Biochemical study

Early fractionation studies with thylakoids that had been fragmented by detergents [54] or mechanical means [55] followed by collection of the thylakoid submembrane fractions by centrifugation provided the first evidence for lateral heterogeneity. Submembrane fractions derived from granal stacks were enriched in PS II but they also contained PS 1, whereas the stroma thylakoids had mainly PS I. Sane et al. [55] proposed that appressed membranes were the site of non-cyclic electron transport, while the non-appressed membranes carried out cyclic photo- 

The aqueous polymer two-phase partition technique pioneered by Albertsson et al. [11] not only provides a method to separate right-side-out from inside-out vesicles (Section 2.2), but also allows the partial separation of appressed and non-appressed membrane fractions. The inside-out vesicles which partition to the lower phase were depleted in PS II activity [59]. Significantly, they were derived from the appressed membranes of the grana stacks as judged by electron microscopy [60] and their mode of formation [61], Futhermore, analysis of the Chl-protein content revealed a substantial depletion of PS I complex, and an enrichment of PS II complex and LHC II in the appressed membrane fraction [62]. In 1980, An-dersson and Anderson postulated that PS II and PS I are mainly laterally segregated, with PS I excluded from the appressed grana partitions, where most PS II-LHC II complexes are located [62,63] (Fig. 5). 

Heterogeneity of PS II is a feature of the current model for molecular organization of the thylakoid multisubunit complexes (Fig. 6). While there is no doubt that PS II is structurally and functionally heterogeneous, this heterogeneity is not yet fully defined. The idea of PS II heterogeneity was first introduced to interpret 

Recently, antibody labelling of ultrathin sections of embedded tissue followed by ferretin or gold provided direct visualization of the in situ distribution of thylakoid complexes. To ensure that this approach is valid, care must be taken to ensure that only specific antibodies are used. 

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The modeling of biomolecules is a very broad and sophisticated field. The description given in this chapter is only meant to provide the connections between the topics in this book and this field. Before embarking on a computational biochemical study, it is recommended that the researcher investigate the literature pertaining to this field more closely. The references provided below should provide a good starting point for such a survey. 

During the course of biochemical studies (138). the mass spectrum of 2-acetamidothiazole was recorded its main peaks are the molecular ion (m/e= 142, relative intensity = 26%) and fragments 100 (100), 58 (2. 5), and 43 (39). For 2-acetamido-5-bromothiazole the main peak results again from the loss of C2H2O by the molecular ion. 2-AcetyIacet-amido-4-methylthiazole (2S) exhibits significant loss of from the... 

Identification, isolation, and removal of (polyhydroxy)benzenes from the environment have received increased attention throughout the 1980s and 1990s. The biochemical activity of the benzenepolyols is at least in part based on thek oxidation—reduction potential. Many biochemical studies of these compounds have been made, eg, of enzymic glycoside formation, enzymic hydroxylation and oxidation, biological interactions with biochemically important compounds such as the catecholamines, and humic acid formation. The range of biochemical function of these compounds and thek derivatives is not yet fully understood. 

Biosynthesis. Biochemical studies on dalbaheptides have been reviewed (92,97). Experiments with and H have shown that in vancomycin (39), D-tyrosine is the precursor of D-/> -hydroxyphenyiglycine and P-hydroxy-y -chlorotyrosine, and acetate the precursor of the two y jy -dihydroxyphenyiglycines (98). Similar results using either or radioactively labeled material have been reported for avoparcin (Table 5) (23), ristocetin (Table 2) (99,100), ardacin (Table 3) (101), and A47934 (102). 

Biochemical studies showed that the a-aminoadipoyl analogue derived from (2,3)-P-methylenepenam was not a substrate for expandase activity but rather, it was a potent reversible inhibitor of the ring expansion of (7.-aminoadipoy1-penici11in into deacetoxycephalosporanic acid by the expandase enzyme... 

Biochemical studies and molecular structure give complementary functional information... 

Many nitroxides are very stable under normal conditions, and heterolytic reactions can be carried out on other fimctional groups in the molecule without destroying the nitroxide group. Nitroxides are very useful in biochemical studies by virtue of being easily detected paramagnetic probes. ... 

Phosphorus-32 is used in biochemical studies to follow the pathway taken by metabolites. Its half-life is 14.3 days. A vial containing 10.0 jug (1 jug = 1 X 10-6 g) of P-32 is accidentally spilled into a sink. 

Hen egg-white lysozyme catalyzes the hydrolysis of various oligosaccharides, especially those of bacterial cell walls. The elucidation of the X-ray structure of this enzyme by David Phillips and co-workers (Ref. 1) provided the first glimpse of the structure of an enzyme-active site. The determination of the structure of this enzyme with trisaccharide competitive inhibitors and biochemical studies led to a detailed model for lysozyme and its hexa N-acetyl glucoseamine (hexa-NAG) substrate (Fig. 6.1). These studies identified the C-O bond between the D and E residues of the substrate as the bond which is being specifically cleaved by the enzyme and located the residues Glu 37 and Asp 52 as the major catalytic residues. The initial structural studies led to various proposals of how catalysis might take place. Here we consider these proposals and show how to examine their validity by computer modeling approaches. 

The elucidation of the X-ray structure of chymotrypsin (Ref. 1) and in a later stage of subtilisin (Ref. 2) revealed an active site with three crucial groups (Fig. 7.1)-the active serine, a neighboring histidine, and a buried aspartic acid. These three residues are frequently called the catalytic triad, and are designated here as Aspc Hisc Serc (where c indicates a catalytic residue). The identification of the location of the active-site groups and intense biochemical studies led to several mechanistic proposals for the action of serine proteases (see, for example, Refs. 1 and 2). However, it appears that without some way of translating the structural information to reaction-potential surfaces it is hard to discriminate between different alternative mechanisms. Thus it is instructive to use the procedure introduced in previous chapters and to examine the feasibility of different... 

Both mechanics and biochemical studies of muscle fibers suggest there is more than one force generating state. Crossbridges are thought to attach initially as... 

Early biochemical studies supported the hypothesis that the HRl and the HR2 peptides would interact to form a helical structure (Chen et al. 1995 Lu et al. 1995). This hypothesis was strengthened when X-ray structures were resolved for co-crystals of HRl and HR2 peptides (Chan et al. 1997 Tan et al. 1997 Weissenhom et al. 1997). The results showed that in the six-helix bundle, three HRl domains were packed tightly together in the center of the bundle, with the HR2 domains bound in an antiparallel manner in grooves formed along the HRl core. 

Subsequent studies provided a wealth of information that appeared to support the hypothesis that the Fepr protein was a genuine [6Fe-6S]-containing protein. In a biochemical study (10) the elemental analysis was meticulously repeated, and, based on an assumed molecular mass of 52 kDa, the prismane protein was found to contain 6.3 Fe atoms, averaged over as many as nine different preparations. Again, no other metals than Fe were detected, suggesting that all... 

Is the paramagnetic adduct between CO and Cluster A a kinetically intermediate in acetyl-CoA synthesis Questions have been raised about whether this adduct is a catalytic intermediate in the pathway of acetyl-CoA synthesis 187, 188) (as shown in Fig. 13), or is formed in a side reaction that is not on the main catalytic pathway for acetyl-CoA synthesis 189). A variety of biochemical studies have provided strong support for the intermediacy of the [Ni-X-Fe4S4l-CO species as the precursor of the carbonyl group of acetyl-CoA during acetyl-CoA synthesis 133, 183, 185, 190). These studies have included rapid ffeeze-quench EPR, stopped flow, rapid chemical quench, and isotope exchange. 

Kiran R, Varma MN. 1988. Biochemical studies on endosulfan toxicity in different age groups of rats. Toxicol Lett 44 247-252. 

This early biological result spurred a variety of biochemical studies of the interactions of various polyamides with the basal transcription machinery and TE-DNA complexes. Two studies have used promoter scanning to identify sites where polyamide binding inhibits transcription [64, 65]. The method uses a series of DNA constructs with designed polyamide binding sites at varying distances from... 

Palytoxin (PTX) is one of the most potent marine toxins known and the lethal dose (LD q) of the toxin in mice is 0.5 Mg/kg when injected i.v. The molecular structure of the toxin has been determined fully (1,2). PTX causes contractions in smooth muscle (i) and has a positive inotropic action in cardiac muscle (4-6). PTX also induces membrane depolarization in intestinal smooth (i), skeletal (4), and heart muscles (5-7), myelinated fibers (8), spinal cord (9), and squid axons (10). PTX has been demonstrated to cause NE release from adrenergic neurons (11,12). Biochemical studies have indicated that PTX causes a release of K from erythrocytes, which is followed by hemolysis (13-15). The PTX-induced release of K from erythrocytes is depress by ouabain and that the binding of ouabain to the membrane fragments is inhibited by PTX (15). 

Barret L, Torch S, Leray CL, et al. 1992. Morphometric and biochemical studies in trigeminal nerve of rat after trichloroethylene or dichloroacetylene oral administration. Neurotoxicology 13 601-614. 

Phenazines — The phenazines are biosynthesized by the shikimic acid pathway, through the intermediate chorismic acid. The process was studied using different strains of Pseudomonas species, the major producers of phenazines. The best-known phenazine, pyocyanine, seems to be produced from the intermediate phenazine-1-carboxylic acid (PCA). Although intensive biochemical studies were done, not all the details and the intermediates of conversion of chorismic acid to PCA are known. In the first step, PCA is N-methylated by a SAM-dependent methyltransferase. The second step is a hydroxylative decarboxylation catalyzed by a flavoprotein monooxygenase dependent on NADH. PCA is also the precursor of phenazine-1-carboxamide and 1-hydroxyphenazine from Pseudomonas species. - - ... 

As mentioned above, the first BVMOs were already purified several decades ago. Subsequent biochemical studies have revealed that these enzymes are typically soluble and often easy to express at high levels in, e.g., E. coli. These features suggest that BVMOs are perfect candidates for X-ray crystallography studies. However, crystallization of several type I BVMOs has been attempted and proven... 

The psubunit has been purified from PGl by ourselves and others and is a heat stable, acidic, heavily glycosylated protein with an apparent molecular mass of 37-39 kD (19, 26). No enzymatic activity has been identified for the protein. The psubunit can be extracted from the cell walls of both green and ripe tomato fruit by high salt buffers (13, 14, 18, 19, 20), and in the latter case is associated with PG2 polypeptide(s) in the form of PGl. Purified psubunit can also associate with and convert PG2 in vitro into an isoenzyme that closely resembles PGl (13, 14, 24). Biochemical studies have shown that in vivo and in vitro formation of PGl by the association of PG2 with the p-subunit alters the biochemical and enzymic properties of the associated catalytic PG2 polypeptide including its pH optima, response to cations and thermal stability (summarized in Table 1). This later property provides a convenient assay for the levels of PGl and PG2 in total cell wall protein extracts. 

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