Tyrosine Hydroxylase from Bovine Striatum: Catalytic Properties of the Phosphorylated and Nonphosphorylated Forms of the Purified Enzyme

Abstract: The properties of purified tyrosine hydroxylase (TH) from bovine corpus striatum, both native and phosphorylated forms of the enzyme, were studied. TH had a tendency toward greater affinity for tetrahydrobiopterin (BH₄) than for the synthetic cofactor 6-methyltetrahydropterin (6-MPH₄), although the maximal velocity of the TH-catalyzed reaction was greater with 6-MPH4. Phosphorylation increased the affinity of TH for cofactor at pH 6.0, with little change in V _max. At pH 7.0, phosphorylation caused increased activation of TH by increasing V _max as well as reducing the K _m for cofactor. The K _m for dopamine was increased twofold by phosphorylation at pH 6.0, but eightfold at pH 7.0. Phosphorylation was not associated with a change in K _m for tyrosine at any pH or with any cofactor studied, although the K _m for tyrosine of TH was cofactor-dependent and seven to eight times greater with 6-MPH₄ than with BH₄ as cofactor. Heparin and NaCl activated native TH at pH 6.0, but not at pH 7.0. Phosphorylated TH was unaffected by heparin or salt at pH 6.0, but was relatively inhibited at pH 7.0. The data are presented in the context of the physiological environment of TH.     

α-N-Acetyl β-Endorphins in the Pituitary: Immunohistochemical Localization Using Antibodies Raised Against Dynorphin(1-13)

Abstract: Intense immunohistochemical staining of the intermediate lobe of the pituitary was observed by using an antiserum raised against synthetic dynorphin(1-13) treated with a water-soluble carbodiimide (CDI). Subsequent studies showed that the immunostaining was blocked by preincubation of the antiserum with acetylated derivatives of both β-endorphin and dynorphin(1-13) as well as by CDI-treated dynorphin(1-13), but only weakly by authentic dynorphin(1-13). Neither nonacetylated β-endorphin nor any other fragments of the ACTH/endorphin precursor blocked the immunostaining of the intermediate lobe. Analysis of the CDI-treated dynorphin(1-13) used as an antigen showed that most of the peptide was acetylated at primary amino groups. CDI treatment of dynorphin(1-13) results in the formation of an acetyl derivative because the commercially available peptide is supplied as the acetate salt. The antibodies responsible for the intermediate lobe staining were isolated by affinity chromatography, using a column containing partially purified intermediate lobe extract linked to an affinity resin and a radioimmunoassay (RIA) was developed with CDI-treated dynorphin(1-13) used as a trace and as a standard. Competition studies showed 0.5-1% cross-reactivity with α-N-acetyl β-endorphin(1-31), α-N-acetyl β-endorphin(1-27), and totally acetylated β-endorphin(1-31). Nonacetylated β-endorphins did not cross-react. Posterior-intermediate lobe extracts from rat and beef were fractionated by gel filtration. Rat posterior-intermediate lobe extracts were also fractionated by cation-exchange chromatography. Fractionated extracts were analyzed by RIAs for β-endorphin, CDI-treated dynorphin(1-13), and authentic dynorphin(1-13). The results suggested that the peptides responsible for the intermediate lobe staining were mainly four different derivatives of β-endorphin bearing an acetyl group at the amino terminus. No immunostaining was seen in the posterior and anterior lobes of the pituitary. This suggests that the intermediate lobe is the main source of acetylated β-endorphins in the pituitary.     

Biogenesis of the protein import channel Tom40 of the mitochondrial outer membrane: intermembrane space components are involved in an early stage of the assembly pathway

Tom40 forms the central channel of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The precursor of Tom40 is encoded in the nucleus, synthesized in the cytosol, and imported into mitochondria via a multi-step assembly pathway that involves the mature TOM complex and the sorting and assembly machinery of the outer membrane (SAM complex). We report that opening of the mitochondrial intermembrane space by swelling blocks the assembly pathway of the beta-barrel protein Tom40. Mitochondria with defects in small Tim proteins of the intermembrane space are impaired in the Tom40 assembly pathway. Swelling as well as defects in the small Tim proteins inhibit an early stage of the Tom40 import pathway that is needed for formation of a Tom40-SAM intermediate. We propose that the biogenesis pathway of beta-barrel proteins of the outer mitochondrial membrane not only requires TOM and SAM components, but also involves components of the intermembrane space.     

Carotenoid radical chemistry and antioxidant/pro-oxidant properties

The purpose of this review is to summarise the current state of knowledge of (i) the kinetics and mechanisms of radical reactions with carotenoids, (ii) the properties of carotenoid radicals, and (iii) the antioxidant/pro-oxidant properties of carotenoids.     

The carboxyl-terminal third of the dicarboxylate carrier is crucial for productive association with the inner membrane twin-pore translocase

The carrier proteins of the mitochondrial inner membrane consist of three structurally related tandem repeats (modules). Several different, and in some cases contradictory, views exist on the role individual modules play in carrier transport across the mitochondrial membranes and how they promote protein insertion into the inner membrane. Thus, by use of specific translocation intermediates, we performed a detailed analysis of carrier biogenesis and assessed the physical association of carrier modules with the inner membrane translocation machinery. Here we have reported that each module of the dicarboxylate carrier contains sufficient targeting information for its transport across the outer mitochondrial membrane. The carboxyl-terminal module possesses major targeting information to facilitate the direct binding of the carrier protein to the inner membrane twin-pore translocase and subsequent insertion into the inner membrane in a membrane potential-dependent manner. We concluded that, in this case, a single structural repeat can drive inner membrane insertion, whereas all three related units contribute targeting information for outer membrane translocation.     

Idiopathic Hypercalciuria and Hyperparathyroidism

With provocative tests of parathyroid activity based on phosphate deprivation and the administration of chlorothiazide 8 out of 19 patients with idiopathic hypercalciuria developed hypercalcaemia. Five of the eight underwent exploration of the neck and four of them had parathyroid adenomas; the fifth patient had “normal” glands but responded satisfactorily to subtotal parathyroidectomy. One patient who failed to develop hypercalcaemia had primary chief cell hyperplasia of the parathyroids.     

Amino acid sequence of bovine gamma E (IVa) lens crystallin.

When electrospray ionization mass spectrometry (ESMS) was used to analyze purified bovine gamma E (gamma IVa)-crystallin, it yielded a relative molecular mass (M(r)) of 20.955 +/- 5. This mass is significantly different from that calculated from the published sequence (M(r) 20.894) (White HE et al., 1989, J Mol Biol 207:217-235). Further, ES-MS analysis of the protein after it had been reduced and carboxymethylated indicated the presence of five cysteine residues, whereas the published sequence contains six (Kilby GW et al., 1995, Eur Mass Spectrom 1:203-208). The entire protein sequence of gamma E crystallin has therefore been studied via a combination of ES-MS, ES-MS/MS, and Edman amino acid sequencing. The corrected sequence gives an M(r) of 20.955.3, which matches that obtained by ES-MS analysis of the purified native protein. The corrected sequence is also in agreement with a recent cDNA sequence obtained for a bovine gamma-crystallin by R. Hay (pers. comm.).     

SOME ASPECTS OF MORPHOGENESIS IN CUSCUTA GRONOVII

More than 100 primary root segments from dormant seeds, 100 radicular segments from immature embryos, 450 whole immature embryos, and a number of whole seedlings of C. gronovii were studied under in vitro cultivation in the presence of such growth-regulating factors as: coconut milk, casein hydrolysate, auxins, kinetin, adenine, gibberellic acid, and additional B vitamins as supplements to White's medium. In normal embryogeny early swelling of the basal pole of the embryo and profuse production of epidermal hairs upon germination are superficial characteristics of Cuscuta which resemble seedling root growth in other plants. At no time is there evidence of a primary-root meristem, nor at any time do cotyledons develop. It was not possible to induce root formation experimentally from any of the starting embryonic materials, nor from stems which were cultured to maturity culminating in the production of flowers after 6 months of in vitro cultivation, nor from callus derived from the radicular pole of the embryo. The undifferentiated radicular pole frequently gave rise to rapidly growing callus from which new shoots were formed spontaneously, although some immature embryos completed normal embryogeny. Embryo size and subsequent type of development were not strictly correlated but there was a tendency toward the following patterns. Embryos less than 0.4 mm in length lost their original form, callused evenly throughout and became masked by development of buds over the entire surface. Embryos 0.4–1.2 mm were most apt to retain their original form; callus developed at the radicular pole but not at the shoot pole. Embryos 1.5 mm and larger frequently gave rise to radicular callus but also grew normal shoots. Embryo inoculants which had begun to coil were most apt to complete normal embryogenesis. No correlation could be drawn between the addition of specific growth regulators or combinations of regulators and specific patterns of development. It is suggested that in the evolution of Cuscuta from autotrophic ancestors, the loss of both cotyledons and roots was a single embryonic event and so complete that even under the influence of a highly modified biochemical environment it would be impossible to derive roots from dodder tissue. The usual interpretation of the dodder haustorium as a highly modified adventitious root in all probability is not valid. Is is further suggested, however, that this loss of root and cotyledon is not a necessary consequence of the evolution of the parasitic mode of nutrition, nor of the gross adult growth form.