C-terminal deletion analogs of a crustacean pigment-dispersing hormone

This study deals with the effect of deamidation and C-terminal truncation on the potency of an octadecapeptide pigment-dispersing hormone (PDH: Asn- Ser-Gly-Met-Ile-Asn-Ser-Ile-Leu-Gly-Ile-Pro-Arg-Val-Met-Thr-Glu-Ala-NH₂), first described as light-adapting distal retinal pigment hormone (DRPH) from Pandalus borealis. Bioassay of synthetic analogs for melanophore pigment dispersion in destalked fiddler crabs (Uca pugilator) showed that deamidation causes a 300-fold decrease in potency. The analogs 1–17-NH₂ and 1–16-NH₂ were about 3 times more potent than 1–18-OH. Further truncation led to decreases in potency, with the peptide 1–9-NH₂ being the smallest C-terminal deletion analog to display activity (0.001% potency). Smaller analogs (1–8-NH₂, 1–6-NH₂ and 1–4-NH₂) were inactive when tested in doses as high as 500 nmoles/crab. On the basis of our earlier work on N-terminal deletion analogs and the present findings the residues 6 to 9 seem to be important for PDH action.     

Biologically active analogs of thymopentin with enhanced enzymatic stability

Thymopentin, a synthetic pentapeptide fragment of thymopoietin (residues 32–36, Arg-Lys-Asp-Val-Tyr) is biologically active but susceptible to proteolytic digestion. Analogs were synthesized and studied for biological activity and susceptibility to peptidases. Amino acid changes were incorporated at positions known to not affect activity adversely and N-terminal acetylation and C-terminal amidation were used to increase resistance to proteolytic degradation by exopeptidases. Ac-Pro²-TP5-NH₂ and Aib²-TP5-NH₂ retained activity and were shown to exhibit a high degree of stability when incubated in human serum.     

IN VITRO PROTEIN SYNTHESIS ON FREE POLYRIBOSOMES ISOLATED FROM THE DEVELOPING MOUSE BRAIN

Abstract— Free polyribosomes were isolated at three ages (newborn, 2 weeks and 8 weeks) from the developing mouse brain. All three preparations were found to be highly polyribosomal in nature and were essentially identical with respect to their chemical composition and sedimentation properties. An estimate of the sedimentation coefficients of the first seven members of these polysome preparations yielded S °_20,w values of 76, 114, 146, 174, 196, 217 and 236. All three preparations were found to be very active when employed in in vitro protein synthesizing systems. An age-dependent response to the concentration of K⁺ was observed in the activities of the in vitro protein synthesizing systems. Optical K⁺ concentrations for the 0, 2 and 8 week old systems were 30, 50 and 65 mm, respectively. No such age dependence was observed when NH⁺₄ was used as the sole monovalent cation, with all systems exhibiting maximal activity at 50mm-NH⁺₄. The highest in uitro activities were consistently observed (at all three ages) when NNH⁺₄ was employed as the sole monovalent cation. Under optimal conditions, the newborn in vitro protein synthesizing system was observed to be approx 40% as active as either the 2 week or the 8 week systems which were equivalent in activity. The reduced activity of the newborn system appeared to be a function of both the polyribosomal and pH 5 enzyme preparations.     

Antibodies to a Segment of Tyrosine Hydroxylase Phosphorylated at Serine 40

Abstract: A synthetic peptide corresponding to residues 32–47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser⁴⁰ and used to generate antibodies in rabbits. Reactivity of the anti-pTH_32–47 antibodies with phospho- and dephospho-Ser⁴⁰ forms of TH protein and peptide TH_32–47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody-capture ELISAs, anti-pTH_32–47 was more reactive with the phospho-TH than with the dephospho-TH forms. Conversely, antibodies against the nonphosphorylated peptide reacted preferentially with the dephospho-TH forms. In western blots, labeling of the ∼60-kDa TH band by anti-pTH_32–47 was readily detectable in lanes containing protein kinase A-phosphorylated native TH at 10–100 ng/lane. In blots of supernatants prepared from striatal synaptosomes, addition of a phosphatase inhibitor was necessary to discern labeling of the TH band with anti-pTH_32–47. Similarly, anti-pTH_32–47 failed to immunoprecipitate TH activity from supernatants prepared from untreated tissues, whereas prior treatment with either 8-bromoadenosine 3',5'-cyclic monophosphate or forskolin enabled removal of TH activity by anti-pTH_32–47. Lastly, in immunohistochemical studies, anti-pTH_32–47 selectively labeled catecholaminergic cells in tissue sections from perfusion-fixed rat brain.     

Evidence for Protein Kinase C Involvement in the Short-Term Activation by Prolactin of Tyrosine Hydroxylase in Tuberoinfundibular Dopaminergic Neurons

Abstract: The mechanism of the short-term activation by prolactin (PRL) of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by 3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a dose-dependent increase within 2 h of incubation of the hypothalamic slices with PRL. To determine whether a phosphorylation process was involved in this increase in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition. In control median eminences, two kinetically different forms of TH coexisted, one exhibiting a K _1(DA) value of 29.92 ± 0.49 μ M , the other being × 15-fold more sensitive to DA inhibition with a K _1(DA) of 1.96 ± 0.09 μ M , likely corresponding to a phosphorylated and active form and to a nonphosphorylated and less active form, respectively. After PRL treatment, the TH form of low K _1(DA) remained unaffected, whereas the K _1(DA) of the purported active form of TH increased to 62.6 ± 0.8 μ M , suggesting an increase in the enzyme phosphorylation. This increase in the K _I(DA) of TH was selectively prevented by GF 109203X, a potent and selective inhibitor of protein kinase C, but not by a specific inhibitor of protein kinase A or calmodulin. Finally, this action of PRL could be mimicked by 12- O -tetradecan-oylphorbol 13-acetate (a direct activator of protein kinase C). These results suggest that PRL, at the median eminence level, activates TH by increasing the enzyme phosphorylation and that this action may involve an activation of protein kinase C.     

Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor

To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His¹,¹²⁵I-Tyr¹⁰,Nle²⁷]hGHRH(1–32)-NH₂ increased linearly with protein concentration (10–45 μg protein/tube). Binding reached equilibrium after 90 min at 30°C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (K_d = 1.04 ± 0.19 nM, B_max = 3.9 ± 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC₅₀) for porcine GHRH (2.8 ± 0.51 nM), rat GHRH (3.1 ± 0.69 nM), [N-Ac-Tyr¹, -Arg²]hGHRH(3–29)-NH₂ (3.9 ± 0.58 nM), and [ -Thr⁷]GHRH(1–29)-NH₂ (189.7 ± 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.     

Intermediates in the refolding of urea-denatured dimeric arginine kinase from Stichopus japonicus

The refolding of urea-denatured dimeric AK was investigated by both equilibrium and kinetic measurements. Both studies indicated that the refolding of dimeric AK is a multiphasic process. The equilibrium studies, monitored by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphtalene-8-sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking showed that there were at least two intermediates involved in this process: I₁ (existing in 1.8–1.4 M urea) and I₂ (existing in 0.8–0.4 M urea). I₁ was a monomeric intermediate and possessed characteristic similar to the globular folding intermediates described in the literature. I₂ was an active native-like intermediate. The kinetic studies suggested that the refolding of AK possessed a burst phase, fast phase and slow phase, which involved at least the burst phase intermediates (I_B). Comparison of the properties of these intermediates suggested that I_B in the kinetic process corresponded to I₁ in the equilibrium process. Based on these results, a scheme for refolding of urea-denatured AK was proposed.     

EFFECTS OF HYPOXIA ON TYROSINE HYDROXYLASE ACTIVITY IN RAT CAROTID BODY

Abstract— Tyrosine hydroxylase (TH) activity was measured in the carotid body. superior cervical ganglion and adrenal glands of the rat under normal conditions and at 48 h following exposure of the animals for 1-3 h in a low O₂ atmosphere. Basal TH levels were 5-6 nmol/h/mg tissue for both the carotid body and the ganglion. Forty-eight hours after hypoxia, there was an increase in enzyme activity in both tissues which paralleled the severity of the hypoxia but was greater in the carotid body than the superior cervical ganglion. Thus, following exposure to 5% O₂ in N₂ for two 30-min periods (20-min interim), TH activity had increased by 50% in the carotid body and 33% in the ganglion; after exposure to 10% O₂ in N₂ for 3 h (continuous), TH levels were increased by 37% in the carotid body and 12% in the ganglion. In the adrenal gland, basal TH activity was 3.42 ± 1.87 nmol/h/mg tissue, and this value was unchanged following either level of hypoxia.     

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.     

High affinity receptors for vasoactive intestinal peptide on a human glioma cell line

Vasoactive intestinal peptide (VIP) bound with high affinity (K_d 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin. PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and [des-His¹]VIP bound with 10 and 100 times lower affinity. The fragment VIP(7–28) displaced 25% of the receptor-bound ¹²⁵I-VIP whereas VIP(16–28) and VIP(1–22-NH₂) were inactive. The binding of ¹²⁵I-VIP could be completely inhibited by 10 μmol/l of the antagonists [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂, [pCl-D-Phe⁶,Leu¹⁷]VIP and VIP(10–28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated ¹²⁵I-VIP was bound to receptors on the cell surface. The internalized ¹²⁵I-VIP was completely degraded to ¹²⁵I-tyrosine which was released from the cells. Degradation of internalized ¹²⁵I-VIP was significantly reduced by chloroquine phenantroline and pepstatin-A. Surface binding and internalization of ¹²⁵I-VIP was increased 3 times by phenantroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound ¹²⁵I-VIP, but caused retention of internalized ¹²⁵I-VIP.     

Short-Term Inhibitory Effect of Estradiol on Tyrosine Hydroxylase Activity in Tuberoinfundibular Dopaminergic Neurons In Vitro

Abstract: The short-term inhibition by estradiol of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by L-3,4-di-hydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a 30–40% decrease within 1 h of incubation with estradiol. To determine whether a dephosphorylation process was involved in this decline in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition: In controls, we observed that two kinetically different forms of TH coexisted, with one exhibiting a K_l(DA) of 26.4 ± 2 μM the other being ∼ 10-fold more sensitive to DA inhibition, with a [k_1{DA)] of 2.56 ± 0.17 μM. likely corresponding to a phosphorylated and active form and to a non-phosphorylated and poorly active form, respectively. Conversely. after estradiol treatment all TH molecules exhibited the same K_1(DA) of 2.5 ± 0.3 μM. This effect was stereospecific, because 17α-estradiol could not promote it. whereas with 17β-estradiol. it could be observed at only 10⁻¹¹M and after a short delay (30 min). Finally, this decrease in the K_1(DA) of the purported active form of TH could be prevented by okadaic acid (an inhibitor of protein phosphatases). These results suggest that estradiol can act directly on the mediobasal hypothalamus to trigger a rapid decline in TH activity and that this action may involve a decrease in TH phosphorylation.     

Angiotensin-(1–7) through AT2 receptors mediates tyrosine hydroxylase degradation via the ubiquitin–proteasome pathway

Hypothalamic norepinephrine (NE) release regulates arterial pressure by altering sympathetic nervous system activity. Because angiotensin (Ang) (1–7) decreases hypothalamic NE release and this effect may be correlated with a diminished NE synthesis, we hypothesize that Ang-(1–7) down-regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis. We investigated the effect of Ang-(1–7) on centrally TH activity and expression. TH activity was evaluated by the release of tritiated water from ³H- l -tyrosine. TH expression and phosphorylation were determined by western blot. Hypothalami from normotensive or spontaneously hypertensive rats pre-incubated with Ang-(1–7) showed a significant decrease in TH specific activity. Ang-(1–7) caused a decrease in TH phosphorylation at Ser19 and Ser40 residues. The heptapeptide induced a decrease in TH expression that was blocked by an AT₂ receptor antagonist and not by an AT₁ or Mas receptor antagonist, suggesting the involvement of AT₂ receptors. The proteasome inhibitor MG132 blocked the Ang-(1–7)-mediated TH reduction. In addition, Ang-(1–7) increased the amount of TH–ubiquitin complexes, indicating that the Ang-(1–7)-mediated TH degradation involves ubiquitin conjugation prior to proteasome degradation. We conclude that Ang-(1–7) down-regulates TH activity and expression centrally leading to a decrease in the central NE system activity.     

Lack of effect of vasoactive intestinal peptide antagonists on blood flow in the rat thyroid

We used three putative vasoactive intestinal peptide (VIP) antagonists: 1) [4Cl-D-Phe⁶,Leu¹⁷]VIP, 2) [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂, and 3) VIP(10–28) to assess the involvement of endogenous VIP in the regulation of thyroid hormone secretion and thyroid blood flow (BF). We measured thyroid BF in ketamine-pentobarbital-anesthetized rats using the microsphere technique. Increases in thyroid BF induced by VIP administration (30 pmol-1.5 nmol/100 g b.wt.) were not affected by any of the three compounds tested at doses 10–100 times higher than that of VIP. These compounds (3–15 nmol/100 g b.wt.) also failed to affect basal thyroid BF or hormone secretion. Increases in pancreatic and salivary gland BFs induced by VIP (30 pmol/100 g b.wt.) were also not affected by [4Cl-D-Phe⁶,Leu¹⁷]VIP or [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂ (3 nmol/100 g b.wt.). These results indicate that the three compounds tested are not effective inhibitors of VIP receptors in the thyroid vasculature and, therefore, they cannot be used in the investigation of the functional significance of endogenous VIP in the regulation of thyroid BF.     

Manganese induces sustained Ser40 phosphorylation and activation of tyrosine hydroxylase in PC12 cells

Manganese (Mn²⁺) is an essential metal involved in normal functioning of a range of physiological processes. However, occupational overexposure to Mn²⁺ causes neurotoxicity. The dopaminergic system is a particular target for Mn²⁺ neurotoxicity. Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis and is regulated acutely by phosphorylation at Ser40 and chronically by protein synthesis. In this study we used pheochromocytoma 12 cells to investigate the effects of Mn²⁺ exposure on the phosphorylation and activity of TH. Mn²⁺ treatment for 24 h caused a sustained increase in Ser40 phosphorylation and TH activity at a concentration of 100 μM, without altering the level of TH protein or PC12 cell viability. Inhibition of protein kinase A and protein kinase C and protein kinases known to be involved in sustained phosphorylation of TH in response to other stimuli did not block the effects of Mn²⁺ on Ser40 phosphorylation. A substantial increase in H₂O₂ production occurred in response to 100 μM Mn²⁺. The antioxidant Trolox^TM completely inhibited H₂O₂ production but did not block TH phosphorylation at Ser40, indicating that oxidative stress was not involved. Sustained TH phosphorylation at Ser40 and the consequent activation of TH both occurred at low concentrations of Mn²⁺ and this provides a potential new mechanism for Mn²⁺-induced neuronal action that does not involve H₂O₂-mediated cell death.     

Straight Oso4 versus Glutaraldehyde-Oso4 In Sequence as Fixatives for the Granular Vesicles in Sympathetic Axons of the Rat Pineal Body

Pineal bodies were removed immediately after death from 6 rats: representing both sexes, and adult and 21-day postnatal ages; cut into 2 or 3 pieces, and subjected to experimental fixations at pH 7.3, 0-4 C as follows: 1-2 hr in 1% OsO₄, with veronal-acetate buffer of phosphate buffer; 3-4 hr in 3% or 6% glutaraldehyde in 0.1 M or 0.2 M phosphate buffer, with or without 1% sucrose. Specimens from OsO₄ were dehydrated, and embedded in epoxy resin; those from glutaraldehyde were allowed to soak in buffer for 12-16 hr, then transferred to 1% OsO₄ at 0-4 C for 2 hr, and embedded in the same manner as the ones fixed directly in OsO₄. Representative electron micrographs of postganglionic sympathetic endings were studied for the morphology and frequency of granular vesicles. No consistent difference was shown between vesicles fixed in OsO₄ buffered by phosphate or by veronal-acetate, nor was there any effect caused by the different concentrations used for the glutaraldehyde solution; however, vesicles fixed by the glutaraldehyde-OsO₄ sequence showed an enhancement in the graininess of their membranes, were slightly larger, and had a much larger dense core than those fixed by OsO₄ alone. After glutaraldehyde-OsO₄, granular vesicles showed a frequency of 81%, whereas after direct fixation in OsO₄, only 40% without significant change their number per unit area. Therefore, glutaraldehyde-OsO₄ seems to be more effective than straight OsO₄ for the demonstration of granular vesicles in the autonomic nervous system.     

Metal complexes of crosslinked chitosans Part II. An investigation of their hydrolysis to chitooligosaccharides using chitosanase

This paper investigates the behavior of crosslinked chitosans and metal-complexed crosslinked chitosans under similar hydrolytic conditions. Crosslinked chitosans with trimellitic anhydride, diisocyanatohexane, and dibromodecane as crosslinking agents under heterogenous reaction conditions were used as metal complexing agents by equilibrating them with metal salts such as ZnCl₂, MnSO₄, CuSO₄, CdSO₄, Pb(NO₃)₂, and HgCl₂. Crosslinked chitosan without metal complexation had the same hydrolytic behavior as uncrosslinked chitosan. However, when the crosslinked chitosans were complexed with metals, their rates of hydrolysis and extent of hydrolysis were significantly reduced. Thus, while for chitosan about 840 μg/ml reducing sugar was produced in 4 h time, and 780 μg/ml was produced for diisocyanatohexane crosslinked chitosan, only 400 μg/ml and 320 μg/ml reducing sugars were produced for cadmium sulfate with crosslinked chitosan and diisocyanatohexane crosslinked chitosan, respectively. Similar results are obtained for other crosslinking agents. Studies on preincubation of the metal with the enzyme show that of the metals studied, Mn has no effect on preincubatioin with the enzyme, Hg, Cd, Pb, and Cu completely deactivates the enzyme, while Zn reduces the enzyme activity by about 43.3%. Preincubation of the metal salts with the chitosan shows that Hg and Cu completely deactivate the molecule from enzyme hydrolysis, Cd and Zn inactivate it to the extent of 56.8% and 43.3%, respectively, while Mn has no effect. Availability of the amino functions seems to be a key feature for the chitosanase to hydrolyze the chitosan polymer. This was also proved by the significant increase in the extent of hydrolysis for chitosan samples with 88% (final value 1120 μg/ml reducing sugar) and 85% deacetylation (final value 840 μg/ml reducing sugar). HPIC studies of the products show that a variety of oligomers are produced in the chitosanase enzyme hydrolytic reaction.     

Influence of different silica derivatives in the immobilization and stabilization of a Bacillus licheniformis protease (Subtilisin Carlsberg)

Alcalase 2T, a commercial preparation of Subtilisin Carlsberg, was covalent immobilized onto physiochemically characterized silica supports. The effect of mean pore diameter and surface chemistry on enzyme activity in the hydrolysis of casein has been examined. Two sets of chemically distinct silica supports were used presenting terminal amino (S_APTES) or hydroxyl groups (S_TESPM-pHEMA). The percentage of immobilized protein was smaller in S_APTES (31–39%) than in S_TESPM-pHEMA (62–71%), but presented higher total and specific activity. Silicas with large pores (S₁₀₀₀, 130/1200 Å) presented higher specific activities relative to those with smaller pore sizes (S₃₀₀, 130/550 Å). The influence of glutaraldehyde concentration and the time of enzyme coupling to the S₁₀₀₀S_APTES supports was examined. The apparent K_m value for the S₁₀₀₀S_APTES immobilized enzyme is lower than the soluble one which may be explained by the partitioning effects of the substrate. No intraparticle diffusion limitations were observed for the immobilized enzyme and therefore the substrate diffusion does not influence the observable kinetics. Finally, the optimum pH, optimum temperature, thermal stability, operational stability, and storage stability of the immobilized and freely soluble enzymes were compared.     

Generation of Reactive Oxygen Species by Tyrosine Hydroxylase: A Possible Contribution to the Degeneration of Dopaminergic Neurons?

Abstract: It has been suggested that idiopathic parkinsonism, characterized by a loss of dopaminergic neurons of the nigrostriatal pathway, is due to the intracellular generation of reactive oxygen species, generated by a nonenzymatic or enzymatic partial reduction of dioxygen. Based on in vitro studies of the iron-containing monooxygenase tyrosine hydroxylase (TH), evidence is presented that this enzyme system may also contribute to such an oxidative stress. Thus, the purified and Fe²⁺-reconstituted recombinant human enzyme shows a time- and temperature-dependent partial uncoupling of the hydroxylation of l -tyrosine with the natural cofactor (6 R )-tetrahydrobiopterin, resulting in the formation of H₂O₂. The degree of uncoupling of the hydroxylation reaction is significantly higher when certain substrate analogues, notably the 7-substituted isomer (7-tetrahydrobiopterin) of the natural cofactor, is used. In the presence of H₂O₂ and Fe²⁺, the addition of TH increases the production of the highly reactive ^•OH radical, probably via a Fenton type of reaction. It is not clear whether this in vitro reaction can mediate cellular injury in vivo. However, it is known that the distribution of TH in the central and peripheral nervous system often corresponds to that of the neuronal degeneration in idiopathic parkinsonism, a finding that is compatible with a pathogenetic effect of TH.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.