Electroosmotic Perfusion-Microdialysis Probe Created by Direct Laser Writing for Quantitative Assessment of Leucine Enkephalin Hydrolysis by Insulin-Regulated Aminopeptidase in Vivo.

There are many processes that actively alter the concentrations of solutes in the extracellular space. Enzymatic reactions, either by soluble enzymes or membrane-bound ectoenzymes, and uptake or clearance are two such processes. Investigations of ectoenzymatic reactions in vivo is challenging, particularly in the brain. Studies using microdialysis have revealed some qualitative information about what enzymes may be present, but microdialysis is a sampling technique so it is not designed to control conditions such as a substrate concentration outside the probe. Micropush-pull perfusion has been used to determine which nitric oxide synthase enzymes are active in discrete regions of the rat retina. Ectopeptidases are a particularly important class of ectoenzymes. As far as it is known, the extracellular activity of active peptides in the brain is controlled by ectopeptidases. To understand ectopeptidase activity, we developed a physical probe and an accompanying method. The probe has a two-channel source that supplies substrate or substrate plus inhibitor using electroosmotic perfusion (EOP). It also has a microdialysis probe to collect products and unreacted substrate. The method provides quantitative estimates of substrate-to-product conversion and the influence of inhibitors on this process. The quantitative estimates are made possible by including a d-amino acid-containing peptide analog of the substrate in the substrate-containing solution infused. Quantitative analysis of substrate, substrate analog, and products is carried out by quantitative, online capillary liquid chromatography-tandem mass spectrometry. The electroosmotic perfusion-microdialysis probe and associated method were used to determine the effect of the selective inhibitor HFI-419 on insulin-regulated aminopeptidase (EC 3.4.11.3) in the rat neocortex.

M42 aminopeptidase catalytic site: the structural and functional role of a strictly conserved aspartate residue.

The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron-shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined by seven conserved residues. Five of them are involved in metal ion binding which is important to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The seventh residue is an aspartate whose function remains poorly understood. This aspartate residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2 binding site. We assess its role in TmPep1050, an M42 aminopeptidase of Thermotoga maritima, through a mutational approach. Asp-62 was substituted with alanine, asparagine, or glutamate residue. The Asp-62 substitutions completely abolished TmPep1050 activity and impeded dodecamer formation. They also interfered with metal ion binding as only one cobalt ion is bound per subunit instead of two. The structure of Asp62Ala variant was solved at 1.5 Å showing how the substitution has an impact on the active site fold. We propose a structural role for Asp-62, helping to stabilize a crucial loop in the active site and to position correctly the catalytic base and a metal ion ligand of the M1 site.

Entropic stabilization of proteins and its proteomic consequences.

Evolutionary traces of thermophilic adaptation are manifest, on the whole-genome level, in compositional biases toward certain types of amino acids. However, it is sometimes difficult to discern their causes without a clear understanding of underlying physical mechanisms of thermal stabilization of proteins. For example, it is well-known that hyperthermophiles feature a greater proportion of charged residues, but, surprisingly, the excess of positively charged residues is almost entirely due to lysines but not arginines in the majority of hyperthermophilic genomes. All-atom simulations show that lysines have a much greater number of accessible rotamers than arginines of similar degree of burial in folded states of proteins. This finding suggests that lysines would preferentially entropically stabilize the native state. Indeed, we show in computational experiments that arginine-to-lysine amino acid substitutions result in noticeable stabilization of proteins. We then hypothesize that if evolution uses this physical mechanism as a complement to electrostatic stabilization in its strategies of thermophilic adaptation, then hyperthermostable organisms would have much greater content of lysines in their proteomes than comparably sized and similarly charged arginines. Consistent with that, high-throughput comparative analysis of complete proteomes shows extremely strong bias toward arginine-to-lysine replacement in hyperthermophilic organisms and overall much greater content of lysines than arginines in hyperthermophiles. This finding cannot be explained by genomic GC compositional biases or by the universal trend of amino acid gain and loss in protein evolution. We discovered here a novel entropic mechanism of protein thermostability due to residual dynamics of rotamer isomerization in native state and demonstrated its immediate proteomic implications. Our study provides an example of how analysis of a fundamental physical mechanism of thermostability helps to resolve a puzzle in comparative genomics as to why amino acid compositions of hyperthermophilic proteomes are significantly biased toward lysines but not similarly charged arginines.

Anticuerpos monoclonales en la evaluación de enzimas del ribete estriado yeyunal de lactantes con diarrea persistente / Monoclonal antibodies in the assessment of the jejunal striatum border enzymes in infants with longstanding diarrhea

En las biopsias de mucosa yeyunal de 10 pacientes con diarrea persistente se estudió expresión de las enzimas lactasa, sacarasa-isomaltasa, maltasa y aminopeptidasa, del ribete estriado, mediante anticuerpos monoclonales y los resultados se contrastaron con los síntomas y signos clínicos, morfológicos (microscopía de luz), actividad disacaridásica (Dahlqvist) y la expresión de lactasa por un método histoquímico. Se obtuvo expresión de aminopeptidasa en criptas y vellosidades, mediante los anticuerpos correspondientes. La expresión por anticuerpos histoquímica y actividad enzimática (Dahlqvist) fueron concordantes para la expresión de lactasa en las vellosidades, mientras en las criptas se registró positividad sólo en 2 casos. En las vellosidades los anticuerpos monoclonales tendieron a producir más reacciones positivas para sacarasa-isomaltasa en los casos con menos daño morfológico, excepto en uno de deficiencia primaria; en las criptas el resultado fue positivo en todos, menos dos pacientes, en los que tampoco hubo positividad en las vellosidades. Los anticuerpos monoclonales pueden aportar información útil para entender mejor los mecanismos de daño y reparación de las enzimas del ribete estriado y estimar el pronóstico de la lesión

Assessment of metabolic activity in aortic vein grafts in rats by histochemical examination of hydrolases.

The metabolic pattern of aortic vein grafts in rats was studied by estimating histochemically the activity of the hydrolytic enzymes alfa-esterase, aminopeptidase, adenosine triphosphatase, acid phosphatase and alkaline phosphatase. The enzyme activity was lowest in the 16 hour and 1 day old grafts, and recovery was noted at three days. Five days after transplantation the enzyme activities were higher than in the non-transplanted veins. The rapid increase in enzymatic activity found in histochemical studies on wound healing was not seen in these vein grafts. At four weeks some grafts showed intimal thickening the activity of which did not exceed that of the other layers of the graft wall. At the end of the observation period of sixteen weeks most of the grafts showed intimal thickening, and this layer stained intensely especially for ATPase. The staining pattern of most of the grafts at sixteen weeks resembled that of the aortic media.

Epididymal and testicular enzymes as monitors for assessment of male antifertility drugs.

PIP: Since the synthesis and maturational processes of sperm are associated with characteristic alterations in different marker enzyme activities in testes and epididymis, it is possible to monitor these enzymes to investigate whether 3 antispermatogenic agents, WIN 18 446, alpha-chlorohydrin (AC), and cyproterone acetate (CA), have any characteristic effects on biochemical events associated with spermatogenesis and maturation of sperm; acid, neutral, and alkaline proteinases, particulate and soluble arylamidases, and sialidase were studied after treatment with 1 of the 3 agents in male albino rats (CIBA strain) by measuring these enzyme levels in rat testicular and epididymal tissues. After WIN treatment, sialidase activity as well as sialic acid content increased in the testis, whereas no such effect occurred in the epididymis at any dose. At low dose, AC (10 mg/kg/day for 7 days) and CA (50 mg/kg/day for 10 days) decreased the sialic acid content and the sialidase activity in the epididymis, whereas the sialic acid and sialidase activity in the testis remained unchanged. At higher dose levels, AC (25 mg) and CA (50 mg) both affected the tissues significantly, i.e., enhancing sialidase activity and lowering sialic acid content. Therefore, the effect of CA and AC is more prominent on the maturational phenomena than the testicular spermatogenesis. AC and CA deserve further investigation for use as a male contraceptive. The relationship between proteinase, sialidase, and arylamidase activities and different phases of spermatogenesis and maturation was established by these test results.^ieng