology and modern co...

Abstract Introduction Physicians experience consistent environmental pressures while doing thier work. To give students practice making clinical decisions in a time- and cost-limited scenario, we created this group learning, case-based activity for first-year medical students during their second semester. During this activity students are tasked wi...

Proteins are stabilised by the difference between the bond energies between interacting amino acids and the energy of interaction between these amino acids and water (several MJ∕mol each). Protein folding decreases not only the enthalpy, but also the entropy of the molecule. Hence the total stabilisation energy is ≈ 20–60kJ∕mol which should be comp...

This introduction to NMR must be necessarily brief. Further information can be found in [344, 436] and references therein.

In equilibrium dialysis, the protein and its ligand are separated by a semi-permeable membrane which allows passage of unbound ligand, but not the protein (see Fig. 43.1). Thus the ligand will diffuse across the membrane until the concentration of free ligand at both sides of the membrane will be identical. Since the RL-complex can not diffuse acro...

Some reactions proceed via an intermediate that is stable enough to be detected with sensitive isotope techniques.One example is the transfer of the γ -phosphate group to aspartic acid in P-type ATPases, forming an acyl-phosphate intermediate, from which ADP is released followed by dephosphorylation:

The microscope is without question the most important instrument available to the biologist. The physiological function of proteins cannot be addressed without taking their localisation in a living cell and their interaction with other proteins into account. To answer these questions the microscope is an invaluable tool. http://micro.magnet.fsu.edu...

For a more detailed, quantitative treatment of electrophoresis see [60, 428]. Simulations for electrophoretic experiments are available on the web (http://www.rit.edu/~pac8612/electro/E_Sim.html or http://www.jvirgel.de/).

An important step in the characterisation of a new protein is the determination of its primary structure (amino acid sequence). This can be determined either by repeated chemical cleavage of terminal amino acids (either from the N- or C-terminal end, see Fig. 30.1 and Fig. 30.2 respectively), or by MS/MS.

The following methods rely on the specific recognition of ligands by a protein. Although they are described here (as in most other textbooks) for antibody-antigen interaction, you should keep in mind that they can also be used for other specific interactions, like ligand-receptor. [146] should be consulted first for experimental details, despite it...

In the last chapter we have seen how to determine the sequence of a protein. For many purposes it would be useful to synthesise proteins of a given sequence. The solid-phase approach of Merrifield (Fig. 31.1) serves this purpose.

An important consideration in protein modification is the length of the spacer-arm. Fluorescence quantum yield of many labels, for example, is reduced if the label is too close to the protein. Steric hindrance may prevent the binding of proteins to affinity matrices if spacer length is insufficient. For special applications labile groups may be int...

Assume two compartments separated by a semipermeable membrane (see Fig. 26.1). One compartment contains pure solvent, which can freely cross the membrane, for example, water. The other compartment contains solvent plus a solute, which is too large to cross the membrane, say, a protein. If both compartments are open to the atmosphere – that is, expo...

The simplest device to measure interaction of a sample with light is the absorbance spectrophotometer. Monochromatic light is passed through the sample (or, in some cases, reflected from it). If absorption occurs, the light intensity that arrives at a detector is lower than what would arrive in the absence of the sample.

Many analytical techniques involve the interaction of a sample with light (IR, visible and UV). While microwave radiation excites rotation of a molecule (ΔE ≈ 4kJ∕mol) and IR-light oscillatory movement of atoms around their position in a molecule (vibration, ΔE ≈ 40kJ∕mol), visible and UV light move electrons to higher energy levels (ΔE ≈ 400kJ∕mol...

Assume a sample of a fairly dilute protein solution, 1nM ( = 50ng∕ml if M r = 50kDa). Then 1μl will contain 1fmol of protein. Multiplied with Avogadro’s number (6. 022e23mol−1) this still corresponds to 6e8 protein molecules.

Molecules move by random Brownian motion so that over time all concentration differences are evened out and the entropy of the system is maximised. Fick’s first law of diffusion (for simplicity here for the 1-D case) states that the net flux J is proportional to the concentration difference and the pathlength x: $$J = -D\left (\frac{\partial c} {\p...

One would like to think of proteins as relatively stable entities, at least in the absence of modifying enzymes. Alas, this view is mistaken. As time passes, proteins can undergo spontaneous modifications that change their structure and function.

However, the precision for the parameters V max and K m determined this way is much lower than by the conventional variation of [ S ]. Also, since all measurements are obtained from a single experiment, they are highly correlated and normal least-square fitting techniques give artificially low error estimates for the parameters. Therefore, this met...

There are several methods available that can be used to gain information on secondary structure, apart from X-ray crystallography, which remains the ‘gold standard’.

Aliphatic amines ( ε -amino group of Lys) are moderately nucleophilic when deprotonated (beyond pH 9 for the free amino acid, but often much lower in proteins); on the other hand, the inactivation of the labelling reagent by water increases with pH. The α -amino group has a pKa of 7. 6–8. 5, thus the N-terminus of a protein can be labelled specific...

One of the most sensitive detection methods available is chemiluminescence. Chemiluminescent substrates undergo reactions, where the product is in an excited state, usually the first triplet (T1) state (see Fig.7.1). By internal systems crossing (spin-reversal, slow!) it is converted to the first singlet (S1) state, and reaches the ground state (S0...

We are looking at the following reaction: R + L ⇌ RL. Since for binding experiments the concentration of protein needs to be much higher than for the determination of enzyme turnover to obtain a measurable signal, the free substrate concentration ([F]) can no longer be assumed to be equal to the total substrate concentration ([ T ]). Thus the bindi...

Cells from plants and animals – including humans – can be grown in culture if their requirements are met with respect to medium composition and temperature. The following paragraphs will focus on the culture of mammalian cells, even though the culture of insect and plant cells is of considerable economic and scientific interest. More detailed infor...

Fluorescence can be used to determine the concentration of a substance quantitatively (see Fig. 6.1). A more thorough introduction into the various fluorescent techniques is given in [219]. Even very specialised techniques, discussed below, can also be performed on microscopes [141, 142].

If you want to study, say, the enzymes present in the liver, the first thing you have to do is obtain fresh liver tissue. This is done at the abattoir immediately after an animal has been slaughtered. The tissue is transported into the laboratory on ice to minimise proteolytic damage.

The reactivity of alcohols in aqueous solutions is low, most reagents prefer to react with more nucleophilic groups like amine or thiol.

There are two principal routes to radio-labelled proteins: metabolic labelling in vivo with labelled amino acids (in particular 35S labelled Cys and/or Met) and chemical introduction in vitro. Metabolic labelling has the advantage that it will not interfere with the proteins function, however, only a small part of the radioactivity used ends up in...

Usually we use mathematical models to describe our data. Regression methods (linear or non-linear) are then used to determine the parameters of such models that result in the best fit, for example by the least-squares criterium. There is one problem with this approach, however: If the model is inappropriate then the fitting procedure will spit out...

Bifunctional reagents may be used to cross-link proteins (see Fig. 22.1). They may be homo-bifunctional (two identical reactive groups) or hetero-bifunctional, for example with a thiol- (maleimides) and an amino-specific end (NHS-ester). Intra-molecular cross-links are favoured over inter-molecular ones by low protein concentration, a high net char...

After homogenisation the various cell organelles need to be separated from each other. This is done by fractionated centrifugation [103]. First, connective tissue, undamaged cells, and other debris are removed by a brief spin at low speed (10min at 500\(\mathfrak{g}\)). In the next step nucle and plasma membranes are spun down (10min at 3 000 \(\ma...

In principle, any technique that separates bound from unbound ligand, or occupied from unoccupied protein, can be used to monitor binding. As a general rule, highest accuracy is achieved when the concentration of the protein in binding studies is approximately equal to K d (within about one order of magnitude either way), this makes it easier to de...

To ease removal of enzymes from the product and their recycling, enzymes are often immobilised. Immobilisation may also be required to convert batch into continuous processes.

Compared to conventional chemical methods, enzymatic processes have the following advantages:Milder reaction conditions (temperature, pressure, pH) making the process cheaper and safer Avoid environmentally unfriendly reactants Fewer reaction steps Fewer side products leading to easier purification Stereoselectivity

Literally thousands of detailed recipes and procedures are given in the handbook started by Romeis [286]—this should be consulted before embarking on such studies.

Radioactivity is a phenomenon that can be detected with high sensitivity which can be matched only by chemiluminescence and mass spectrometry. This makes radio-labelling a favourite technique in biochemistry. For this reason the required equipment is almost universally available.

Protein precipitation is a very crude method for purification and it rarely achieves enrichment by more than a factor of 2–3. However, it is quick and cheap. If applied to crude homogenisates, it may remove material that would interfere with later purification steps. It is essential to perform these reactions in the cold because proteins are rapidl...

For a brief history of the field and more detailed discussions of the method see [65], [62–64, 68]. Isotope effects can be used to investigate reaction mechanism and transition state structure.

This type of investigation is quite important for the characterisation of an enzyme: $$\Delta {G'}^{0} = -R {_\ast} T {_\ast}\ln (1/{K}_{\mathrm{ d}})\qquad \mathrm{at}\,\,25\mathrm{\!\circ \mathrm{C}}$$ (44.1) $$\ln ({K}_{\mathrm{d}}) = -\dfrac{\Delta {H'}^{0}} {R} {_\ast} \dfrac{1} {T} + A$$ (44.2) $$\Delta {S'}^{0} = \dfrac{\Delta {H'}^{0} - \De...

A nice introduction into membrane biology is [247].

Interaction between molecules slows down their movement; this intermolecular friction is observed macroscopically as viscosity.

Centrifugation is used for preparative and analytical separation of molecules and organelles (see [103]); centrifugation is one of the techniques used to determine the molecular mass from first principle, rather than by comparison with standard proteins. The other two are osmometry (see Chap. 26 on p. 245) and laser light scattering (see Sect. 29.1...

K d and K m values are relatively easy to determine, but the underlying rate constants often require special equipment to measure fast and very fast reactions. This is usually available only in specialised departments.

A more detailed description of Electron spin resonance (ESR) than possible here may be found in [334].

The theoretical aspects of enzyme activity, inhibition, and inactivation have been discussed in a separate volume [44]. More extensive treatments of both theory and practice of enzyme kinetics may be found in [27, 68, 69]. Ready-to use rate equations for most mechanisms may be found in the otherwise dated monography by Segel [372], rate equations m...

Quality control and management is an important function for the head of a laboratory, even in places not regulated by GLP. Several cases in the last years where respected labs had to withdraw publications because of measurement error or data forgery by subordinates, demonstrate this need. Textbooks on quality management should be consulted [298, 31...

Undergraduate biochemistry courses cover what proteins do, as enzymes, receptors, hormones, motors or structural components. The much more interesting question is how can proteins achieve all these functions? Presented here is an overview of the methods used in such projects, their possible applications, and their limitations. Focusing on the bioph...

We propose a novel strategy applicable to case‐based learning during the initial years of a medical curriculum. Step 1 presents a clinical vignette that requires students to obtain additional information. Step 2: Students are provided access to an on‐line database containing results of physical exams (PE). Students must pay “PE bucks” to obtain add...

This book serves as an introduction to the fundamentals of protein structure and function. Starting with their make up from simple building blocks called amino acids, the 3-dimensional structure of proteins is explained. This is followed by an introduction into enzymology and modern concepts of enzyme kinetics, taking into account the physiological...

Metazoans consist of many different cell types, whose functions are closely interlinked. Only together can they perform all the functions that characterise a living organism: Attaining shape, movement, perception, metabolism and reproduction.

Amino acids are compounds which have a carboxy group at one end and an amino group at the carbon atom next to the carboxy group, the so called α-carbon (see fig. 1.1). Several amino acids contain additional acidic or basic groups.

Enzymatic turnover of substrates requires the formation of an enzyme substrate complex and finally the dissociation of the enzyme-product complex

Mitochondria and plastids were derived from endosymbiont bacteria about 1-1.5 x 109 a ago, and contain their own DNA (see fig. 15.1). There may be several mtDNA molecules in each mitochondrium, the exact number is species specific. The mtDNA is replicated and the mitochondria divide during the entire interphase. Division of mitochondria starts with...