Release of acid hydrolases in spectrum of human leprosy.

Release of acid hydrolases by blood monocytes (BM) of leprosy patients both before and after 6 months of chemotherapy was measured fluorimetrically. Monocyte cultures were set up for spontaneous as well as zymosan dependent enzyme release measured after 2 hrs and 24 hrs of culture. In the untreated multibacillary group (BL/LL) a significantly higher (P < 0.001) release of both B-glucuronidase (BG) and N-acetyl glucosaminidase (NAG) was observed compared to the paucibacillary group (BT/TT) and healthy controls. On comparing the BT/TT group with controls a significant decrease (P < 0.001) in zymosan dependent NAG release was observed in the former group at 2 hrs culture. After 6 months of antileprosy therapy, a significant decrease (P < 0.05) in BG release was observed from BM of multibacillary patients, whereas NAG activity increased significantly (P < 0.05) in the paucibacillary group compared to the controls. The results of the present study suggest that non-oxidative metabolic status of BM vary within the leprosy spectrum.     

Lysosomes in lymphoid tissue. II. Intracellular distribution of acid hydrolases

Differential centrifugation and density gradient isopycnic centrifugation have been used to fractionate homogenates of rat spleen and, in a few experiments, of rat thymus and cervical lymph nodes. The fractions have been analyzed for proteins, DNA, RNA, cytochrome oxidase, esterase, and up to 11 acid hydrolases. The results obtained indicate that the hydrolases are associated, at least largely, with cytoplasmic particles of lysosomal nature, and suggest further that these particles belong to two, and possibly three, distinct populations, perhaps reflecting the cellular heterogeneity of the tissues. The populations are identified as: (a) the L(19) population, the most important group, containing all 12 hydrolases and characterized by a modal density of about 1.19 in a sucrose-0.2 M KCl gradient; (b) the L(15) population with a modal density of 1.15, a group of apparently incomplete lysosomes containing cathepsin D and a few other enzymes, but very poor in, or entirely devoid of, several acid hydrolases, including cathepsins B and C; (c) the L(30) population, comprising all 12 enzymes and banding together with the nuclei at a density of 1.30 or higher. Lack of success in separating the latter group from the nuclei renders its significance unclear.     

Determination of acid hydrolases in human platelets

A method is described which allows the preparation of pure cinnamoyl-CoA thiolesters in high yields. This procedure utilizes a partially purified cinnamoyl-CoA ligase obtained from a strain of Pseudomonas putida and some properties of this new enzyme are described. Product isolation involves polyamide column chromatography which allows the purification of 50-mg batches of thiolesters. The method is applicable to a range of cinnamic acids, and is particularly suitable in preparing the biologically important CoA esters of p-coumarate, ferulate, and caffeate.     

The quantitative histochemistry of multiple sclerosis plaques: acid proteinase and other acid hydrolases.

Abstract— Sensitive micromethods were used to study the plaques, adjacent white matter and remote, grossly normal white matter from two cases of multiple sclerosis and to compare them with white matter from normal controls. Lipid-free dry wt/unit of volume was found to be similar for plaques and for normal white matter, reflecting a high water content of plaque tissue and establishing a base for comparison of enzyme activities. Elevations of acid proteinase in and around plaques were confirmed, but they were far exceeded by the increases in acid phosphatase; other acid hydrolases (β-galactosidase, β-glucuronidase and dipeptidyl arylamidase II) showed no significant or consistent changes. However, an acid lipase-esterase hydrolysing 4-methylumbelliferyl oleate was about 30% as active in plaques as in normal-appearing white matter. Glucose-6-phosphate dehydrogenase was unchanged except in one plaque, but lactic dehydrogenase was markedly elevated both in plaques and adjacent white matter. The grossly normal white matter of MS patients, although histologically far from normal (showing gliosis, perivascular infiltrations and small plaques), did not differ significantly from controls with regard to the activity of any of the enzymes studied. DNA levels were much reduced in plaques, but comparisons were difficult because of the apparent gliosis in normal white matter. Decreases in dry wt/unit vol, reflecting partial demyelination, could be shown to extend in a gradient to a distance of about 2 mm. from the edge of certain plaques.     

Plant fatty acid (ethanol) amide hydrolases

Fatty acid amide hydrolase (FAAH) plays a central role in modulating endogenous N-acylethanolamine (NAE) levels in vertebrates, and, in part, constitutes an "endocannabinoid" signaling pathway that regulates diverse physiological and behavioral processes in animals. Recently, an Arabidopsis FAAH homologue was identified which catalyzed the hydrolysis of NAEs in vitro suggesting a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, we provide evidence to support this concept by identifying candidate FAAH genes in monocots (Oryza sativa) and legumes (Medicago truncatula), which have similar, but not identical, exon-intron organizations. Corresponding M. truncatula and rice cDNAs were isolated and cloned into prokaryotic expression vectors and expressed as recombinant proteins in Escherichia coli. NAE amidohydrolase assays confirmed that these proteins indeed catalyzed the hydrolysis of 14C-labeled NAEs in vitro. Kinetic parameters and inhibition properties of the rice FAAH were similar to those of Arabidopsis and rat FAAH, but not identical. Sequence alignments and motif analysis of plant FAAH enzymes revealed a conserved domain organization for these members of the amidase superfamily. Five amino-acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the FAAH sequences of six plant species. Homology modeling of the plant FAAH proteins using the rat FAAH crystal structure as a template revealed a conserved protein core that formed the active site of each enzyme. Collectively, these results indicate that plant and mammalian FAAH proteins have similar structure/activity relationships despite limited overall sequence identity. Defining the molecular properties of NAE amidohydrolase enzymes in plants will help to better understand the metabolic regulation of NAE lipid mediators.     

Alpha-amino acid ester hydrolases: Properties and applications

The review describes two major groups of α-amino acid ester hydrolases (AEHs)—enzymes with a similar active center structure, which determines their unique specificity to esters containing an amino group in the α position to the carbonyl. The first group comprises microbial AEHs of the β-lactam acylase type. Technical biocatalysts based on this group of enzymes are used for the production of semi-synthetic amino-β-lactam antibiotics. The second AEH group includes eukaryotic valacyclovirases, which activate in vivo a number of antiviral and anticancer prodrugs. The directed activity of these enzymes is used for the development of target pharmaceutical preparations for the therapy of viral and oncological diseases. The review summarizes and compares the available data on the structure and properties, substrate specificity, and the kinetic parameters of enzymes of these two groups. Experiments identifying the AEH active site and providing the molecular basis for the unique specificity of these enzymes are discussed. The data from the available scientific and patent publications concerning the aminopenicillin and aminocephalosporin synthesis catalyzed by β-lactam acylase AEHs are reviewed and systematized.     

Membranes carrying acid hydrolases in pea seedling roots

Subcellular localization of acid hydrolases in pea seedlingroots was studied by differential and sucrose density gradientcentrifugations. Significant parts of hydrolase activities inthe tissue were recovered in mitochondrial and microsomal fractions.Sedimentable phosphatase was separated into two subtractions:denser and lighter membrane fractions. The distribution of phosphataseactivity after sucrose density gradient centrifugation of thedenser fraction coincided with that of antimycin AinsensitiveNADH-cytochrome c reductase activity. Electron microscopic observationssuggested that the fraction contained only microsomes. RNasein the denser fraction seemed to associate with ribosomes. Phosphataseand RNase were solubilized by sonic treatment in the presenceof high concentrations of salt. On the other hand, a-amylasewas tightly bound to a membrane. The results are discussed withspecial regard to the relationship between the membranes andlysosomes. (Received May 4, 1973; )     

Latency of acid hydrolases in rat kidney cortex

1. Some lysosomal populations in the rat kidney cortex appear to be mechanically weak and are readily disrupted by gentle homogenization, while other populations remain intact even after repeated homogenization. 2. Lysosomes in the rat kidney cortex appear to be resistant to hypertonic media but are readily disrupted under hypotonic conditions. 3. Lysosomes in rat kidney cortex are readily disrupted when incubated in isotonic sucrose at 37 degrees C. 4. Measurement of total and free activity of three acid hydrolases: N-acetyl-beta-D-glucosaminidase (NAG), acid beta-galactosidase and acid beta-glycerophosphatase, indicates that the latency of these enzymes is relatively low in the homogenate (10-29%) and the ML-fraction (14-42%), but high (60-95%) in the purified large lysosomes (protein droplets). 5. The latency of purified small lysosomes is relatively lower (30-60%) than that of large lysosomes, suggesting that small lysosome populations are relatively permeable to the acid hydrolase substrates. 6. Latency variations of acid hydrolases amongst subcellular fractions appear to reflect the heterogeneity of lysosomal populations present in the kidney cortical homogenate.