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The Chlamydomonas cell wall degrading enzyme, lysin, acts on two substrates within the framework of the wall

The Chlamydomonas cell wall is a multilayered, extracellular matrix containing 20-25 proteins and glycoproteins, many of which are highly enriched in hydroxyproline. 80-90% of the wall protein is located in a crystalline portion of the wall that is soluble in sarkosyl-urea solutions as well as in chaotropic salts. Although the wall has no cellulose it contains a noncrystalline, highly insoluble framework portion that is responsible for the integrity and overall shape of the wall. In the present report we show that the framework of the wall is composed of two components that are acted upon by lysin, a wall degrading enzyme released by mating gametes. One, which makes up the major portion of the framework, is insoluble upon boiling in SDS-PAGE sample buffer. Lysin treatment of this portion leads to its physical degradation and the concomitant appearance of several SDS-dithiothreitol-soluble polypeptides ranging in relative molecular mass from greater than 400,000 to less than 60,000. The second component is the flagellar collar. This hollow cylinder composed of striated fibers aligned in parallel array serves as the tunnel in the wall through which the flagella protrude. Our evidence indicates that the primary collar polypeptide is a 225,000-Mr molecule that itself has at least two functional domains. One domain, contained in a 185,000-Mr fragment, permits the self-association of the molecules to form the main body of the collar. The second part of the molecule anchors the collar to the wall framework via sarkosyl-urea-insensitive, SDS-dithiothreitol-sensitive linkages.     

The release of alanine by rat diaphragm muscle in vitro.

Z discs were isolated from Lethocerus flight muscle by removing the contractile proteins from myofibrils with a solution of high ionic strength. The protein composition of the Z discs was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the major proteins were alpha-actinin, actin and tropomyosin. Z lines were selectively removed from intact myofibrils by digestion with crude lipase and chymotrypsin, but not by purified lipase.     

Enzymes of vitamin B6 degradation. Purification and properties of 5-pyridoxic-acid oxygenase from Arthrobacter sp

5-Pyridoxic-acid oxygenase, a cytoplasmic enzyme formed when Arthrobacter Cr-7 is grown with pyridoxine as a sole source of carbon and nitrogen, was purified about 190-fold to homogeneity from fully induced cells. The enzyme catalyzes Reaction a, (Formula: see text) the essential ring-opening step in the degradation of pyridoxine, and provides a second example of an FAD-dependent oxygenase that adds both two hydrogen and two oxygen atoms to its substrate. 5-Pyridoxic-acid oxygenase has an isoelectric point of 4.6, functions optimally between pH 7 and 8, appears to contain a single subunit of Mr = 51,000 and one FAD (but no iron) per subunit, and is readily resolved by precipitation with ammonium sulfate at pH 3.0. FMN and riboflavin do not replace FAD as coenzyme, but their presence enhances a normally minor side reaction (Reaction b) NAD(P)H + H+ + O2----NAD(P)+ + H2O2 (b) catalyzed by the holoenzyme. Reaction b also is enhanced when the poorly utilized analogues, 3-hydroxy-2-methylpyridine-5-carboxylic acid or NADH, replace 5-pyridoxic acid or NADPH, respectively, as substrates in Reaction a. Each of the enzymes required in two different pathways for degradation of pyridoxine to anabolic intermediates has now been studied. A comparison of these two pathways and their enzymes is provided.     

Purification and properties of pyridoxal-5''-phosphate-dependent histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes.

Histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes were purified to homogeneity and compared with the histidine decarboxylase from Morganella morganii. All three enzymes required pyridoxal 5'-phosphate as a coenzyme, showed optimal activity at pH 6.5, decarboxylated only histidine among the amino acids derived from protein, and were tetramers or dimers of identical subunits. Amino-terminal sequences of the three enzymes showed up to 81% homology through residue 33, but the enzymes differed sufficiently in amino acid composition and sequence so that no cross-reaction occurred between the K. planticola or E. aerogenes enzymes and antibodies to the decarboxylase from M. morganii. All three enzymes were inhibited by carbonyl reagents; by amino-, carboxyl-, and some methyl-substituted histidines; and by alpha-fluoromethylhistidine. These decarboxylases, all from gram-negative organisms, differed greatly in subunit structure, biogenesis, and other properties from the pyruvoyl-dependent histidine decarboxylases from gram-positive organisms described previously.     

L-Histidinol phosphate aminotransferase from Salmonella typhimurium. Kinetic behavior and sequence at the pyridoxal-P binding site

A coupled assay with alpha-hydroxyglutarate dehydrogenase was used to analyze the kinetic behavior of histidinol phosphate aminotransferase from Salmonella typhymurium. Data obtained from studies of initial velocity, inhibition by products or substrate analogues, isotope exchange rates, and the determination of the equilibrium constant were consistent only with a Ping-Pong Bi Bi mechanism. Variations in inhibition patterns by different substrate analogues indicate that the microenvironment about the pyridoxal phosphate and the pyridoxamine phosphate forms of histidinol phosphate amino-transferase are different, and favor the presence of one active site with partially overlapping substrate-binding subsites for these 2 forms of the enzyme. Histidinol phosphate aminotransferase also catalyzes decomposition of beta-chloro-L-alanine to pyruvate, NH3 and Cl-; no transamination of this substrate occurs and inactivation of the enzyme accompanies this reaction. After reduction of histidinol-P aminotransferase with [3H]NaBH4, carboxymethylation, and tryptic digestion, one major radioactive peptide absorbing at 325 nm was isolated. Its primary structure was determined to be TLSK*AFALAGLR, where K* is the P-pyridoxyllysine residue. Although this peptide is only 30-40% homologous with the corresponding segment reported for other transaminases, all of these peptides are similar in placement of an hydroxyamino acid residue three residues upstream from the lysine residue, and in the cluster of hydrophobic amino acid residues immediately following the lysine residue.     

Control analysis of mammalian serine biosynthesis. Feedback inhibition on the final step.

The flux of serine biosynthesis in the liver of the normal rabbit, and of the rat on a low protein diet, is most sensitive to the activity of phosphoserine phosphatase (flux control coefficient up to 0.97), the last of the three enzymes in the pathway after it branches from glycolysis. The concentration of the pathway product, serine, has a strong controlling influence on the flux (response coefficient up to -0.64) through feedback inhibition at this step. The pathway is therefore controlled primarily by the demand for serine rather than the supply of the pathway precursor, 3-phosphoglycerate. Under conditions where there is a lower biosynthetic flux, the flux control coefficients of the first two enzymes of the pathway are increased, and are probably dominant in the rat on a normal diet. In rabbit liver, when ethanol is used to inhibit serine biosynthesis, control can be distributed between the three enzymes, even though the reactions catalysed by the first two remain close to equilibrium. Apart from their intrinsic value in aiding the understanding of the regulation of mammalian serine metabolism, our findings illustrate the danger of assuming that there are invariant design principles in the regulation of metabolic pathways, such as feedback control on the first step after a branch.