Occurrence and properties of a macroglobulin dimer in some hyperglobulinemic sera.

A 22S human gammaM-globulin component has been subjected to various study. It appears to be a dimer of 19S proteins and can undergo reversible molecular transitions which are both pH and temperature dependent. The 22S material regenerated from its 8S subunits no longer undergo similar transformations. Efforts to demonstrate that the 22S material was an antigen-antibody complex of 19S molecules were unsuccessful. Complex precipitin reactions in gels are given by macroglobulins containing this component with antibody to L chains.

Cytotoxic effects of daunomycin-fatty acid complexes on rat hepatoma cells.

The anthracycline antibiotic drug daunomycin has been coupled to fatty acids through formation of a peptide bond with the amino group of its lyxose residue. The arachidonic (C20:4) and docosahexaenoic (C22:6) acid derivatives showed potent in vitro and in vivo cytotoxic activity against rat hepatoma cells producing alpha-fetoprotein. With the exception of the acetate derivative, the complexes with saturated fatty acids showed no activity. The in vivo toxicity of the free daunomycin is markedly diminished when present in the form of these derivatives. The antitumor activities of the polyene fatty acid complexes appear to depend upon the high degree of affinity of alpha-fetoprotein for these acids. These results strongly suggest the use of daunomycin or other cytotoxic drug:polyene fatty acid complexes in the therapy of human tumors producing alpha-fetoprotein.

Chemistry and biology of alpha-fetoprotein.

Alpha-Fetoprotein (AFP) is a product of specific fetal tissues and of neoplastic cells of hepatocyte or germ cell origin in adults. This protein belongs to a gene family that is phylogenetically most closely related to serum albumin. Its primary, secondary, and tertiary structural aspects appear similar to the three-domain concept proposed for the latter protein. The primary sequence of AFP departs most widely from serum albumin in the first 135 amino acid residues, with about 42% of the remaining 590 residues of the human proteins being identical. Some evidence exists that there are limited sequence differences in the AFP of a given animal species. AFP shows considerable charge heterogeneity that appears to relate mostly to its glycoid moiety. The proteins of some species such as the rat show more pronounced heterogeneities than that of humans. The variations in extent and type of glycosylations are evidenced by differences in the binding to various lectins. These interactions are being extensively explored in attempts to differentiate the sources of the protein produced by various normal and neoplastic cells and may provide valuable diagnostic methods. AFP, like serum albumin, shows relatively strong binding affinities for a variety of ligands. The most notable difference is the strong preferential binding of polyunsaturated fatty acids by AFP. This protein may play a role in transporting these substances to developing and to malignant cells. Various agents affect the synthesis of this protein both by specific fetal tissues and by neoplastic cells. Marked differences in the responses of cells, particularly those of neoplastic types, are indicative of variations in the genetic factors responsible for control of its synthesis. The subject of the genomic repression of the synthesis of AFP seen in fetal life upon maturation of the liver and the reoccurrence of synthesis upon malignant conversion of hepatocytes and of certain germ cells are of particular interest. The regulation of the closely related AFP and albumin genes is providing a powerful and attractive model to examine molecular events in the activation and inactivation of specific genes during development and in oncogenic processes. Extensive measurements of AFP during pregnancy and in the course of neoplasias, notably hepatoma, are being made to aid in following changes in such developments. Various specific physiological roles for this protein are also being proposed. One of these is its possible action in the regulation of immune processes.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of the distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein by concanavalin A affinity chromatography.

The distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein was measured in vitro by a new method based on the separation of the two proteins by virtue of the binding specificity of concanavalin A for the carbohydrate moiety of alpha-fetoprotein. Human and bovine proteins were investigated. It was found that palmitate and oleate were distributed almost equally between albumin and alpha-fetoprotein, while docosahexaenoate and diethylstilbestrol bound preferentially to alpha-fetoprotein even at an albumin: alpha-fetoprotein ratio of 10:1. The results confirm the binding specificity of alpha-fetoprotein for polyunsaturated fatty acids and also show that alpha-fetoprotein binds diethylstilbestrol much more strongly than albumin does. This suggests that alpha-fetoprotein may play a role in the fetal uptake of diethylstilbestrol.

Preparation of human manganese superoxide dismutase by tri-phase partitioning and preliminary crystallographic data.

Human liver manganese superoxide dismutase has been purified by a short procedure that includes a tri-phase partitioning step to provide materials that can be crystallized from ammonium sulfate. X-ray diffraction studies at 3 A resolution show that the crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with cell dimensions a = b = 81.1 A, c = 242.2 A. Manganese superoxide dismutase levels as determined by enzymatic assay as well as by enzyme-linked immunosorbent assay indicated that considerable variations occur in different livers but the total superoxide dismutase activity (Mn superoxide dismutase plus Cu,Zn superoxide dismutase) seems to be kept at constant values.

Primary structure of a human lambda-chain (Weir) of the Mcg type.

The amino acid sequence of the Bence-Jones protein Weir has been determined. This lambda II protein is of the Mcg type. Since a mixed dinner of Weir and Mcg can be prepared in a form suitable for crystallographic analysis it was of interest to compare the sequences of these two lambda-chains. A total of 37 differences, which include one previously determined constant-region substitution, have been found.

Variable-region sequences of five human lambda-chain proteins reacting with an idiotypic antibody to the MCG Bence-Jones protein.

An antibody to the V-region of BJ protein MCG has been used to detect other human lambda-chains with similar idiotypic specificities. Five such proteins have been found and sequenced. They show relatively strong sequence homologies to some segments of the MCG V-region. The results of this study indicate that L-chains which contain short segments of their V-regions that are identical can be readily detected by the use of appropriate antisera.

Unexpected similarities in the crystal structures of the Mcg light-chain dimer and its hybrid with the Weir protein.

The covalently linked hybrid of two human lambda-type light chains (Mcg and Weir) crystallizes as trigonal bipyramids in ammonium sulfate [Ely et al., Molec. Immun. 22, 85-92 (1985)]. While markedly different in appearance from the barrel-shaped crystals of the parental Mcg dimer, the bipyramids of the hybrid have the same space group: trigonal P3(1)21. Moreover, the unit cell dimensions are practically identical: a = 72.3 A in both proteins; c = 188.1 A in the hybrid and 185.9 A in the Mcg dimer. These observations imply that the crystal packing and the main features of the three-dimensional structures are closely similar in the Mcg X Weir hybrid and the Mcg dimer. The "constant" domains of the Mcg and Weir proteins belong to the same genetic subclass and were expected to interact in comparable ways in hybrids and parental dimers. However, the overall similarities in the "variable" domain pairs in the hybrid and Mcg dimer were completely unpredicted, since the amino acid sequences of the heterologous variable domains differ by 36 residues. By difference Fourier analysis the Weir light chain has been tentatively identified as monomer 1 (heavy-chain analogue) and the Mcg protein as monomer 2 (light-chain analogue) in the hybrid dimer. Substitutions in key positions in the hypervariable loops explain the differences in binding activity of the Mcg and Weir dimers. In the Mcg dimer bis(dinitrophenyl)lysine spans two relatively spacious subsites (A and B), with primary contacts involving tyrosines 34 and 38 of monomer 2. The Weir dimer, which does not bind dinitrophenyl ligands, has serine and phenylalanine in homologous positions. Moreover, the bilateral replacement of valine 48 and serine 91 in Mcg by leucine and methionine in the Weir dimer should effectively block access to subsite B. In the hybrid binding activity for bis(dinitrophenyl)lysine is restored because the Mcg light chain is present as the monomer 2 subunit.

Daunomycin-arachidonic acid complex as a potential new antitumor agent.

A daunomycin-arachidonic acid complex (DM-C20:4) administered IV had a marked antitumor effect on the intraperitoneal growth of the AFP-producing hepatoma cell line, AH66. However, the daunomycin-arachidic acid (DM-C20:0) saturated fatty acid complex showed only the same antitumor effect on the AH66 cells as free daunomycin. It was demonstrated that the DM-C20:4 preparation maintained its chemical properties for some time in both plasma and liver homogenate.

Isolation and biological activity of aspidospermine and quebrachamine from an Aspidosperma tree source.

The indolealkaloids aspidospermine and quebrachamine have been isolated in crystalline form by a relatively rapid fractionation from the extract of a powdered material designated "Quebracho" derived from an Aspidosperma tree species. We present a novel isocratic LC method that provides baseline resolution of these two compounds and of the structurally related yohimbine in less than 15 min. Gas chromatography-mass spectrometry was employed to identify these compounds as well as several minor derivatives of aspidospermine during and after the purification process. Aspidospermine and quebrachamine like yohimbine have been found to possess adrenergic blocking activities for a variety of urogenital tissues.

Simplified methods for isolation of ubiquitin from erythrocytes. Generation of ubiquitin polymers.

1. Ubiquitin has been isolated from bovine erythrocytes by procedures in which the hemoglobin was removed by denaturation with either ethanol-chloroform mixtures or by heating. 2. The proteins soluble to the denaturation step were removed by 3% sodium trichloroacetate (TCA) at pH 2.0-2.5 or by 5% TCA. 3. Ubiquitin was isolated in relatively high yield from the TCA insoluble fraction by use of single ion-exchange chromatographic and gel permeation steps. 4. Ubiquitin shows relatively little cross-linking upon treatment with glutaraldehyde or with dimethyl suberimidate. Heating of the glutaraldehyde treated material in 4 M guanidine, however, leads to marked aggregation. 5. The polymers of ubiquitin react strongly with antibody in an immunoblot assay.

Carbonic anhydrases.

Some of the current studies of carbonic anhydrases are directed to the genetic mechanisms underlying their synthesis. Determination of the structure of their genes will probably most readily resolve the question of whether the membrane bound forms of the enzyme represent products of additional loci other than those of the three well-known soluble forms. Extensions of our knowledge of the sequences of these isozymes as well as those from lower animals and from plants will make possible a more precise evaluation of the extent of the multigene aspects of these proteins and their evolutionary backgrounds. Studies of the interrelationships of the regulation of the transcriptional and translational processes of the well-known isozymes and in particular the effects of hormones will be of interest. Insights into modifications of the isozymes' synthetic processes occurring in various diseases should also be forth-coming from these studies. In addition to the above the applications of what are perhaps today somewhat classical methods of protein chemistry will be needed to explore the reasons for the changes in activity accompanying the sequence variations of the different isozymes, the decreases or increases in activity accompanying derivatizations of specific residues and the reasons for the differences in the activity of different inhibitors on the various isozymes. The broad specificity of these enzymes for different substrates and the ability of CA-III to hydrolyze various phenyl esters and in some cases to become derivatized also present problems in protein structural chemistry. In terms of the latter reactions, the meaning of the relationships of these activities to those of the protein ubiquitin, which is homologous to CA-III, needs clarification. It would appear that various of the protein structural studies will be aided by crystallographic investigations of not only CA-III but of various of its derivatives which undergo either increases or decreases in activity. The above areas of studies present a wide variety of problems for workers in various disciplines and backgrounds who are interested in the carbonic anhydrases.

Variable-region sequence homologies of human kappa-chains with idiotypic serologic similarities.

An antibody to the V-region of a kappa I light chain has been used to detect other human kappa-chains with similar idiotypic specificities that appear to be related to complementarity-determining regions (CDR). Four such proteins, three of which react with this antibody, have been sequenced. While the proteins show relatively strong sequence similarities in their CDR2 regions, the relatively high degree of homology in a localized segment of the V-region of lambda-chains detected by this method was not observed here.