I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli

Although cancer is a diverse set of diseases, cancer cells share a number of adaptive hallmarks. Dysregulated pH is emerging as a hallmark of cancer because cancers show a 'reversed' pH gradient with a constitutively increased intracellular pH that is higher than the extracellular pH. This gradient enables cancer progression by promoting proliferation, the evasion of apoptosis, metabolic adaptation, migration and invasion. Several new advances, including an increased understanding of pH sensors, have provided insight into the molecular basis for pH-dependent cell behaviours that are relevant to cancer cell biology. We highlight the central role of pH sensors in cancer cell adaptations and suggest how dysregulated pH could be exploited to develop cancer-specific therapeutics.

Dysregulated pH: a perfect storm for cancer progression.

Short-chain dehydrogenases/reductases (SDR) constitute a large protein family. Presently, at least 57 characterized, highly different enzymes belong to this family and typically exhibit residue identities only at the 15-30% level, indicating early duplicatory origins and extensive divergence. In addition, another family of 22 enzymes with extended protein chains exhibits part-chain SDR relationships and represents enzymes of no less than three EC classes. Furthermore, subforms and species variants are known of both families. In the combined SDR superfamily, only one residue is strictly conserved and ascribed a crucial enzymatic function (Tyr 151 in the numbering system of human NAD(+)-linked prostaglandin dehydrogenase). Such a function for this Tyr residue in SDR enzymes in general is supported also by chemical modifications, site-directed mutagenesis, and an active site position in those tertiary structures that have been characterized. A lysine residue four residues downstream is also largely conserved. A model for catalysis is available on the basis of these two residues. Binding of the coenzyme, NAD(H) or NADP(H), is in the N-terminal part of the molecules, where a common GlyXXXGlyXGly pattern occurs. Two SDR enzymes established by X-ray crystallography show a one-domain subunit with seven to eight beta-strands. Conformational patterns are highly similar, except for variations in the C-terminal parts. Additional structures occur in the family with extended chains. Some of the SDR molecules are known under more than one name, and one of the enzymes has been shown to be susceptible to native, chemical modification, producing reduced Schiff base adducts with pyruvate and other metabolic keto derivatives. Most SDR enzymes are dimers and tetramers. In those analyzed, the area of major subunit contacts involves two long alpha-helices (alpha E, alpha F) in similar and apparently strong subunit interactions. Future possibilities include verification of the proposed reaction mechanism and tracing of additional relationships, perhaps also with other protein families. Short-chain dehydrogenases illustrate the value of comparisons and diversified research in generating unexpected discoveries.

Short-chain dehydrogenases/reductases (SDR).

Fluorescence spectroscopy, one of the most informative and sensitive analytical techniques, has played and continues to play key roles in modern research. Indeed, unraveling the inner workings of biomolecules, cells and organisms relied on the development of fluorescence-based tools. As many of the players in these sophisticated interactions and exceedingly complex systems are not inherently emissive, researchers have relied on synthesizing fluorescent analogs of the building blocks found in biological macromolecules. These are the constituents of the cell surface and cell membrane, as well as proteins and nucleic acids. This review article is dedicated to emissive analogs of these relatively small molecules.

For organizational purposes, we have arbitrarily selected to approach these diverse families of biomolecules by imagining “a journey into the center of the cell”. Approaching the exterior of a cell, one first encounters oligosaccharides that decorate the cell surface and are involved in cell recognition and signaling. Next, we arrive at the cell membrane itself. This semi-permeable envelope sets the cell boundaries and regulates its traffic. Several types of building blocks assemble this membrane, most notably among them are the phospholipids. Upon entering the cell, the cytosol reveals a plethora of small and large molecules, including proteins, as well as soluble RNA molecules and RNA-rich ribosomes. Within the cytosol of eukaryotes and prokaryotes lies the nucleus or nucleoid, respectively. This membrane-enclosed control center contains most of the cells’ genetic material. DNA, the cellular blueprint, is permanently found in the nucleus, which also hosts diverse RNA molecules. Accordingly, we first discuss emissive carbohydrate derivatives. We then present fluorescent membrane constituents, followed by emissive amino acids. Our journey ends by focusing on emissive analogs of nucleosides and nucleotides, the building blocks of nucleic acids.

The common biomolecular building blocks, excluding a few amino acids, lack appreciably useful fluorescence properties. This implies that structural modifications are required to impart such photophysical features. Ideally, a designer probe should closely resemble its natural counterpart in size and shape without the loss of the original function (a feature we refer to as “isomorphicity”). This presents a fundamental predicament, as any modification attempting to alter the electronic nature of a molecule, typically by including aromatic residues or extending conjugation, will also alter its steric bulk and therefore the interactions with its surroundings.

Clearly not all biomolecular building blocks can or need to accommodate strict isomorphic design criteria. The heterocycles found in nucleosides already provide a platform that facilitates the extension of π-conjugation, which is also true for some aromatic amino acids. In contrast, employing fluorescence spectroscopy to membrane research requires very creative probe designs. Saccharides can be viewed as the most restrictive in this context, as no chemical modification is conceivable without a major structural disruption and likely loss of function. Such aliphatic biomolecules accommodate labeling only, where an established fluorophore is covalently conjugated to provide an emissive derivative. We therefore reserve the term probe to molecular designs that are expected to furnish useful modified biomolecules capable of reliable reporting. Understandably, fluorescent probes must meet the most stringent isomorphic design principles to ensure a biologically meaningful read-out. The isomorphic design principle is therefore a central theme of this review.

This article focuses on designing fluorescent probes for the four major families of macromolecular building blocks discussed above. Although not necessarily in chronological order, it spans roughly four decades of probe design with emphasis, when justified, on recent contributions. As the reader may imagine, this topic encapsulates a vast research field and cannot be comprehensively reviewed within the space limitation of Chemical Reviews. Nevertheless, we have attempted to summarize the most important and general contributions discussing fluorescent probes that were designed to shed light on biological processes and refer the reader to other resources.1 Although a few examples have found their way into the text, we do not generally address here the development of small molecule fluorophores and sensors that are not part of biomolecular assemblies. We open this article with a brief overview of the key features of fluorescence spectroscopy, where essential theoretical, experimental, and practical elements are discussed.

/pdf/fluorescent-analogs-of-biomolecular-building-blocks-design-11xiwhr1gd.pdf

Fluorescent Analogs of Biomolecular Building Blocks: Design, Properties, and Applications

Amino acid sequence comparisons reveal that tyrosine-152 and lysine-156 of Drosophila alcohol dehydrogenase (ADH) are conserved in homologous dehydrogenases, suggesting that these residues are important in catalysis. To test this hypothesis, we used site-directed mutagenesis to substitute tyrosine-152 with phenylalanine, histidine, or glutamic acid or to substitute lysine-156 with isoleucine. All of these mutants are catalytically inactive. Two mutants were active: A cysteine mutation of tyrosine-152 has 0.25% of wild-type ADH activity, while an arginine substitution of lysine-156 retains 2.2% of wild-type ADH activity. Kinetic analysis shows that the cysteine mutant increases Km(ethanol) 56-fold and Km(propan-2-ol) 100-fold, while Km(NAD) values are essentially unaltered. The arginine mutant also shows the significant enlargement of Km(ethanol), but not of Km(NAD). Furthermore, the cysteine mutant and arginine mutant have different substrate specificity and behave differently on competitive inhibition than wild-type ADH. These results suggest that both tyrosine-152 and lysine-156 have essential roles in catalysis by Drosophila ADH.

Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis

Drosophila alcohol dehydrogenase (ADH), an NAD(+)-dependent dehydrogenase, shares little sequence similarity with horse liver ADH. However, these two enzymes do have substantial similarity in their secondary structure at the NAD(+)-binding domain [Benyajati, C., Place, A. P., Powers, D. A. & Sofer, W. (1981) Proc. Natl Acad. Sci. USA 78, 2717-2721]. Asp38, a conserved residue between Drosophila and horse liver ADH, appears to interact with the hydroxyl groups of the ribose moiety in the AMP portion of NAD+. A secondary-structure comparison between the nucleotide-binding domain of NAD(+)-dependent enzymes and that of NADP(+)-dependent enzymes also suggests that Asp38 could play an important role in cofactor specificity. Mutating Asp38 of Drosophila ADH into Asn38 decreases Km(app)NADP 62-fold and increases kcat/Km(app)NADP 590-fold at pH 9.8, when compared with wild-type ADH. These results suggest that Asp38 is in the NAD(+)-binding domain and its substituent, Asn38, allows Drosophila ADH to use both NAD+ and NADP+ as its cofactor. The observations from the experiments of thermal denaturation and kinetic measurement with pH also confirm that the repulsion between the negative charges of Asp38 and 2'-phosphate of NADP+ is the major energy barrier for NADP+ to serve as a cofactor for Drosophila ADH.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1991.tb16371.x

Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase.

Three amino acid residues (glycine-14, cysteine-135, and cysteine-218) previously speculated to be important for the structure and function of Drosophila melanogaster alcohol dehydrogenase have been investigated by using site-directed mutagenesis followed by kinetic analysis and chemical modification. Mutating glycine-14 to valine (G14V) virtually inactivates Drosophila ADH, and substitution of alanine at this position (G14A) causes a 31% decrease in activity. Thermal denaturation and kinetic and inhibition studies further demonstrate that replacing glycine-14 with either alanine or valine leads to structural changes in the NAD binding domain. These results provide direct evidence for the role played by glycine-14 in maintaining the correct conformation in the NAD binding domain. On the other hand, changing of cysteine-135, -218, or both to alanine (C135A, C218A, and C135A/C218A) causes no decrease in the catalytic activity of the enzyme, indicating that neither of the cysteinyl residues is essential for catalysis. C135A and wild-type enzyme are both inactivated by DTNB. In contrast, C218A and C135A/C218A are unaffected by DTNB treatment. DTNB modification of cysteine-218 can be prevented by the substrates NAD and 2-propanol, suggesting that cysteine-218 may be in the vicinity of the active site. Cysteine-135 which is normally insensitive to DTNB becomes accessible in the presence of 2-propanol and/or NAD, suggesting a conformational change induced by binding of these substrates.

Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase.

http://users.man.poznan.pl/wojtekr/pdf/wr-lit-57.pdf

The Crystal Structures of Eukaryotic Phosphofructokinases from Baker's Yeast and Rabbit Skeletal Muscle.

http://www.jbc.org/content/264/1/131.full.pdf

Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase: Mutation at the substrate-binding site affects allosteric behavior

Simon H. Chang

Papers

Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis

Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase.

Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase.

The Crystal Structures of Eukaryotic Phosphofructokinases from Baker's Yeast and Rabbit Skeletal Muscle.

Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase: Mutation at the substrate-binding site affects allosteric behavior