ATP sulfurylase from Penicillium chrysogenum: measurements of the true specific activity of an enzyme subject to potent product inhibition and a reassessment of the kinetic mechanism

Homogeneous ATP sulfurylase from Penicillium chrysogenum has been reported to have an extremely low activity toward its physiological inorganic substrate, sulfate. This low activity is an artifact resulting from potent product inhibition by 5'-adenylylsulfate (APS) (Ki less than 0.25 microM). Assays based on 35S incorporation from 35SO4(2-) into charcoal-adsorbable [35S]APS are nonlinear with time, even in the presence of a large excess of inorganic pyrophosphatase. However, in the presence of excess APS kinase (along with excess pyrophosphatase), the ATP sulfurylase reaction is linear with time and the enzyme has a specific activity (Vmax) of 6 to 7 units mg protein-1 corresponding to an active site turnover number of at least 400 min-1. Monovalent oxyanions such as NO3-, ClO3-, ClO4-, and FSO3- are competitive with sulfate (or molybdate) and essentially uncompetitive with respect to MgATP. However, thiosulfate (SSO3(2-)), a true sulfate analog and dead-end inhibitor of the enzyme (competitive with sulfate or molybdate), exhibited clear noncompetitive inhibition against MgATP. Furthermore, APS was competitive with both MgATP and molybdate in the molybdolysis assay. These results suggest (a) that the mechanism of the normal forward reaction may be random rather than ordered and (b) that the monovalent oxyanions have a much greater affinity for the E X MgATP complex than for free E. In this respect, FSO3-, ClO4-, etc., are not true sulfate analogs although they might mimic an enzyme-bound species formed when MgATP is at the active site. The nonlinear ATP sulfurylase reaction progress curves (with APS accumulating in the presence of excess pyrophosphatase or PPi accumulating in the presence of excess APS kinase) were analyzed by means of "average velocity" plots based on an integrated rate equation. This new approach is useful for enzymes subject to potent product inhibition over a reaction time course in which the substrate concentrations do not change significantly. The analysis showed that ATP sulfurylase has an intrinsic specific activity of 6 to 7 units mg protein-1. Thus, the apparent stimulation of sulfurylase activity by APS kinase results from the continual removal of inhibitory APS rather than from an association of the two sulfate-activating enzymes to form a "3'-phospho-5'-adenylylsulfate synthetase" complex in which the sulfurylase has an increased catalytic activity. The progress curve analyses suggest that APS is competitive with both MgATP and sulfate, while MgPPi is a mixed-type inhibitor with respect to both substrates. The cumulative data point to a random sequence for the forward reaction with APS release being partially rate limiting.     

Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

Improving the clinical assessment of consciousness with advances in electrophysiological and neuroimaging techniques

In clinical neurology, a comprehensive understanding of consciousness has been regarded as an abstract concept - best left to philosophers. However, times are changing and the need to clinically assess consciousness is increasingly becoming a real-world, practical challenge. Current methods for evaluating altered levels of consciousness are highly reliant on either behavioural measures or anatomical imaging. While these methods have some utility, estimates of misdiagnosis are worrisome (as high as 43%) - clearly this is a major clinical problem. The solution must involve objective, physiologically based measures that do not rely on behaviour. This paper reviews recent advances in physiologically based measures that enable better evaluation of consciousness states (coma, vegetative state, minimally conscious state, and locked in syndrome). Based on the evidence to-date, electroencephalographic and neuroimaging based assessments of consciousness provide valuable information for evaluation of residual function, formation of differential diagnoses, and estimation of prognosis.     

Immunotherapy with beta-amyloid for Alzheimer's disease: a new frontier.

Alzheimer's disease (AD) represents the fourth leading cause of death in the U.S. and the leading cause of dementia in the elderly population. Until recently, there was little hope of finding a way to prevent the underlying brain pathology from progressing toward the inevitable conclusion of the disease. However, new immunotherapeutic approaches have been described that are based on vaccination with the beta-amyloid 1-42 peptide (Abeta). The encouraging efficacy and safety of Abeta immunization in reducing neuropathology in animal models of AD has opened up new therapeutic possibilities for patients. Immunization with Abeta is aimed at reducing the Abeta-associated pathology of AD. It is hypothesized that this approach will also reduce the cascade of downstream events leading to neuronal cell loss and, ultimately, dementia. The ensuing articles in this issue describe various aspects of the Abeta immunization strategy and their potential relevance to AD treatment.     

Identification of a mutation in the ovine uroporphyrinogen decarboxylase (UROD) gene associated with a type of porphyria

Porphyria is a group of at least eight diseases caused by abnormalities in the chemical steps that lead to haeme production. The different types of porphyria show different signs and symptoms and can be strongly influenced by environmental factors. Mutations of the uroporphyrinogen decarboxylase (UROD) gene have been shown to be causative for porphyria cutanea tarda (PCT) in humans. Porphyria is a rare disorder in livestock. Although disorders of haeme biosynthesis have been described in cattle, pigs, sheep and cats, PCT has only been reported in pigs. We observed typical signs of porphyria (photosensitivity and porphyrinuria) in a flock of German Blackface sheep and postulated that the porphyria could be caused by a mutation in the UROD gene. To investigate this, we cloned and sequenced the ovine UROD gene. We identified a single point mutation (C --> T) in UROD which leads to an amino acid substitution at Leu 131 Pro, which is located within the active cleft site of the UROD protein.     

Effects of alpha-synuclein immunization in a mouse model of Parkinson's disease

Abnormal folding of alpha-synuclein (alpha-syn) is thought to lead to neurodegeneration and the characteristic symptoms of Lewy body disease (LBD). Since previous studies suggest that immunization might be a potential therapy for Alzheimer's disease, we hypothesized that immunization with human (h)alpha-syn might have therapeutic effects in LBD. For this purpose, halpha-syn transgenic (tg) mice were vaccinated with halpha-syn. In mice that produced high relative affinity antibodies, there was decreased accumulation of aggregated halpha-syn in neuronal cell bodies and synapses that was associated with reduced neurodegeneration. Furthermore, antibodies produced by immunized mice recognized abnormal halpha-syn associated with the neuronal membrane and promoted the degradation of halpha-syn aggregates, probably via lysosomal pathways. Similar effects were observed with an exogenously applied FITC-tagged halpha-syn antibody. These results suggest that vaccination is effective in reducing neuronal accumulation of halpha-syn aggregates and that further development of this approach might have a potential role in the treatment of LBD.     

Streptococcus pyogenes SpyCEP Influences Host-Pathogen Interactions during Infection in a Murine Air Pouch Model

Streptococcus pyogenes is a major human pathogen worldwide, responsible for both local and systemic infections. These bacteria express the subtilisin-like protease SpyCEP which cleaves human IL-8 and related chemokines. We show that localization of SpyCEP is growth-phase and strain dependent. Significant shedding was observed only in a strain naturally overexpressing SpyCEP, and shedding was not dependent on SpyCEP autoproteolytic activity. Surface-bound SpyCEP in two different strains was capable of cleaving IL-8. To investigate SpyCEP action in vivo, we adapted the mouse air pouch model of infection for parallel quantification of bacterial growth, host immune cell recruitment and chemokine levels in situ. In response to infection, the predominant cells recruited were neutrophils, monocytes and eosinophils. Concomitantly, the chemokines KC, LIX, and MIP-2 in situ were drastically increased in mice infected with the SpyCEP knockout strain, and growth of this mutant strain was reduced compared to the wild type. SpyCEP has been described as a potential vaccine candidate against S. pyogenes, and we showed that surface-associated SpyCEP was recognized by specific antibodies. In vitro, such antibodies also counteracted the inhibitory effects of SpyCEP on chemokine mediated PMN recruitment. Thus, α-SpyCEP antibodies may benefit the host both directly by enabling opsonophagocytosis, and indirectly, by neutralizing an important virulence factor. The animal model we employed shows promise for broad application in the study of bacterial pathogenesis.