Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.     

Inhibited light activation of fructose and sedoheptulose bisphosphatase in spinach chloroplasts exposed to osmotic stress

The light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) was inhibited in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Decreases in the velocity and magnitude of light activation correlated with the overall reduction in CO₂ fixation rates. Responses of osmotically stressed chloroplasts to both varying pH and exogeous dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerete (PGA) were examined. In the presence of DHAP, the absolute rate of CO₂ fixation was increased and this increase was most pronounced at alkaline pH. Enhanced light activation of these enzymes was also observed under these conditions. However, in the presence of PGA, similar increases in photosynthetic rate and enzyme activation were not evident. Light-dependent stromal alkalization was unaffected by the stress treatments. Inhibition of light activation under hypertonic conditions is discussed in terms of substrate availability, possible alterations of the redox state of ferredoxin and associated electron carriers, and inhibited enzyme-enzyme or enzyme-substrate interactions involved in the light activation process.Abbreviations and symbols DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate - _s osmotic potential     

Protoplast production from filamentous fungi with their own autolytic enzymes

Abstract Production of protoplasts in different genera of filamentous fungi with their own lytic enzymes obtained from autolyzed cultures, as well as the regeneration of these protoplasts, has been studied. The results support the idea that the use of these autolytic enzymes could be a general method of production of protoplasts from filamentous fungi.     

The site of carotenogenic enzymes in chromoplasts from Narcissus pseudonarcissus L.

The membranes from the chromoplasts of Narcissus pseudonarcissus L. which are derived from the inner envelope membrane are the site of -carotene synthesis from [1-¹⁴C]isopentenyl diphosphate. The enzymes involved are partly peripheral membrane proteins (prenyltransferase, phytoene synthase) and partly integral membrane proteins (cis-trans isomerase, dehydrogenase(s), cyclase(s)). Metabolic channeling is suggested.Abbreviations IPP isopentenyl diphosphate - GGPP geranylgeranyl diphosphate     

Exchange and interactions between lipid layers at the surface of a liposome solution

Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. p] Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.     

Effects of 2.45-GHz microwave radiation on embryonic quail hearts

Although exposure to nonionizing electromagnetic radiation has been reported to cause a variety of systemic alterations during embryonic development, there are few reports of the induction of specific physiologic or morphologic changes in the myocardium. This study was designed to examine the effects of microwave radiation on cardiogenesis in Japanese quail embryos exposed during the first eight days of development to 2.45-GHz continuous-wave microwaves at power densities of 5 or 20 mW/cm². The specific absorption rates were 4.0 and 16.2 mW/g, respectively. The ambient temperature for each exposure was set to maintain the embryonated eggs at 37.5 °C. This did not preclude thermal gradients in the irradiated embryos since microwaves may not be uniformly absorbed. The test exposure levels did not induce changes in either the morphology of the embryonic heart or the ultrastructure of the myocardial cells. Analysis of the enzymatic activities of lactate dehydrogenase, glutamic oxaloacetic transaminase, and creatine phosphokinase failed to reveal any statistically significant differences between the nonexposed controls and those groups exposed to either 5 or 20 mW/cm². The data indicate that 2.45-GHz microwave radiation at 5 or 20 mW/cm² has no effect on the measured variables of the Japanese quail myocardium exposed during the first eight days of development.     

Genetic studies among Kanet and Koli of Kinnar district in Himachal Pradesh,India

Data are presented on serological and electrophoretic variants of 18 systems of red cells in 228 individuals belonging to a scheduled tribe (Kanet) and a scheduled caste (Koli) of Kinnar district in Himachal Pradesh, India. Differences in gene frequencies clearly indicate biological distinction in the local population. The possible cause of this genetic heterogeneity is discussed.     

Photosynthetic induction in wheat protoplasts and chloroplasts. Autocatalysis and light activation of enzymes

Abstract Unlike wheat chloroplasts, wheat protoplasts showed a pronounced restoration of the induction phase after a short period of darkness. This difference was used to investigate the relative roles of light-induced reductive activation of enzymes and the auto-catalytic increase in the level of substrates in the control of the rate of photosynthesis during induction. Light activation and dark inactivation of ribulose 5-phosphate kinase, fructose 1,6-biphosphatase and NADP⁺-specific glyceraldehydephosphate dehydrogenase were measured. In this respect there was no appreciable difference between protoplasts and chloroplasts. In contrast, the level of photosynthetic intermediates remained constant in darkened isolated chloroplasts, but declined rapidly in chloroplasts isolated from darkened protoplasts. When fructose 1,6-bisphosphatase was pre-activated by treating protoplasts with dithiothreitol the lag was only slightly shortened. These results are discussed in terms of control of the rate of the photosynthesis during the lag by substrates rather than limitation imposed by activity of any of the enzymes measured.     

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Properties of enzymes from bacteria grown in the 70–100°C range

In chemostat cultures of Bacillus caldolyticus, adaptation in a single step from 70–100°C was followed under aerobic and oxygen-limited conditions and was found to proceed more smoothly under the latter circumstances. Variations of the medium (e.g. yeast extract or silicate concentrations) showed that growth at 100°C is in all respects similar to that of cultures at moderate temperatures.Enzyme preparations derived from cultures at 5°C intervals between 70 and 100°C were used to determine the temperature range. For all nine enzymes tested, the optimum temperature was found to be 67°C; the latter was independent of the growth temperature. Differences were found, however, with respect to the maximum temperature of individual enzymes, and three groups, with maxima between 70 and 80°C, 80 and 90°C and 90 and 100°C can be distinguished. Again, there was no correlation with the growth temperature.Stability experiments also revealed that enzymes from the same organism can have different thermal properties: Some were found to be quite thermolabile (e.g. the pyruvate kinase), while others (e.g. hexokinase and glutamate-pyruvate transaminase) exhibited a high thermostability. These properties were not related to the growth temperature within the 70–100°C range, too.Six of the enzymes tested could be stabilized by their respective substrates, but the degree of protection varied for individual enzymes. Three enzymes (acetate kinase, glutamate dehydrogenase and myokinase) could not be stabilized by their substrates.Comparative experiments with the hexokinase suggested, that the thermal integrity of the enzymes is better protected within the cell as compared to the stability of the enzyme preparations.Abbreviations used AK acetate kinase - Ala-DH alanine dehydrogenase - Ald aldolase - GIDH glutamate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - GTP glutamate-pyruvate transaminase - HK hexokinase - ICDH isocitrate dehydrogenase - MK myokinase - PK pyruvate kinase