Distinct Subunit Types Research Articles

Isolation and partial characterization of a lectin from ground elder (Aegopodium podagraria) rhizomes

A lectin has been isolated from rhizomes of ground elder (Aegopodium podagraria) using a combination of affinity chromatography on erythrocyte membrane proteins immobilized on cross-linked agarose and hydroxyapatite, and ion-exchange chromatography. The molecular structure of the lectin was determined by gelfiltration, sucrose density-gradient centrifugation and gel electrophoresis under denaturing conditions. It has an unusually high Mr (about 480000) and is most probably an octamer composed of two distinct types of subunits with slightly different Mr (about 60000). Hapten inhibition assays indicated that the Aegopodium lectin is preferentially inhibited by N-acetylgalactosamine. Nevertheless, it does not agglutinate preferentially blood-group-A erythrocytes. The ground-elder lectin is a typical non-seed lectin, which occurs virtually exclusively in the underground rhizomes. In this organ it is an abundant protein as it represents up to 5% of the total protein content. The lectin content of the rhizome tissue varies strongly according to its particular location along the organ. In addition, the lectin content changes dramatically as a function of the seasons. The ground-elder lectin differs from all other plant lectins by its unusually high molecular weight. In addition, it is the first lectin to be isolated from a species of the family Apiaceae.

Rat lung glutathione S-transferases. Evidence for two distinct types of 22000-Mr subunits

Two immunologically distinct types of 22000-Mr subunits are present in rat lung glutathione S-transferases. One of these subunits is probably similar to Ya subunits of rat liver glutathione S-transferases, whereas the other subunit Ya' is immunologically distinct. Glutathione S-transferase II (pI7.2) of rat lung is a heterodimer (YaYa') of these subunits, and glutathione S-transferase VI (pI4.8) of rat lung is a homodimer of Ya' subunits. On hybridization in vitro of the subunits of glutathione S-transferase II of rat lung three active dimers having pI values 9.4, 7.2 and 4.8 are obtained. Immunological properties and substrate specificities indicate that the hybridized enzymes having pI7.2 and 4.8 correspond to glutathione S-transferases II and VI of rat lung respectively.

Assembly and calcium-induced cooperativity of Limulus IV hemocyanin: a model system for analysis of structure-function relationships in the absence of subunit heterogeneity

Hemocyanins, the high molecular weight copper proteins which serve as oxygen carriers in many arthropods and molluscs, are representative of multisubunit complexes which are capable of reversible dissociation and assembly. Although reversible, in many hemocyanins these processes are not in true thermodynamic equilibria, and it has been suggested that there is "microheterogeneity" among the molecules in solution. An alternative explanation is that their complex behavior is due to the existence of quaternary interactions between structurally distinct types of subunits within the native molecule which have varying pH and ionic strength sensitivity. Limulus IV hemocyanin was used as a model system to examine structure-function relationships in the absence of subunit heterogeneity. Purified subunit IV of Limulus polyphemus hemocyanin is homogeneous by a number of electrophoretic and immunological criteria and is capable of undergoing pH-dependent self-assembly into hexamers. The monomer-hexamer transition was found to be an equilibrium whose rate is dependent on the presence or absence of calcium ions. The observation that the assembly of this homopolymer behaves as a true equilibrium suggests that the nonequilibrium dissociation profiles observed for native Limulus hemocyanin are related to the extensive subunit heterogeneity of the native protein. In calcium-containing buffers, the monomer-hexamer transitions of Limulus IV hemocyanin can be described by a cooperative mechanism with approximately six protons per hexamer lost on assembly from acid pH and three protons gained on assembly from alkaline pH. Increased ionic strength or increased temperature favors dissociation. Like the native molecule, Limulus IV hemocyanin behaves as an allosteric protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity associated with individual subunits of pig heart NAD-specific isocitrate dehydrogenase

NAD-specific pig heart isocitrate dehydrogenase is composed of three distinct types of subunits: α, β, and γ, which have molecular weights of about 40,000 but differ in amino acid composition and in isoelectric points. When the native enzyme is subjected to polyacrylamide gel electrophoresis under nondenaturing conditions, two major protein bands with M r values of about 360,000 (band 1) and 100,000 (band 2) and two minor bands (bands 3 and 4) with M r values of about 40,000 are consistently present. Enzymatic activity, as detected from NADH fluorescence, is distributed throughout the protein-staining region. Analytical isoelectric focusing in urea reveals that band 1 is composed of all three subunits in roughly the normal ratio of 2α:1β:1γ, and is probably an octamer, band 2 of an equal amount of α and β and is probably dimer, while bands 3 and 4 each consist of only the monomeric α subunit. The highest enzymatic specific activity is associated with a region intermediate between octamer and dimer, which includes the 160,000 tetramer. The protein pattern resulting from isoelectric focusing under nondenaturing conditions consists of protein bands comparable in pattern to those in the presence of urea along with bands of intermediate p I values, many of which are associated with enzymatic activity. Analysis of the subunit composition of these bands supports the activity of the α species in isolation and establishes the activity of the separated β component. No activity of the isolated γ subunit species has thus far been demonstrated. However, the highest apparent specific activity is observed when at least two types of subunits are present. These studies indicate that a range of oligomeric species of the enzyme are enzymatically active and that at least three of the four subunit chains comprising the minimum complete enzyme molecule (2α:1β:1γ) possess an active site.

Chemical modification A probe of the structure and function of the subunits of DPN-dependent isocitrate dehydrogenase.

DPN-dependent isocitrate dehydrogenase is composed of three distinct types of subunits: alpha, beta, and gamma which have molecular weights of about 40,000 but differ in isoelectric points. The relationship of subunit diversity to function was probed by use of chemical modification. 3-Bromo-2-ketoglutarate, a substrate and affinity label for the active site of isocitrate dehydrogenase, was shown to cause significant modification of all types of subunits. The substrate affinity label, 3-ene-2-keto-glutarate, labels each of the subunits equally. Approximately equal labeling of subunits was also found upon modification by cyanate of an essential lysyl residue in the isocitrate binding site. When enzyme was inactivated by a carbodiimide in the presence of glycine ethyl ester, both glutamate and aspartate residues reacted, and labeling of each type of subunit occurred. These studies suggest that the structurally distinct subunits of DPN-dependent isocitrate dehydrogenase are functionally similar and each type of subunit contains a substrate binding site.

Studies on enolase isoenzymes in normal and pathological human tissues

Enolase has three immunologically distinct subunit types a, p and y (Fletcher et al., 1976; Pearce et al., 1976). In the rat the /$subunit is essentially confined to the muscular tissues and the ysubunit to neurons and cells of the APUD (amine precursor uptake and decarboxylation) system (Schmechel et al., 1978). Immunological titration with monospecific antisera suggests that the distribution of these subunits in human tissue extracts is rather less specific and that the occurrence of the uphybrid may be widespread. These findings have important implications if enolase isoenzymes are to be used in diagnostic tests and particularly in determining damage to specific tissues (HerraezDominguez et al., 1975). The problem has been further investigated by immunohistolocalization studies on normal human tissues. The findings for the cellular distribution of a-, pand ysubunits of enolase are consistent with the results of immunological titration of tissue extracts. Preliminary work to assess the potential use of enolase isoenzymes as markers of pathological change has been carried out using the peroxidase/anti-peroxidase technique of immunohistolocalization on a wide range of tumours from tissues of neural-crest origin. The results of the studies on cerebral tumours show that the enolase isoenzyme composition of a tumour is similar to that of the cell type from which it is derived. Thus astrocytomas, ependymomas and acoustic neuromas show strong staining for a-enolase, oligodendrogliomas only weak staining for this isoenzyme, whereas neuroblastomas and medulloblastomas showed no enolase staining. No cerebral tumour was found to contain yenolase. Ischaemic and necrotic tissues showed diminished enolase activity and this might be expected to result in a leakage of enolase into the cerebrospinal fluid, accounting for the raised concentrations found in the cerebrospinal fluid of many patients with cerebral tumours (Royds et al., 1981). Melanomas are tumours derived from cells that originate from the neural crest, and immunohistolocalization studies of the aand yenolases show that the malignant melanocytes of the dermis normally contain both of these isoenzymes. No enolase could be detected either in normal or malignant melanocytes that are found in the epidermis. This contrasts with the finding that benign intradermal naevi show strong staining for both aand yisoenzymes with a-enolase usually present in excess. The a/y ratio appears to increase with the degree of malignancy with some of the highly de-differentiated cells having no demonstrable yenolase. Tumours of APUD cells have been studied using the same immunological staining technique. Most tumours stained for both aand yenolase with the exception of the oat-cell carcinoma, where five out of 11 cases showed only a-enolase.

A Mutant A-Type Lactate Dehydrogenase Subunit in Fundulus heteroclitus (Pisces: Cyprinodontidae)

The rarity of mutant alleles at the loci coding for the polypeptide subunits of the tetrameric enzyme lactate dehydrogenase (LDH) is emphasized in all vertebrate classes. Fundulus heteroclitus synthesizes three physicochemically distinct types of LDH subunits (A, B and E) under the direction of three genetic loci. Two codominant alleles are present at the B locus which code for the synthesis of two B-type subunits (B and B'). Antisera specific to fish LDH subunits selectively precipitate LDH isozymes from tissue homogenates of F. heteroclitus. Coupled with starch gel electrophoresis and enzyme specific staining techniques, it is possible to determine the LDH isozyme composition of tissues and the genetic interrelationships of the isozymes. Employing this procedure, we have discovered a rare A-locus allele coding for a subunit designated A'. At alkaline pH's, the A' subunit is less negatively charged than the A subunit and migrates more slowly toward the anode. This allele was found only in one out of more than 300 specimens examined. This specimen was an AA' heterozygote.

An investigation of bacteriophage lambda, its protein ghosts and subunits

The sedimentation coefficients and molecular weights of bacteriophage lambda and its ghost were determined as S o 20,w = 416 × 10 −13 second and M = 57 × 10 6 for the virus, and S o 20,w = 141 × 10 −13 second and M = 21 × 10 6 for the ghost. The protein portion of the virus consists of at least two distinct types of subunits, with molecular weights of 55,000 and 110,000, which differ markedly in their amino acid content.