Felix Friedberg

Co++ binding by plasma proteins

Abstract 50Co++ added to human or rabbit plasmain vitro combines selectively with the albumin fraction. At pH 7.1, the Scatchard plot yields a curve that may be resolved for two classes of sites: one, wheren1 = 2 andK1 = 6.5 × 103, the other wheren2 = 23 andK2 = 1.6 × 102, Mn++ cannot compete for these sites. The affinity for the cobaltous ion displays a steep increment between pH 6.7 and 8.6. If injected intravenously 50% of the cobalt tracer is cleared from the plasma in 25 minutes. An additional complex of higher molecular weight than that of albumin is formedin vivo.

EFFECT OF IRRADIATION ON SOME LYOPHILIZED PROTEINS.

Exposure of lyophilized native ribonuclease or lysozyme in vacuo to about 30 Mrads causes aggregation of the protein molecules. There is no evidence of extensive degradation of these globular molecules. Guanidine-HCl (4 M) does not disassemble the aggregated material. If the S-carboxymethylated form of ribonuclease or lysozyme (that is, a form devoid of disulfide linkages) is exposed to the gamma ray, there is also no indication of marked degradation of the polypeptide chain, and again the molecules are prone to aggregate. On the other hand, lathyritic collagen exhibits a definite lowering of molecular weight, under the same conditions. Equivalent irradiation in an hydrogen sulfide atomosphere rather than in vacuo causes a diminished aggregation of the S-carboxymethylated ribonuclease and less degradation of the collagen.

On the primary structure of amylases

There may be a family of enzymes (such as the serine proteases) of carbohydrate hydrolases where certain amino acid residues compose the active site, and while the sequence may not be homologous, the arrangement of the catalytic site may be (as is the case for trypsin and subtilisin). This question is even more intriguing because of the nuances in specificity of action that exist for the amylases (e.g., cy and ,B, saccharifying and liquefying, etc.). dispensible for enzyme action can be more finely defined.

Calmodulin genes in zebrafish (revisited).

Calmodulin (CaM), a ubiquitous protein, ancestral in early eukaryotes, regulates a large number of physiologically important functions by activating other proteins, some of them enzymes, usually in response to changes in the local concentration of calcium ions. Invertebrates possess one gene that codes for CaM. Among vertebrates, mammals display three genes that code for a 100% identical CaM molecule, while for zebra fishes etc., a non-mammalian vertebrate, we reported earlier the existence of four such genes. The number of multiple genes coding for a 100% identical CaM molecule present in the zebra fish genome, however, is corrected here, from the four, as previously suggested, to six (alpha, alpha2, beta, beta2, gamma and gamma 2). Identification of each of these genes is readily achieved upon examination of the characteristic 5′ and 3′ UTRs within their respective mRNAs even though we do not know at present what role these UTRs might play. A scanning of the 3′ UTRs for short homology elements among the six genes (and a comparison with the human type I, II, and III CaM 3′ UTRs) also suggests that duplication processes for three genes resulted in the formation of six such genes. As they become available, the promoter regions for these six genes should be scanned for possible identification of putative regulatory elements if we are to understand the need for the uniquely rigid evolutionary maintenance of these six genes. A comparison of the promoter regions for the beta and beta 2 genes is presented in this paper. A few common short homologous elements appear to be retained in these generally highly variant two regions, but conclusions about differential expression controls must be delayed until the promoter regions for all the other CaM genes have been examined.

Alternative splicing for members of human mosaic domain superfamilies. I. The CH and LIM domains containing group of proteins.

In this paper we examine (restricted to homo sapiens) the products resulting from gene duplication and the subsequent alternative splicing for the members of a multidomain group of proteins which possess the evolutionary conserved calponin homology CH domain, i.e. an “actin binding domain”, as a singlet and which, in addition, contain the conserved cysteine rich double Zn finger possessing Lim domain, also as a singlet. Seven genes, resulting from gene duplications, were identified that code for seven group members for which pre-mRNAs appear to have undergone multiple alternative splicing: Mical 1, 2 and 3 are located on chromosomes 6q21, 11p15 and 22q11, respectively. The LMO7 gene is present on chromosome 13q22 and the LIMCH1 gene on chromosome 4p13. Micall1 is mapped to chromosome 22q13 and Micall2 to chromosome 7p22. Translated Gen/Bank ESTs suggest the existence of multiple products alternatively spliced from the pre-mRNAs encoded by these genes. Characteristic indicators of such splicing among the proteins derived from one gene must include containment of some common extensive 100% identical regions. In some instances only one exon might be partly or completely eliminated. Sometimes alternative splicing is also associated with an increased frequency of creation of an exon or part of an exon from an intron. Not only coding regions for the body of the protein but also for its N– or –C ends could be affected by the splicing. If created forms are merely beginning at different starting points but remain identical in sequence thereafter, their existence as products of alternate splicing must be questioned. In the splicings, described in this paper, multiple isoforms rather than a single isoform appear as products during the gene expression.

COMPARISON OF GAMMA-RADIATION EFFECTS ON SOLID ATP-CREATINE PHOSPHOTRANSFERASE AND GELATIN

Recent studies (1-4) have shown that differences in radiosensitivity of various amino acid residues within proteins irradiated by y-rays in the dry state are small and that there is no selective breakage of sulfur-sulfur bonds or other covalent structures. In irradiated gelatin and in other fibrous proteins (and also in polyamino acids) the main polypeptide chain is broken, but such degradative action is not apparent with globular proteins. This investigation was undertaken to further substantiate the differences in the properties of these two types of proteins after exposure to y-rays in the dry state.

Single and multiple CH (calponin homology) domain containing multidomain proteins in Dictyostelium discoideum: an inventory

We present an inventory of single or multiple calponin homology (CH) domain containing proteins of Dictyostelium discoideum. A multiple alignment and a phylogenetic tree of all 60 CH domains found in 36 proteins showed that most CH domains can be assigned to one of 6 types. We have then distributed the proteins into several classes according to the type and arrangement of the CH domains. Most proteins belong to the class of ABD (actin-binding domain)-forming CH tandems (CH1–CH2) of the α-actinin and fimbrin families or to the class of CH3 domain-bearing proteins. There are a few examples of proteins with a single CH1 or CH2 domain, one with a CH1–CH1 doublet and a single representative of the CHe class of microtubule-binding proteins. A comparison with CH domain proteins in Homo sapiens suggests that while the individual domains are available in both species, the existence of identical multidomain proteins in toto is rare. Fimbrin 1, α-actinin and EB1 appear as perfect orthologs in both species, whereas filamin and interaptin may represent ancestral forms of human filamin and nesprins. In four more cases (NAV/Unc-53-, smoothelin-, transgelin- and Gas2-related proteins) functional data are needed in order to establish a potential relationship with a human counterpart. Although extensive data exist for a few of the D. discoideum CH proteins, most remain to be characterized and our analysis may help predicting some of their properties.

Singlet CH domain containing human multidomain proteins: an inventory

The actin cytoskeleton presents the basic force in processes such as cytokinesis, endocytosis, vesicular trafficking and cell migration. Here, we list 30 human singlet CH (calpononin homology/actin binding) containing multidomain molecules, each encoded by one gene. We show the domain distributions as given by the SMART program. These mosaic proteins organize geographically the placement of selected proteins in proximity within the cell. In most instances, their precise location, their actin binding capacity by way of the singlet CH (or by other domains?) and their physiological functions need further elucidation. A dendrogram based solely on the relationship for the human singlet CH domains (in terms of AA sequences) for the various molecules that possess the domain, implies that the singlet descended from a common ancestor which in turn sprouted three main branches of protein products. Each branch bifurcated multiple times thus accounting for a cornucopia of products. Wherever, additional (unassigned), highly homologous regions exist in related proteins (e.g., in LIM and LMO7 or in Tangerin and EH/BP1), these unrecognized domain regions await assignment as specific functional domains. Frequently genes coding multidomain proteins duplicated. The varying modular nature within multidomain proteins should have accelerated evolutionary changes to a degree not feasible to achieve by means of mere post-duplication mutational changes.

Multiple calmodulin genes in fish

In mammals, identical calmodulin (CaM) proteins are encoded by three nonallelic genes that differ in their promoter regions and untranslated regions (UTRs). The UTRs of each of these three genes are specific for each gene and are highly conserved. In this study, sequences obtained from the GenBank and EST databases and sequencing were examined for several species of fish to ascertain whether this multi-gene one protein system exhibited in mammals extends to other vertebrates. Three genes in zebrafish (Danio rerio) designated α, β, and γwere identified. As in mammals, these genes differ in the 3′-UTR region but encode completely identical CaMs. PCR primers spanning the coding and the 3′-UTR regions were designed based on the assembled sequences and used to confirm the presence of each gene in the cDNA library. Other species of fish were also found to contain homologous genes that were closely related as indicated by phylogenetic analysis. The 3′-UTR of the α, βand particularly the γCaM gene of fish were not found to be as conserved as the corresponding genes of mammalian species possibly due to the span of evolutionary time. Only a few short elements in the 3′-UTR were observed to be similar in fish and mammals. These short regions of identity are shared primarily between the mammalian CaM II and CaM I and the αgene and βgene of fish, respectively. Thus, the multi-gene one protein system occurs among fish as well as among mammals.

The amino acid composition of adenosine triphosphate-creatine transphosphorylase.

Abstract The full amino acid composition of adenosine triphosphate-creatine transphosphorylase has been measured by using ion-exchange chromatography. In addition, other methods were employed for the estimation of tyrosine, tryptophan, arginine, and amide ammonia. Cystine values were calculated on the assumption that the difference between total sulfur and measured methionine sulfur represents cystine sulfur.