Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.     

A sensitive method for quantitative analysis of phospholipid molecular species by high-performance liquid chromatography

A simple and sensitive method was developed for quantitative analysis of phospholipid molecular species. Diacylglycerols were prepared from phospholipids by phospholipase C treatment and converted to the corresponding dinitrobenzoyl derivatives, which could be sensitively detected at 254 nm. The derivatives of 21 molecular species were resolved by high-performance liquid chromatography with an octadecylsilyl reversed-phase column. All the derivatives had the same peak area per mol, and peak areas were proportional to the amounts of the derivatives. Quantification was carried out at the picomole level.     

Inactivation and sedimentation of mercury from organomercurials by Klebsiella pneumoniae

Abstract A susceptibility of 63 clinical isolates of Klebsiella pneumoniae to inorganic and organic mercuric compounds was determined. 18 of them were found to be resistant to fluorescein mercuric acetate (FMA) and merbromin (MB). Moreover, all the resistant strains inactivate the antibacterial effect of FMA. The changes in the amount of organic mercury at the time of inactivation of the drug and the structures of the end products were examined in detail with the plasmid-bearing strain JK9 and its transconjugants of Escherichia coli .
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent.     

A new hereditary single band variant of the Gc system

Summary A new single band variant (Gc Ar) or the Gc subtypes not identical with the known Gc variants has been detected in the plasma of a healthy blood donor by isoelectric focusing. Using this technique the variant is represented by a single band which has a similar isoelectric point to the Gc 1C2 anodal band. It is well known that the single band Gc phenotypes remain unaltered after neuraminidase treatment. Nevertheless, the new single band variant (Gc Ar) is altered after neuraminidase treatment as is Gc 2A3. After neuraminidase treatment, the Gc Ar band is affected and moved to the nearby position of the Gc 2 band. Investigation of the proband's family shows that the variant occurs combined with the common alleles Gc 1F, Gc 1S and that it has an autosomal dominant inheritance.     

Demonstration of Aujeszky's disease virus antigen in trypsin-digested paraffin-embedded tissue sections by immunofluorescence

After trypsin digestion of buffered formalin-fixed, paraffin-embedded brain section, immunofluorescent indication of Aujeszky's disease virus antigen was successful in experimentally infected cow and goat. This result was comparable to those obtained with light and electron microscopic examinations. This method will be diagnostically useful where fresh-frozen tissue is not available.     

Improvement of first-service pregnancy rate in cows with gonadotropin-releasing hormone analog

The effect of an intramuscular injection of a new analog of gonadotropin-releasing hormone (GnRH), fertirelin, on the first-service pregnancy rate in cows was investigated by a double blind experiment. A total of 1,194 cows was injected intramuscularly either with 100 mug of GnRH or placebo (physiological saline solution) at the time of first insemination postpartum. Pregnancy rate (number of cows calved/ number of cows serviced) was 57.2 % in 605 cows treated with GnRH, while the performance was 49.7 % in 589 cows of the placebo group. The difference of pregnancy rates in both groups was significant (P<0.05). GnRH injected at insemination was effective, especially in cows at the first and third lactations, cows at 101 days postpartum or later, cows with daily milk yield of 26-30 kg, and also in cows from the area where a regional average fertility was relatively low.     

Topological considerations in protein structure. I. Disulfide linkage

The patterns of disulfide bridges in proteins were considered by using the concept of topological information content. It was proposed that the difference between topological informal ion content in the half-cystine residues of the native state and of the fully reduced form of a protein is related to the problems of the renaturation of the fully reduced and denatured protein. Specifically, when there is no difference between the topological information contents of the two states, the reduced protein is able to recover its native conformation. The concept reported in the present paper is consistent without exception with reported experimental results.