20K Dalton Research Articles

Osteo-anabolic effects of human growth hormone with 22K- and 20K Daltons on human osteoblast-like cells.

Human growth hormone with 22,000 Dal (22K-hGH) stimulates proliferation and differentiation of osteoblasts as well as production of interleukin-6 in vitro and bone formation and remodeling in vivo. To investigate whether hGH isoform with 20Kd (20K-hGH), which accounts for 10% of circulating hGH, elicits similar metabolic effects on skeletal tissues, we studied the biological effects of 20K-hGH in cultured human osteoblast-like cells (HOB). HOB were obtained from trabecular bone explants and cultured in alpha-MEM supplemented with 10% FCS. In subconfluent cultures, 22K- and 20K-hGH stimulated [3H]thymidine incorporation by 62 +/- 27% and 63 +/- 23%, respectively (mean +/- SD, n=8, P>0.1). In confluent cultures, 22K- and 20K-hGH increased alkaline phosphatase activity by 38 +/- 23% and 41 +/- 23% (P>0.1), respectively, and increased the osteocalcin concentration in the presence of 10(-9) M 1,25-(OH)2D3 by 50% and 47% (P>0.1), respectively. Furthermore, both hGHs doubled the interleukin-6 (IL-6) concentration in the conditioned medium. RT-PCR analysis revealed that 22K- and 20K- hGH increased IL-6 gene expression 2.2 +/- 0.6 and 2.4 +/- 0.7 -fold, respectively. In summary, we have demonstrated that 20K-hGH elicits equipotent anabolic effects on HOB and stimulates to the same extent the production of IL-6, a cytokine which initiates osteoclastogenesis. These in vitro findings suggest that 22K- and 20K-hGH may equipotently stimulate bone remodeling and elicit anabolic effects on skeletal tissue when administered in vivo.

Anti-Platelet Action of GU-7, A 3-Arylcoumarin Derivative, Purified from Glycyrrhizae Radix

We have purified GU-7, a 3-arylcoumarin derivative, from glycyrrhizae radix, which is a crude drug of kampo herbal medicines. This was identified to be a new chemical compound and was found to have an anti-platelet action. GU-7 inhibited platelet aggregation, phosphorylation of 40K and 20K dalton proteins, inositol 1,4,5-trisphosphate production, intraplatelet calcium increase and phosphodiesterase activity in vitro. The data indicate that GU-7 inhibits platelet aggregation by increasing intraplatelet cAMP concentration. We have already reported the existence of aldose reductase inhibitors in some kampo medicines. In addition to the aldose reductase inhibiting action, anti-platelet action may also explain the mechanism by which kampo medicines are effective to diabetic neuropathy.

Biologic Properties of the 20K-Dalton Variant of Human Growth Hormone: A Review

1. The hGH20K variant accounts for 10-15% of the hGH in the pituitary gland and is, therefore, the second most abundant hormone in the gland.2. Although no specific RIA exists for hGH20K, which must be measured by a combination of electrophoretic and immunological techniques, the following conclusions appear warranted. During provocative in vivo secretion, the ratio of hGH20K to hGH22K in blood is similar to that found in the pituitary gland. During basal in vivo secretion of hGH, the ratio of hGH22K to hGH20K can be quite different. A differential secretion rate for hGH22K and hGH20K was noted during in vitro stimulation of pituitary cells with GHRH. A six-fold rise in hGH 20K contrasted with only a twofold rise in hGH22K.3. The 20K dalton variant has full growth promoting activity and stimulates production of somatomedins even though binding hGH 20K to membrane receptors for hGH is lower than that observed with hGH22K.4. In vitro insulin-like activity of hGH20K on adipose tissue is either absent or at best 10-15% of that seen with hGH22K, whereas in vivo insulin-like activity of hGH 20K is 30-40% of that observed with hGH22K.5. At high doses, hGH 20K exhibits about the same anti-insulin activity as does hGH22K.

Demonstration of high molecular weight forms of inhibin in bovine follicular fluid (bFF) by using monoclonal antibodies to bFF 32K inhibin

Monoclonal antibodies specifically recognizing each 20K and 13K subunit of bovine follicular fluid (bFF) 32K inhibin have been prepared. By immunoblotting procedures using these antibodies, we have demonstrated in bFF at least six inhibin forms, the apparent molecular weights of which are estimated to be 120K, 108K, 88K, 65K, 55K and 32K daltons, respectively. Amongst them 65K, 55K and 32K inhibin forms comprise two polypeptide subunits linked by disulfide bridge(s). In these inhibin forms a polypeptide of 13K daltons, a shorter subunit of bFF 32K inhibin, is attached by disulfide linkage(s) to polypeptides of 57K, 44K and 20K daltons, which are immunologically related to a larger subunit of the 32K inhibin, to yield 65K, 55K and 32K inhibins, respectively. On the other hand the higher molecular weight forms, 120K, 108K and 88K inhibins, are composed of three polypeptide subunits. In these forms a polypeptide of 62K daltons, immunologically related to the shorter subunit of bFF 32K inhibin, is attached by disulfide bridge(s) as the third component to the respective smaller inhibin forms which are composed of two subunits. These findings suggest that the complex formation of the subunit precursors may be extremely important and that restricted proteolytic cleavages following the complex formation may yield such divergent forms of inhibin in bFF.

Monoclonal Antibodies Distinguish between the Head Portions of Two Myosin Isozymes from Chicken Breast Muscle

Monoclonal antibodies against chicken breast myosin and its subfragment-1(S-1) were produced. One antibody, 2G41, reacted with S-1 containing a light chain 3 (LC3), but not with another S-1 containing a light chain 1 (LC1) or a mixture of the light chains. A structural difference can be assumed to exist between the head portions of the two myosin isozymes. Antigenicity of S-1 toward 2G41 could not be detected after tryptic digestion into three fragments of 50K, 27K, and 20K daltons. Another monoclonal antibody, M68, was obtained from mice immunized with myosin. M68 preferably recognized the heavy chain from S-1 containing LC3 rather than that from that containing LC1 or S-1. M68 reacted with the 27K fragment among the three.

Phosphorylation of cytoskeletal proteins tightly associated with the vascular smooth muscle membranes.

A study was performed to determine whether the proteins phosphorylated by cAMP dependent and calmodulin dependent protein kinase in vascular smooth muscle membrane fractions represent integral membrane proteins or are tightly associated cytoskeletal proteins. In the unextracted microsomal fraction cAMP dependent protein kinase phosphorylated proteins of 240K, 105K, and 48K daltons. Similarly, calmodulin dependent protein kinase phosphorylated 65K, 60K, 48K, and 20K dalton bands. The 48K dalton band represented a major protein in the microsomal fraction, and it was a common substrate for both cAMP dependent and calmodulin dependent protein kinase, and the extent of phosphorylation by two kinases was additive. The 48K dalton band showed immunological reaction with monoclonal antibodies raised against human umbilical artery F actin, and it also comigrated with arterial smooth muscle actin on SDS gel electrophoresis. Plasma membranes prepared from vascular smooth muscle microsomes after extraction with actomyosin extraction buffer consisting of 2 mmol X litre-1 TRIS-maleate, pH 6.8, 0.25 mol X litre-1 sucrose, 0.5 mmol X litre-1 ATP, 0.2 mmol X litre-1 CaCl2, 0.1 mmol X litre-1 phenylmethylsulphonylfluoride, and 1 mmol X litre-1 dithiothreitol yielded membranes that were substantially free from cytoskeletal protein contamination. These membranes were enriched 60-80 fold in plasma membrane marker enzymes and showed energy dependent calcium uptake. In the extracted plasma membranes none of the proteins was phosphorylated by cAMP dependent or calmodulin dependent protein kinase. Thus these results show that protein phosphorylated in the unextracted microsomal fraction or unextracted plasma membranes are not integral plasma membrane proteins but represent tightly associated cytoskeletal proteins.

Correlation between the papain digestibility and the conformation of 10s-myosin from chicken gizzard.

In our previous reports, ATP was shown to induce a drastic change in the conformation of gizzard myosin molecules. For example, the sedimentation constant of unphosphorylated myosin (UM) increased from 6S to 10S although an ATP-induced change in the sedimentation constant did not occur with phosphorylated myosin (Suzuki et al. (1978) J. Biochem. 84, 1529). We now report the finding that the ATP-induced formation of 10S-myosin is associated with a drastic change in the papain digestibility of gizzard UM. With 10S-myosin, the cleavage by papain was strongly inhibited at two regions on heavy chains and at one region on light chains; that is, the junction between the 72K dalton and 22K dalton fragments (i.e., a cleavable site in myosin head), the one between the 22K dalton and 130K dalton fragments (i.e., a head-tail junction), and the one between the 3K dalton and 17K dalton fragments of 20K dalton light chains. An even more intimate correlation between the myosin conformation and the papain digestibility of myosin was demonstrated by using thiophosphorylated myosin (thioPM); the cleavages by papain at the 72K-22K dalton junction and the 22K-130K dalton junction were not inhibited when thioPM was digested.

Alteration of plasma membrane of drug-resistant tumor cells: 230-Kilodalton protein identified by monoclonal antibody

A number of hybridomas producing antibodies to plasma membrane of an aclarubicin-resistant subline of L5178Y cells were obtained. Among the hybridoma antibodies, one was found by agglutination tests to react with the aclarubicinresistant cell line, but not significantly with the parental and adriamycin-, bleomycin- and macromomycin-resistant cell lines. The monoclonal antibody was designated SC438, and showed complement-dependent cytolytic activity more markedly against the aclarubicin-resistant cells than against the parental cells. Fluorographs of [ 14C] leucine-labeled aclarubicin-resistant cells demonstrated two protein bands of 230k and 20k daltons, which were precipitated by the SC438 antibody. The former seemed to be specific for the aclarubicin-resistant cells.

Separation and Assignment of the Tryptic Peptides of Human Growth Hormone (hGH) and the 20K Dalton hGH Variant by Reversed Phase High Performance Liquid Chromatography

Abstract Reversed-phase high performance liquid chromatography (RP-HPLC) has been used to separate the peptides generated by tryptic cleavage of human growth hormone (hGH) and the 20K dalton human growth hormone variant. The total amino acid compositions of both these pituitary proteins has been accounted for on the basis of these chromatographic mapping procedures. Structural analyses of the peptides isolated from semi-preparative RP-HPLC separations has confirmed that the primary structure of the variant differs from that of the sequence of hGH by deletion of the amino acid residues 32–46.

Characterization of radiolabeled components of human sperm surface and seminal plasma.

Externally oriented components on the human sperm cell surface and components in human seminal plasma were labeled by enzymatic iodination with lactoperoxidase and [125I] NaI. SDS-7.5% PAGE of labeled sperm surface resolved one minor and four major components with approximate molecular weights of 92, 72, 46, 30, and 20K daltons, respectively. SDS-7.5% PAGE of labeled seminal plasma resolved five components with approximate molecular weights of 74, 51, 43, 28, and 20K daltons. Three of the five moieties seen on the sperm surface and in seminal plasma were similar in molecular weight. This suggested that these surface components were adsorbed from seminal secretions. Because the iodination procedure used labels both proteins and lipids, labeled sperm surface and labeled seminal plasma were subjected to isopycnic density gradient centrifugation to identify the chemical composition of the radioiodinated components. With human sperm surface, two areas of radioactivity were resolved in CsCl gradients, one corresponding to protein and the other to lipid. With human seminal plasma, only one area of radioactivity, corresponding to protein, was identified. Electrophoretic analysis of each peak of radioactivity obtained from the gradients demonstrated that all of the sperm surface and four of five seminal plasma components were in the protein fractions. All three of the seminal plasma components which correspond to sperm surface components were recovered in the protein fraction. This observation supports our hypothesis that some of the proteins labeled on the human sperm cell surface are adsorbed from seminal secretions.

Involvement of 17K dalton light chain of smooth muscle myosin in substrate-induced conformational change.

Conformational changes of the region involving 17K dalton light chain (G2) in gizzard myosin induced by ATP and its analogs were studied by a combination of light chain exchange and fluorescence spectroscopy. The cysteinyl residues of myosin light chain mixture, 20K dalton G1 and 17K dalton G2 chain, were labeled with a fluorescent thiol reagent, N-(1-anilionon-aphthyl-4ymaleimide (ANM). Chicken gizzard myosin was incubated with a large excess of the fluorescence labeled exogenous light chains in 2 M urea. After removal of urea and unbound light chains, a strong fluorescent band was observed at the position of the 17K dalton light chain (G2) of myosin on SDS PAGE. There was no difference in enzymatic activity and light chain composition between myosins before and after light chain exchange. The emission spectrum of ANM label in 17K dalton light chain in the bound state in myosin showed a fluorescence enhancement and a blue shift on adding ATP but not ADP. The spectral change disappeared after conversion of ATP to ADP. Various nucleoside triphosphates other than ATP had similar effects on the fluorescence spectrum. The relative effectiveness of most nucleotides on the fluorescence spectral change of ANM in the light chain bound in myosin agreed well with that on tryptophan residues in myosin, except in the case of ADP and CTP. These results suggest that 17K dalton light chain of gizzard myosin is closely related to the ATPase site of myosin.

Active calcium treatment transport via coupling between the enzymatic and the ionophoric sites of Ca2+ + Mg2+-ATPase.

The 20K dalton fragment of Ca2+ + Mg2+-ATPase obtained from th tryptically digested sarcoplasmic reticulum has been further purified using Bio-Gel P-100. This removed low-molecular-weight UV-absorbing and positive Lowry-reacting contaminants. The ionophoric activity of the 20K fragment in both oxidized cholesterol and phosphatidylcholine:cholesterol membranes is unaltered by this further purification. The 20K selectivity sequence in phosphatidylcholine:cholesterol membrane is Ba2+ greater than Ca2+ greater than Sr2+ greater than Mn2+ Mg2+. Digestion of intact sarcoplasmic reticulum vesicles with trypsin, which results in the dissection of the hydrolytic site (30K) from the ionophoric site (20K), is shown to disrupt energy transduction between ATP hydrolysis and calcium transport. This further implicates the 20K dalton fragment as a calcium transport site. These data and previous evidence are discussed in terms of a proposed model for the ATPase molecular structure and the mechanisms of cation transport in sarcoplasmic reticulum.