Tryptophanase from Sphaerophorus funduliformis. Purification, molecular weight and subunit properties.

A crystalline tryptophanase can be obtained from extracts of Spaerophorus funduliformis using a heat treatment, hydroxyapatite chromatography and solubility in solutions of (NH4)2SO4 as a function of pH and temperature. The purified enzyme is homogeneous by several criteria. S. funduliformis tryptophanase has a specific activity of 11.5-13.5 and requires pyridoxal 5'-phosphate for enzymatic activity. Like other tryptophanases that have been studied, the S. funduliformis enzyme is a tetramer protein consisting of four apparently identical subunits. The native enzyme has a sedimentation coefficient of 11.2 S and a molecular weight of 244 000. In solutions of 5 M guanidine - HCl, 8 M urea, and sodium dodecylsulfate, at high pH or in the presence of thiols, the enzyme dissociates to 59 000 molecular weight species which are homogeneous by the criterion of weight. Peptide maps of the reduced holo-tryptophanase show one pyridoxal-containing peptide and, lacking agreement with the determined amino acid composition, suggest that the subunits of the enzyme contain a high degree of internal sequence homology.     

Photosystem II particles from Chlamydomonas reinhardtii. Purification, molecular weight, small subunit composition, and protein phosphorylation

PSII particles from Chlamydomonas reinhardtii were purified according to the protocol of Diner and Wollman (Diner, B.A., and Wollman, F.-A. (1980) Eur. J. Biochem. 110, 521-526) followed by ion-exchange chromatography. They contained the psbA, psbB, psbC, and psbD gene products in a 1/1/1/1 stoichiometry, cytochrome b559, and several small polypeptides, and exhibited electron transfer from donor Z to acceptor QA (40-50 chlorophylls/reducible QA). Upon ultracentrifugation and molecular sieving in the presence of either lauryl maltoside or octaethylene glycol dodecyl ether (C12E8), they behaved as monomers of 440-510 kDa, including the detergent. C12E8 preparations also contained a small proportion of a partially interconvertible dimeric form. Four small subunits were identified by N-terminal sequencing, namely a 6.1-kDa nuclear-encoded subunit and three chloroplast-encoded subunits homologous to psbE, psbK, and psbM gene products. Cytochrome b559 subunit alpha (psbE) of C. reinhardtii, but not subunit beta (psbF), was recognized by an antiserum raised against higher plant cytochrome b559. The products of the psbF, psbI, and psbN genes remained undetected, presumably because of blocked N termini. At least four polypeptides presented both phosphorylated and unphosphorylated forms (psbC, psbD, and psbH gene products, as well as an unidentified 5-kDa subunit).     

Complete amino acid sequence of a subunit from rapeseed high molecular weight protein

A subunit (Mr 15,600) from the high molecular weight protein from rapeseed was separated and isolated; its purity and homogeneity were ascertained. The subunit was cleaved with cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease. The fragments were separated and isolated by polyacrylamide gel electrophoresis, gel filtration, column chromatography on Dowex 1 x 2, and paper electrophoresis. The amino acid compositions of the intact subunit and different fragments obtained from enzymatic and chemical cleavages were determined. The subunit and its fragments were sequenced by manual Edman method. The phenylthiohydantoin amino acids obtained after each step were identified by thin-layer chromatography and ultraviolet spectroscopy. The complete amino acid sequence of the subunit consisting of 125 amino acid residues has been established by the overlapping method.     

Malate dehydrogenase from the thermophilic green bacterium Chloroflexus aurantiacus: purification, molecular weight, amino acid composition, and partial amino acid sequence.

Malate dehydrogenase (MDH; EC 1.1.1.37) from the thermophilic green nonsulfur bacterium Chloroflexus aurantiacus was purified by a two-step procedure involving affinity chromatography and gel filtration. The enzyme consists of identical subunits which had molecular weights of approximately 35,000. In its active form at 55 degrees C, it formed tetramers. At lower temperatures, inactive dimers and trimers existed. Antibodies against the purified enzyme were produced, and immunotitration and enzyme-linked immunosorbent assays showed that there was an immunochemical homology between the MDH from C. aurantiacus and MDHs from several other bacteria. The amino acid composition of C. aurantiacus MDH was similar to those of other MDHs. The N-terminal amino acid sequence was enriched with hydrophobic amino acids, which showed a high degree of functional similarity to amino acids at the N-terminal ends of both Escherichia coli and Thermus flavus MDHs. The activity of the native enzyme was inhibited by high concentrations of substrate and had temperature and pH optima consistent with the optimal growth conditions for the organism.     

Complete amino acid sequence of arachin subunit of molecular weight 21,000

A subunit of molecular weight 21,000 from arachin, the major peanut protein, was isolated in pure form and primary structure was determined. The subunit was fragmented with CNBr, trypsin, and NBS; the fragments were separated and isolated by PAGE, gel filtration, Dowex treatment, and paper electrophoresis, and Edman degradation on each fragment, including the intact subunit, was performed. The PTH-amino acids thus obtained were identified by UV spectroscopy and TLC. The complete sequence of 176 residues was established by overlapping technique.     

Purification, characterization and partial amino acid sequences of carnitine palmitoyl-transferase from human liver

Carnitine palmitoyl-transferase has been extracted with 0.5% Tween-20 from human liver homogenate and purified to homogeneity. The purified enzyme has a native Mr of 274 kDa. The subunit Mr is of 66 kDa, as shown by SDS-PAGE and immunoblots obtained with antibodies raised against human CPT. Purified CPT shows high affinity for palmitoyl-CoA and palmitoyl-carnitine and is not inhibited by malonyl-CoA. Seven tryptic peptides and the N-terminal of purified human CPT have been sequenced, and found homologous to rat CPT sequence. Both antibodies and peptide sequences are important tools for the investigation of the molecular basis of CPT deficiency in man.     

Structure, subunit composition, and molecular weight of RD-114 RNA.

The properties and subunit composition of the RNA extracted from RD-114 virions have been studied. The RNA extracted from the virion has a sedimentation coefficient of 52S in a nondenaturing aqueous electrolyte. The estimated molecular weight by sedimentation in nondenaturing and weakly denaturing media is in the range 5.7 X 10(6) to 7.0 X 10(6). By electron microscopy, under moderately denaturing conditions, the 52S molecule is seen to be an extended single strand with a contour length of about 4.0 mum corresponding to a molecular weight of 5.74 X 10(6). It contains two characteristic secondary structure features: (i) a central Y- or T-shaped structure (the rabbit ears) with a molecular weight of 0.3 X 10(6), (ii) two symmetreically disposed loops on each side of and at equal distance from the center. The 52S molecule consists of two half-size molecules, with molecular weight 2.8 X 10(6), joined together within the central rabbit ears feature. Melting of the rabbit ears with concomitant dissociation of the 52S molecule into subunits, has been caused by either one of two strongly denaturing treatments: incubation in a mixture of CH3HgOH and glyoxal at room temperature, or thermal dissociation in a urea-formamide solvent. When half-size molecules are quenched from denaturing temperatures, a new off-center secondary structure feature termed the branch-like structure is seen. The dissociation behavior of the 52S complex and the molecular weight of the subunits have been confirmed by gel electrophoresis studies. The loop structures melt at fairly low temperatures; the dissociation of the 52S molecule into its two subunits occurs at a higher temperature corresponding to a base composition of about 63% guanosine plus cytosine. Polyadenylic acid mapping by electron microscopy shows that the 52S molecule contains two polyadenylic acid segments, one at each end. It thus appears that 52S RD-114 RNA consists of two 2.8 X 10(6) dalton subunits, each with a characteristic secondary structure loop, and joined at the 5' ends to form the rabbit ears secondary structure feature. The observations are consistent with but do not require the conclusion that the two 2.8 X 10(6) dalton subunits of 52S RD-114 RNA are identical.     

Studies in metachromatic leukodystrophy: XV. Purification of normal and mutant arylsulfatase A from human liver

In this report we describe a method to purify both normal and abnormal (inactive) arylsulfatase A. The abnormal enzyme protein was isolated both from cases of late infantile and early juvenile forms of metachromatic leukodystrophy. Conventional protein isolation methods reported earlier were followed by size exclusion high-performance liquid chromatography in the final purification stages. Both the mutant enzyme and the normal enzyme had the same HPLC elution behavior. They thus appeared to self-associate in a similar pH-dependent fashion. Both could be followed by their reaction to a rabbit antibody to normal human arylsulfatase A. The amount of homogenous protein obtained from about 500 grams of liver was 300-400 micrograms.